scholarly journals Differential Effects of Iberdomide Versus Revlimid on Leukocyte Trafficking, Immune Activation and DLBCL Tumor Cell Killing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 718-718
Author(s):  
Yumi Nakayama ◽  
Hsiling Chiu ◽  
Rama K. Narla ◽  
Arvind Shakya ◽  
Jim Gamez ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Mouse Model ◽  
Nk Cells ◽  
Immune Cells ◽  
Stock Options ◽  
Substrate Degradation ◽  
Privately Held

Abstract Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev. Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p<0.01) were higher in PBMCs treated with Iber compared to Rev. Additionally, functional chemotaxis assays demonstrated that Iber and Rev increased the trafficking capacity of T-cells compared to DMSO alone, with Iber demonstrating a greater increase than Rev (46% vs 21%, p<0.01). Furthermore, Iber increased the proliferative capacity of CD8+ T and NK cells compared to Rev (10 and 3.6-fold vs 4 and 2.8-fold, respectively). Functional co-culture assays with DLBCL cells showed that Iber induced NK cell mediated killing of DLBCL cells to a greater extent than Rev and each molecule enhanced ADCC with Rituximab compared to vehicle controls. Translational data from clinical trials of a related CELMoD, Avadomide, revealed significant trafficking of immune cells such a T cells, NK cells and monocytes to the tumor microenvironment (TME). To examine the effects of Rev and Iber in an in vivo DLBCL GEMM model, we developed a humanized CRBN (hCRBN) mouse capable of facilitating proteasomal degradation of target substrates upon treatment with a CELMoD. The hCRBN mouse was then crossed with the Eμ-Myc DLBCL mouse model resulting in Eμ-Myc/hCRBN progeny that then developed disease. Splenocytes were collected and transplanted to recipient hCRBN mice. The tumor cells were allowed to engraft for 5 days upon which 3 daily doses of vehicle, Rev and Iber were given prior to the mice being sacrificed. Non-transplanted hCRBN mice served as controls. Similar to human disease, DLBCL cells remodeled the myofibroblast-immune network within lymph node and the splenic tissues including activated podoplanin (PDPN)-expressing fibroblastic reticular cells (FRCs) and diminished CD8+ T cells and CD11c+ DCs within the lymphoid TMEs. Treatment with Iber resulted in significantly enhanced infiltration of DCs and notably, cytolytic granzyme B positive T cells into the TME compared to Rev or vehicle treated mice (Figure 1). Additional characterization of the immune (T cell, NK and monocyte)-stroma TME is on-going and will be presented. Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. Figure 1 Figure 1. Disclosures Nakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

scholarly journals NL-201, a De Novo Agonist of IL-2 and IL-15 Receptors, Demonstrates Synergistic Antitumor Activity with Anti-PD-1 Checkpoint Inhibitor Therapy in a Preclinical Non-Hodgkin Lymphoma Model

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4560-4560
Author(s):  
Justin Huard ◽  
Laurie Tatalick ◽  
Carl Walkey ◽  
Ryan Swanson
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Hodgkin Lymphoma ◽  
Stock Options ◽  
Checkpoint Inhibitor ◽  
Publicly Traded ◽  
Non Hodgkin Lymphoma ◽  
Checkpoint Inhibitor Therapy ◽  
Privately Held

Abstract NL-201 is a potent, selective, and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors that is being developed as an immunotherapy for cancer. NL-201 binds to the beta and gamma subunits, selectively stimulating dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and natural killer (NK) cells, thereby enhancing the immune response to the tumor. Absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. In this abstract, we demonstrate that NL-201, alone or in combination, demonstrates robust antitumor activity in preclinical models of non-Hodgkin lymphoma (NHL). We have previously demonstrated that NL-201 has marked antitumor activity in multiple syngeneic tumor models, including the A20 lymphoma model. These observations in lymphoma have been extended to explore the effects of NL-201 in combination with anti-mPD-1 checkpoint inhibitor therapy in vivo. In this model, NL-201 and anti-mPD-1 demonstrated tumor growth inhibition and increased median survival (21 days each vs 17 days as observed in control) when given alone. In combination, NL-201 and anti-mPD-1 resulted in increased antitumor activity and significant prolongation of survival (>51 days). We have also demonstrated that NL-201 does not directly induce signaling or cell death in B cell−derived NHL, suggesting that the observed antitumor activity is due to activation of non-malignant host immune cells. Additional in vitro and in vivo NHL models are being tested to enhance understanding of the interaction between NL-201 and other approved therapies within the hematopoietic tumor microenvironment. These data will be used to design future clinical trials of NL-201 in novel regimens to treat hematological malignancies. Disclosures Huard: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Tatalick: Neoleukin Therapeutics, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Independent paid nonclinical consultant for Neoleukin. Walkey: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Swanson: Neoleukin Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

