scholarly journals Blood DCs activated with R848 and poly(I:C) induce antigen-specific immune responses against viral and tumor-associated antigens

2021 ◽  
Author(s):  
Gerulf Hänel ◽  
Caroline Angerer ◽  
Katja Petry ◽  
Felix S. Lichtenegger ◽  
Marion Subklewe
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Poly I:C ◽  
Tumor Associated Antigens ◽  
Cell Induction

AbstractMonocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer entities. However, objective clinical responses have only been achieved in a minority of patients. Additionally, generation of GMP-compliant DCs requires time- and labor-intensive cell differentiation. In contrast, Blood DCs (BDCs) require only minimal ex vivo handling, as differentiation occurs in vivo resulting in potentially better functional capacities and survival. We aimed to identify a protocol for optimal in vitro activation of BDCs including the three subsets pDCs, cDC1s, and cDC2s. We evaluated several TLR ligand combinations and demonstrated that polyinosinic:polycytidylic acid [poly(I:C)] and R848, ligands for TLR3 and TLR7/8, respectively, constituted the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets. In addition, TLR3 and TLR7/8 activation led to high secretion of IFN-α and IL-12p70. Simultaneous as opposed to separate tailored activation of pDCs and cDCs increased immunostimulatory capacities, suggesting that BDC subsets engage in synergistic cross-talk during activation. Stimulation of BDCs with this protocol resulted in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.We conclude that simultaneous activation of all BDC subsets with a combination of R848 + poly(I:C) generates highly immunostimulatory DCs. These results support further investigation and clinical testing, as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.

scholarly journals Tumor-Related Exosomes Contribute to Tumor-Promoting Microenvironment: An Immunological Perspective

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Wuzhen Chen ◽  
Jingxin Jiang ◽  
Wenjie Xia ◽  
Jian Huang
Keyword(s):  
T Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Suppressor Cells ◽  
Effector T Cell ◽  
T Cell Apoptosis ◽  
And Function

Exosomes are a kind of cell-released membrane-form structures which contain proteins, lipids, and nucleic acids. These vesicular organelles play a key role in intercellular communication. Numerous experiments demonstrated that tumor-related exosomes (TEXs) can induce immune surveillance in the microenvironment in vivo and in vitro. They can interfere with the maturation of DC cells, impair NK cell activation, induce myeloid-derived suppressor cells, and educate macrophages into protumor phenotype. They can also selectively induce effector T cell apoptosis via Fas/FasL interaction and enhance regulatory T cell proliferation and function by releasing TGF-β. In this review, we focus on the TEX-induced immunosuppression and microenvironment change. Based on the truth that TEXs play crucial roles in suppressing the immune system, studies on modification of exosomes as immunotherapy strategies will also be discussed.

T-cell redirected killing and cure of pancreatic and gastric cancer xenografts by targeting Trop-2.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 262-262
Author(s):  
David M. Goldenberg ◽  
Edmund A. Rossi ◽  
Diane L Rossi ◽  
Thomas M. Cardillo ◽  
Chien-Hsing Chang
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Control Group ◽  
Calcium Signal ◽  
Target Cells ◽  
Normal Tissues

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

scholarly journals Enhanced Dendritic Cell Maturation by TNF-α or Cytidine-Phosphate-Guanosine DNA Drives T Cell Activation In Vitro and Therapeutic Anti-Tumor Immune Responses In Vivo

2000 ◽  
Vol 165 (11) ◽  
pp. 6278-6286 ◽  
Author(s):  
Christoph Brunner ◽  
Julia Seiderer ◽  
Angelika Schlamp ◽  
Martin Bidlingmaier ◽  
Andreas Eigler ◽  
...  
Keyword(s):  
T Cell ◽  
Dendritic Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Dendritic Cell Maturation ◽  
Cell Maturation ◽  
Tnf Α

scholarly journals 886 Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A928-A928
Author(s):  
Steve Sazinsky ◽  
Phuong Nguyen ◽  
Mohammad Zafari ◽  
Ryan Phennicie ◽  
Joe Wahle ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immune Cell ◽  
Ex Vivo ◽  
Syngeneic Mouse ◽  
Pathogen Clearance

BackgroundVSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism.1–3 VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion.3–6 Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response.MethodsThe ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce T cell activation was assessed in vitro and ex vivo, by measuring production of inflammatory mediators. In vitro assays were performed primarily with M-CSF plus IL-10 driven monocyte-derived M2c macrophages from healthy donors. Ex vivo assays were performed with fresh, patient-derived tumor samples in culture. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies and characterized for changes in tumor volume and immune cell populations.ResultsIn in vitro and ex vivo assays anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Targeting VSIG4 upregulates pro-inflammatory cytokines in M2c macrophages, as well as upregulates pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines in M2c macrophages co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation. To assess targeting VSIG4 in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). In vivo, tumor growth inhibition is observed in syngeneic mouse models dosed with anti-mouse-VSIG4 alone and in combination with anti-PD-1.ConclusionsTaken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms.Referencesvan Lookeren Campagne M, Verschoor A. Pathogen clearance and immune adherence “revisited”: immuno-regulatory roles for CRIg. Semin Immunol 2018;37:4–11.Xu S, Sun Z, Li L, Liu J, He J, Song D, Shan G, Liu H, Wu X. Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus. Immunol Lett 2010;128(1):46–50.Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, Fei L, Ni B, Ruan Z. VSIG4 expression on macrophages facilitates lung cancer development. Lab Invest 2014;94(7):706–715.Roh J, Jeon Y, Lee A, Lee S, Kim Y, Sung C, Park C, Hong J, Yoon D, Suh C, Huh J, Choi I, Park C. The immune checkpoint molecule V-set Ig domain-containing 4 is an independent prognostic factor for multiple myeloma. Oncotarget 2017;8(35):58122–58132.Xu T, Jiang Y, Yan Y, Wang H, Lu C, Xu H, Li W, Fu D, Lu Y, Chen J. VSIG4 is highly expressed and correlated with poor prognosis of high-grade glioma patients. Am J Transl Res 2015;7(6):1172–1180.Byun J, Jeong D, Choi I, Lee D, Kang M, Jung K, Jeon Y, Kim Y, Jung E, Lee K, Sung M, Kim K. The significance of VSIG4 expression in ovarian cancer. Int J Gynecol Cancer 2017;27(5):872–878.Ethics ApprovalAll legal and ethical requirements were met with regards to the humane treatment of animals described in the study. The animal study was conducted in compliance with CRL IACUC under IACUC No. I033.

scholarly journals DOCK2 sets the threshold for entry into the virtual memory CD8+ T cell compartment by negatively regulating tonic TCR triggering

2019 ◽  
Author(s):  
Ezana Demissie ◽  
Vinay S Mahajan ◽  
Faisal Alsufyani ◽  
Sudha Kumari ◽  
Grace J Yuen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Virtual Memory ◽  
Actin Dynamics ◽  
Immune Functions ◽  
Cell Compartment

AbstractThe control of cytoskeletal dynamics by Dedicator of cytokinesis 2 (DOCK2), a hematopoietic cell-specific actin effector protein, has been implicated in TCR signaling and T cell migration. Biallelic mutations in Dock2 have been identified in patients with a recessive form of combined immunodeficiency with defects in T, B and NK cell activation. Surprisingly, we show here that certain immune functions of CD8+ T cells are enhanced in the absence of DOCK2. Dock2-deficient mice have a pronounced expansion of their memory T cell compartment. Bone marrow chimera and adoptive transfer studies indicate that these memory T cells develop in a cell-intrinsic manner following thymic egress. Transcriptional profiling, TCR repertoire analyses and cell surface marker expression indicate that Dock2-deficient naive CD8+ T cells directly convert into virtual memory cells without clonal effector T cell expansion. This direct conversion to memory is associated with a selective increase in TCR sensitivity to selfpeptide MHC in vivo and an enhanced response to weak agonist peptides ex vivo. In contrast, the response to strong agonist peptides remains unaltered in Dock2-deficient T cells. Collectively, these findings suggest that the regulation of the actin dynamics by DOCK2 enhances the threshold for entry into the virtual memory compartment by negatively regulating tonic TCR triggering in response to weak agonists.

scholarly journals Functionalisation of Virus-Like Particles Enhances Antitumour Immune Responses