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

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

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

scholarly journals Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer Immunotherapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2771-2771
Author(s):  
Mark A Wallet ◽  
Toshinobu Nishimura ◽  
Christina Del Casale ◽  
Andriana Lebid ◽  
Brenda Salantes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stock Options ◽  
Γδ T Cells ◽  
Differentiation Process ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T ◽  
Privately Held

Abstract Introduction Allogenic CAR-T cell therapies for cancer provide a new option to reduce barriers faced by autologous cell therapies, but several challenges remain. One challenge is the risk of graft versus host disease (GvHD) caused by the infused T cells. A potential solution is the use of a subset of gamma delta (γδ) CAR-T cells whose T cell receptors (TCRs) recognize invariant antigens rather than hypervariable MHC molecules. Here we describe an off-the-shelf, induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ CAR-iT) for treatment of cancer and a process for deriving such cells. Methods T cell-derived iPSCs (TiPSC) are generated by reprogramming γδ T cells to yield pluripotent stem cells. For proof-of-concept studies, TiPSC were engineered using CRISPR gene editing to deliver a CD19 CAR transgene. TiPSC are then subjected to a two-stage differentiation process. First, TiPSC are differentiated into CD34-expressing hematopoietic progenitor cells (HPCs). HPCs are then exposed to a feeder-free differentiation process that results in uniform γδ CAR-iT cells. The purity and identity of γδ CAR-iT cells were assessed by flow cytometry and the ability of γδ CAR-iT cells to respond to homeostatic growth factors was determined by intracellular staining of phosphorylated signaling proteins and mRNA transcriptome analysis. Cytokine production by CAR-iT cells was measured by immunoassays following stimulation of the CAR. Tumor cell killing by γδ CAR-iT cells was performed using IncuCyte cytotoxicity assays. In vivo control of tumors by γδ CAR-iT in immunodeficient mice was determined using a NALM-6 B cell lymphoblastic xenograft model. Results A research-grade γδ TiPSC line was used to develop an iT differentiation process. This γδ TiPSC line was engineered to express a CD19 CAR molecule and then subjected to the differentiation process after which >95% of cells were CD3 + γδ TCR + CAR + iT cells. These γδ CAR-iT cells responded to IL-2 and IL-15. STAT5 phosphorylation levels were similar but STAT3 phosphorylation levels were greater in response to IL-15 compared to IL-2 at equimolar concentrations of cytokine. IL-2 and IL-15 elicited qualitatively similar transcriptional responses, but the magnitude of cytokine-induced gene expression was generally greater in IL-15-treated cells. Upon activation, γδ CAR-iT cells released markedly less IFN-γ and other inflammatory cytokines than conventional blood-derived ab CAR-T cells. In an IncuCyte serial killing assay, γδ CAR-iT cells exhibited sustained killing of NALM-6 tumor cells for at least one week in the presence of IL-15. In vivo, γδ CAR-iT cells caused a significant reduction in NALM-6 tumor burden with a single dose of γδ CAR-iT resulting in >95% tumor growth inhibition. To establish an efficient method for derivation of clinical grade γδ TiPSC lines, we investigated methods to isolate, expand, and reprogram human γδ T cells. When γδ T cells were expanded by exposure to the chemical zoledronic acid (zoledronate) and IL-2, we found a large disparity between donors; some donors exhibit robust expansion while others are seemingly resistant to zoledronate. In order to enhance γδ T cell expansion we screened dozens of activation conditions and eventually established a universal activation protocol that can elicit robust expansion of γδ T cells from all donors tested. When expanded γδ T cells were subjected to reprogramming conditions, dozens to hundreds of individual TiPSC colonies were obtained from each donor. The identity of the rearranged γδ TCR locus was confirmed using molecular assays. New γδ TiPSC lines were engineered with a CD19 CAR molecule and killing activity was confirmed in an in vitro serial killing assay. Conclusions γδ CAR-iT cells provide a new opportunity to treat cancers with an off-the-shelf universal T cell platform without the risk for GvHD. γδ CAR-iT cells are readily manufacturable, and we have derived an end-to-end process that enables new TiPSC line reprogramming, genetic modification of TiPSC lines, and feeder-free differentiation. γδ CAR-iT cells exhibit potent antigen-specific tumor killing and they release less inflammatory cytokine than conventional CAR-T cells, potentially reducing the risk for cytokine-mediated toxicities. We believe that this off-the-shelf platform will enable safer and more accessible allogenic cell therapies for hematologic and solid cancers. Disclosures Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Nishimura: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Del Casale: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lebid: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Salantes: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Santostefano: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bucher: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Thompson: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company.

698 Targeting HLA-G-mediated immunosuppression with a first-in-class antagonist antibody

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A740-A740
Author(s):  
Douglas Hodges ◽  
Christina Kochel ◽  
Michael Totagrande ◽  
Jeffrey Jones ◽  
Megan Welch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Solid Tumors ◽  
Immune Cells ◽  
Target Cells ◽  
Human Antibody ◽  
Advanced Solid Tumors ◽  
Tumor Types

BackgroundHuman leukocyte antigen-G (HLA-G) is an immune checkpoint molecule that belongs to the non-classical HLA-class I family of receptors. HLA-G restrains immune cell activation and effector function by engaging with inhibitory receptors ILT2 and ILT4. While expression of HLA-G is highly restricted under normal healthy conditions, we have demonstrated that its expression in cancer is aberrantly upregulated and broadly detected across a variety of tumor types. Tizona Therapeutics has generated a novel, fully human antibody that specifically targets HLA-G and reverses HLA-G-mediated immunosuppression. Here we present in vitro and in vivo data demonstrating the functional impact of HLA-G blockade on immune cells and evidence to support the use of TTX-080 in the clinic to treat patients with advanced solid tumors.MethodsEvaluation of HLA-G expression in cancer was performed using immunohistochemistry, flow cytometry, and gene profiling. Expression of ILT2 and ILT4 was assessed on tumor infiltrating leukocytes by flow cytometry. To demonstrate the suppressive function of HLA-G, primary human NK cells, T cells, and monocyte-derived macrophages were cultured with target cells expressing HLA-G. TTX-080 was then evaluated for its ability to reverse this suppression. In addition, TTX-080 was investigated in vivo using a disseminated xenograft tumor model.ResultsExpression of HLA-G was detected on tumor cells and tumor infiltrating leukocytes across a variety of solid tumor types. TTX-080 blocked interaction of HLA-G with both ILT2 and ILT4 and restored cytotoxicity in multiple assays using either primary NK cells or NKL cell lines. Monocyte-derived macrophages expressing ILT2 and ILT4 exhibited decreased phagocytosis of HLA-G+ target cells; this inhibition was reversed with an antigen-binding fragment of TTX-080. TTX-080 was also able to reverse HLA-G-mediated suppression of ILT2+ CD8+ T cells as assessed by degranulation and proinflammatory cytokine secretion. Notably, mice with disseminated tumors had extended median survival when treated with a single dose of TTX-080.ConclusionsTTX-080 reverses HLA-G-mediated suppression of ILT2+ and ILT4+ immune cells that are found within the tumor microenvironment. Blockade of HLA-G using TTX-080 therefore has the potential to reverse broad immune suppression in patients with advanced solid tumors by reinvigorating CD8+ T cells, enhancing NK cytolytic activity, and increasing macrophage phagocytosis.