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Katrin Kramer ◽  
Farah Al-Barwani ◽  
Margaret A. Baird ◽  
Vivienne L. Young ◽  
David S. Larsen ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mannose Receptor ◽  
Virus Like Particles ◽  
Rabbit Haemorrhagic Disease

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025–33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

scholarly journals Group BStreptococcusInduces a Robust IFN-γResponse by CD4+T Cells in anIn VitroandIn VivoModel

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Damian Clarke ◽  
Corinne Letendre ◽  
Marie-Pier Lecours ◽  
Paul Lemire ◽  
Tristan Galbas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Capsular Polysaccharide ◽  
Ex Vivo ◽  
Specific Cell ◽  
Proinflammatory Response

Group BStreptococcus(GBS) serotype III causes life-threatening infections. Cytokines have emerged as important players for the control of disease, particularly IFN-γ. Although potential sources of this cytokine have been proposed, no specific cell line has ever been described as a leading contributor. In this study, CD4+T cell activation profiles in response to GBS were evaluated throughin vivo,ex vivo,andin vitroapproaches. Total splenocytes readily produce a type 1 proinflammatory response by releasing IFN-γ, TNF-α, and IL-6 and actively recruit T cells via chemokines like CXCL9, CXCL10, and CCL3. Responding CD4+T cells differentiate into Th1 cells producing large amounts of IFN-γ, TNF-α, and IL-2.In vitrostudies using dendritic cell and CD4+T cell cocultures infected with wild-type GBS or a nonencapsulated mutant suggested that GBS capsular polysaccharide, one of the major bacterial virulence factors, differentially modulates surface expression of CD69 and IFN-γproduction. Overall, CD4+T cells are important producers of IFN-γand might thus influence the course of GBS infection through the expression balance of this cytokine.

scholarly journals Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1690-1690
Author(s):  
Xiaomeng Hu ◽  
Mo Dao ◽  
Kathy White ◽  
Corie Gattis ◽  
Ryan Clarke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T

Abstract Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction as well as the ability to generate a final product from healthy T cells. While TCR editing can effectively prevent graft-versus-host reactions, the significant host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells (N Engl J Med. 2021;384(7):673-674). We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced by this strategy. Since CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to delete b2m, CIITA, and TCR expression and lentiviral transduction was used to overexpress CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CAR T cells. Control T cells were unmanipulated except for lentiviral transduction used to overexpress the same CD19CAR and the deletion of the TCR. When transplanted into allogeneic humanized mice, hypoimmune CD19CAR T cells evade immune recognition by T cells even in previously sensitized animals as evidenced by a lack of T cell activation measured using ELISPOT analysis. In contrast, transplantation of non-hypoimmune-edited CD19CAR T cells generated from the same human donor resulted in a significant T cell activation (see figure: mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and non-edited CD19CAR T cells, respectively; p<0.0001 unpaired T-test). In addition to evading T cells, immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmune CAR T cells susceptible to macrophage and NK cell killing in vitro and in vivo, confirming the importance of CD47 overexpression to evade innate immune clearance. The hypoimmune CD19 CAR T cells retained their antitumor activity in both the Daudi and Nalm-6 B cell leukemia models, in vitro and in vivo. This indicated that the hypoimmune technology-i.e. isolated CD47 overexpression, deletion of b2m, CIITA, and TCR- did not show any effect on the cytotoxic potential of CD19 CAR T cells (see figure). These studies demonstrate that in vivo clearance of leukemic cells in NSG mice occurs across a range of tumor cell toCD19 CAR T cell ratios in a manner comparable to control, unedited CD19 CAR T cells (see figure). This result was validated using T cells from 3 different donors These findings show that, in these models, hypoimmune CD19 CAR T cells are functionally immune evasive in allogeneic humanized mouse recipients and have cytotoxic anti-tumor capacity. They suggest that hypoimmune CAR T cells could provide universal CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that hypoimmune CD19 CAR T cells can be used in sensitized patients and for re-dosing strategies. Figure 1 Figure 1. Disclosures Hu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dao: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. White: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Gattis: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Clarke: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Landry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Basco: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tham: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tucker: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Luo: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Bandoro: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Chu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Young: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dowdle: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Rebar: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Schrepfer: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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