scholarly journals Pre-Clinical Development of Gene Modification of Hematopoietic Stem Cells with Chimeric Antigen Receptors for Cancer Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4430-4430
Author(s):  
Laurel Christine Truscott ◽  
Sarah Larson ◽  
Amie Patel ◽  
Roy L. Kao ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
T Cells ◽  
Immune Cells ◽  
Suicide Gene ◽  
Antigen Specificity ◽  
Hematopoietic Stem ◽  
Cancer Activity

Abstract Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Innovative approaches are needed to decrease their morbidity and mortality. Chimeric Antigen Receptors (CAR) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Building upon the standard of care and seeking an approach that could foster persistence of the CAR-modified cells, we have published studies using anti-CD19 CAR-modified human hematopoietic stem cells (HSC) engrafted in NSG immunodeficient mice. We hypothesize that modification of HSC with CD19-specific CAR will generate persistent multi-lineage anti-tumor activity for immunotherapy of CD19+ hematological malignancies. To increase the safety of the modification of HSC, a suicide gene can be inserted into the vector to eradicate the modified cells in the setting of toxicity. Thorough evaluation of this approach in relevant study models is required for advancement to clinical trials. Significance: This approach is untested in clinical translation to this date, and implies harnessing a patient's own HSC to create a whole self-renewing immune system directed to destroy cancer, a concept that can be applied to different cancers just by adjusting the target specificity. The prospect of modifying autologous cells to enhance graft-versus-cancer activity bears the possibility of decreased morbidity and mortality, being desirable for specifically vulnerable populations, as children and elderly patients, and offering alternative therapy for those without cell sources available for allogeneic HSC transplantation, benefiting patients from ethnic minorities. This approach can be easily adopted in the clinical setting for patients planned to receive autologous HSC transplant as their standard therapy, enhancing graft-versus-cancer activity with anti-CD19 specificity. Methods: High-titer third-generation lentiviral constructs were produced carrying second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC isolated from umbilical cord blood and G-CSF-mobilized apheresed peripheral blood stem cells (PBSC) were transduced with such lentiviral vectors and injected into NSG pups after irradiation for in vivo evaluation of engraftment, function and suicide gene activation. Results: We have consistently achieved engraftment of human cells in about 95% of study mice, with engraftment of CAR+ cells in about 80% of the animals. Human HSC were successfully transduced with lentiviral vectors carrying anti-CD19 CAR with no impairment of differentiation or proliferation in vitro and in vivo. Immune cells differentiated in vivo from CAR+ HSC had antigen-specific cytotoxicity directed by CAR. CAR+ human cells were detected in BM, spleen, blood and thymus of injected mice. CAR+ T cells were stably detected in the blood of engrafted mice up to 40 weeks post-injection, demonstrating lymphopoiesis of CAR+ T cells successfully escaping thymic deletion and persisting throughout murine lifetime. As a surrogate of the antigen specificity and efficacy, CD19+ cells were significantly decreased in all mice engrafted with anti-CD19 CAR demonstrating that CAR+ immune cells were not inactivated or developed tolerance. Mice humanized with at least 4% of CAR+ cells in blood had significant protection against challenge with CD19+ tumor cell line, with inhibition or elimination of tumor development and consequent survival advantage. Activation of HSV-sr39TK suicide gene by ganciclovir treatment successfully led to ablation of gene-modified cells in vitro and in vivo. Conclusions: Our results demonstrate feasibility of CAR modification of human HSC for cancer immunotherapy. It could be easily employed in the context of HSC transplantation to augment the anti-cancer activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSC. Disclosures Larson: BMS: Consultancy.

scholarly journals Anti-FLT3 CAR T Cells in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1703-1703
Author(s):  
Sara Sleiman ◽  
Olga Shestova ◽  
Francisco Santiago ◽  
Elina Shrestha ◽  
Raymond Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Stock Options ◽  
Research Funding ◽  
Short Term ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T ◽  
Privately Held

Abstract INTRODUCTION In patients with AML who are eligible for intensive therapy, the goal of treatment is the achievement of complete response followed by consolidation chemotherapy (in favorable risk disease) or hematopoietic stem cell transplantation (in intermediate or adverse risk disease). Patients who do not attain this initial goal lack effective therapeutic options. Extensive experience with chimeric antigen receptor (CAR) T cells in B-ALL has shown that CART cells can deliver potent and durable antigen-specific leukemia control, and that targeting a single antigen (CD19 for B-ALL) is associated with antigen-negative relapse. In this context, we sought to expand the existing preclinical CART armamentarium in AML by developing FLT3-specific CART cells and comparing them to our existing gold standard CD123-specific CART cells. Since activating mutations in FLT3 occur commonly in AML, we reasoned that this molecule would serve as an "Achilles heel" in AML immunotherapy. METHODS Novel fully humanized anti-human FLT3 receptor single chain variable fragments (scFV) were fused to CD28 and CD137 (41BB) costimulatory molecules and the CD3zeta signaling domain and cloned into a lentiviral expression vector. Based on recently published data, we tested linker lengths ranging from 5 to 20 amino acids between the light and heavy chains of the CAR. We used a FLT3-ITD mutated AML cell line (MOLM14) expressing luciferase for in vitro function studies including an exhaustion assay. For in vivo function studies, we engrafted MOLM14 expressing luciferase into NSG mice and treated with CART-FLT3 or untransduced T cells (negative control). RESULTS All FLT3 and CD123-specific CART cells degranulated and produced the effector cytokines IL-2, INFg, TNF and GM-CSF in an antigen-specific manner, with some variability between the different linker lengths and with some superiority of the CAR123 likely resulting from the higher expression of CD123 compared with FLT3 in this model (p < 0.0001, one way ANOVA) (Figure 1). Short-term killing assays (24 hours) revealed that all CART cells killed MOLM14 with equivalent efficiency at low effector:target ratios (Figure 2A). Since short-term killing assays likely do not replicate the physiological situation in vivo wherein CART cells encounter cancer cells repeatedly over many days, we next developed an in vitro exhaustion assay. We incubated MOLM14 cells with CAR T cells at 1:10 E:T ratio and added MOLM 14 tumor cells along with fresh media every other day. Killing was quantified every 48 hours. Interestingly, all CAR constructs showed equivalently efficient cytotoxicity from days 5-15. However, after day 15 there was progressive dysfunction and loss of cytotoxic activity. This exhaustion "stress test" revealed some superiority of the FLT3 CAR 10AA construct (p = 0.042 on day 17, two way ANOVA) (Figure 2B). NOD/SCID gamma chain KO (NSG) mice were then engrafted with 1x10 6 luciferized MOLM14 cells and treated with 0.5x10 6 CAR T cells 7 days later, randomized to treatment groups based on tumor burden. CAR T cells expansion was monitored in peripheral blood by flow cytometry. (Fig 3A). Serial BLI revealed prompt and durable leukemia remissions and survival (Figure 3B,C). CONCLUSIONS We have developed CART-FLT3 for AML using novel human anti-FLT3 targeting domains and demonstrated preclinical efficacy similar to that of CART-123 in an AML model with substantially lower expression of FLT3 compared to CD123 (data not shown). Since inhibition of FLT3 leads to upregulation of surface FLT3 expression, future experiments will explore combinatorial FLT3 inhibition with CART-FLT3. If successful, these experiments will provide a strong rationale for a combination clinical trial in AML where leukemia control by small molecules is followed by a coup-de-grace delivered by CART cells. Figure 1 Figure 1. Disclosures Sleiman: Hemogenyx Pharmaceuticals LLC: Research Funding. Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Santiago: Hemogenyx Pharmaceuticals LLC: Research Funding. Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang: Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda: Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler: Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

CS1-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells and T Cells Enhance In Vitro and In Vivo Anti-Tumor Activity Against Human Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 14-14
Author(s):  
Jianhong Chu ◽  
Youcai Deng ◽  
Don M. Benson ◽  
Shun He ◽  
Tiffany L. Hughes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Trials ◽  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Variable Region ◽  
Advisory Committees ◽  
Empty Vector

Abstract Multiple myeloma (MM) is a B-cell malignancy characterized by the aberrant clonal expansion of plasma cells (PCs) within the bone marrow (BM). Despite the use of proteasome inhibitors and immune-modulating drugs, which have improved overall survival, MM remains an incurable malignancy for which novel therapeutic approaches are urgently needed. Immunotherapy that specifically targets antigens expressed by MM would be a promising approach to treat MM patients refractory to any current treatments. Chimeric antigen receptors (CARs) are engineered fusion proteins containing tumor antigen-recognition moieties and immune cell activation domains. CAR-expressing T cells have been demonstrated successful in the clinic to treat chronic lymphocytic leukemia (CLL) and acute lymphoid leukemia (ALL). However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells or T cells to target MM-expressed CS1 to treat MM has not been previously explored, and is the focus of our study. CS1 is a surface glycoprotein and represents an ideal target for the treatment of MM. CS1 is highly, and nearly ubiquitously, expressed on MM cells, while expression remains very low on NK cells, some T-cell subsets, and normal B cells, and also it is almost undetectable on myeloid cells. In addition, monoclonal antibody directed against CS1, elotuzumab, has already been proven safe in phase 1 and 2 clinical trials, and phase 3 trials are ongoing. Therefore, it should be safe to target CS1 for the treatment of MM. We successfully generated a specific CS1-CAR construct with a lentiviral vector backbone, sequentially containing a signal peptide (SP), a heavy chain variable region (VH), a linker, a light chain variable region (VL), a hinge, CD28 and CD3ζ. Flow cytometry analysis with an antibody against VH and VL regions indicated that CS1-CAR was successfully expressed on the surface of NK cells and T cells transduced with the CAR construct. In vitro, CS1-CAR NK cells and T cells displayed enhanced MM cytolysis (detected by Cr51 release assay) and augmented production of cytokines (determined by enzyme-linked immunosorbent assay, ELISA), such as IFN-g for NK and T cells and IL-2 for T cells, when co-cultured with CS1-expressing MM cell lines. These effects relied on CS1-dependent recognition of MM cells because CS1-CAR NK or T cells possessed higher activity when they were co-cultured with CS1-expressing cells, but remained much lower activity when they were co-cultured with CS1-negative cells. However, CS1-CAR NK or T cells launched significantly higher killing of MM cells and secrete more abundant cytokines when the CS1-negative MM cells ectopically overexpressed CS1 and were co-cultured with the CAR cells. Ex vivo, compared to NK or T cells transduced with the empty vector, NK or T cells transduced with CS1-CAR also showed significantly enhanced effector functions when responding to purified primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, when compared to untreated mice or mice treated with empty vector-tranduced NK or T cells, adoptive transfer of 5 × 106 NK or T cells expressing CS1-CAR once every five days efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged survival of mice bearing IM9 MM cells. Our efforts to translate these findings into clinical trials are ongoing. In summary, CS1 is a promising target for using CAR NK or T cells for MM treatment, and we have generated a CAR that recognizes CS1. We demonstrate that NK cells or T cells armed with this CS1-CAR can recognize and eradicate myeloma cells in vitro and in vivo. Autologous or allogeneic transplantation of these CS1-specific CAR NK cells or CAR T cells may be a promising strategy to treat MM. Disclosures: Caligiuri: Innate Pharma: Membership on an entity’s Board of Directors or advisory committees. Hofmeister:Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

scholarly journals A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katherine E. Harris ◽  
Kyle J. Lorentsen ◽  
Harbani K. Malik-Chaudhry ◽  
Kaitlyn Loughlin ◽  
Harish Medlari Basappa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Bispecific Antibody ◽  
T Regulatory Cells ◽  
Tumor Regression ◽  
Interleukin 2 ◽  
In Vivo Studies ◽  
Preferential Binding

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A113-A113
Author(s):  
Mireia Bachiller García ◽  
Lorena Pérez-Amill ◽  
Anthony Battram ◽  
Alvaro Urbano-Ispizua ◽  
Beatriz Martín-Antonio
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cord Blood ◽  
Immune Cells ◽  
In Vivo Models ◽  
Cell Clusters ◽  
Cytotoxicity Assays ◽  
Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

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