scholarly journals A TCR Mimic Antibody-Directed CAR T Cell Specific for Intracellular NDC80 Is Broadly Cancer Reactive and Displays High Activity Against Hematological Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21
Author(s):  
Martin G. Klatt ◽  
Zhiyuan Yang ◽  
Jianying Liu ◽  
Tatyana Korontsvit ◽  
Sung Soo Mun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Private Company ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Hla Ligand ◽  
Car T

Chimeric antigen receptor (CAR) T cells represent a novel class of FDA-approved drugs with high efficacy against refractory B cell derived malignancies and potentially other cancer types. However, target selection for CAR T cell therapy remains challenging as cell surface proteins are not cancer-specific and therefore often not adaptable for CAR T cell therapy. In contrast, many intracellular proteins can be highly tumor specific and are targetable after proteasomal degradation and presentation on human leukocyte antigen (HLA) complexes recognized by T cell receptor mimic antibodies. This class of antibodies recognizes peptide:HLA complexes with a similar mode of recognition as a TCR, but with the clinical versatility and applicability of an antibody. To identify a tumor specific target that is presented as a peptide in conjunction with the highly prevalent HLA allele A*02:01, we immunopurified peptide:HLA complexes from various cancer cell lines of different origins, separated HLA ligands from complexes and identified their peptide sequences via mass spectrometry. Network analysis of the resulting HLA ligand datasets identified shared biological processes among the tumor cell lines that were not present in network analyses of published datasets of healthy human tissue HLA ligandomes. Through this filtering process several potential targets were identified and an HLA ligand derived from kinetochore NDC80 protein homolog (NDC80) was selected as a target. The NDC80 derived peptide was detected in over 90% of the A*02 positive cell lines tested and never reported to be present in HLA ligand datasets of healthy human tissues. Furthermore, NDC80 has been shown to be differentially expressed in malignant compared to adjacent non-malignant tissues and is associated with poor prognosis in many cancer types. After utilizing E-ALPHA®phage library screening, one clone (NDC80-L1) was selected as the lead TCR mimic antibody. Overall, NDC80-L1 showed high specificity for the target HLA:peptide complex in both antibody and CAR T cell format in vitro and demonstrated binding primarily to the central region of the HLA ligand as determined by alanine screening assays. The exquisite specificity of NDC80-L1 was further illustrated by NDC80 knockdown experiments as well as successful immunopurification of the target peptide together with no relevant off-targets from BV173 ALL cells in mass spectrometry assays. Given the high specificity, sensitivity was assessed primarily in a potent CAR T cell format: Multiple tumor cell lines of different origin (e.g. ALL, AML, lymphoma, melanoma, mesothelioma, pancreatic and thyroid cancer) were successfully killed in vitro by NDC80-L1 CAR T cells, but no toxicity towards A*02:01 positive CAR T cells, healthy PBMCs or NDC80 target negative cell lines was observed. Interestingly, NDC80-L1 CAR T cells demonstrated highest efficacy in hematological malignancies most likely correlating with elevated expression of antigen presentation machinery and rapid cell division which leads to strong surface expression of NDC80 peptides. In summary, CAR T cells directed against peptide/HLA-A*02 derived from the NDC80 protein effectively kill multiple cancer cell lines in vitro without evidence of relevant off-target killing. However, the improved killing especially against ALL, AML and lymphomas highlights the potential of these CAR T cells to preferentially eliminate cancer cells with high proliferative capacity. Future in vivo studies with CAR T cell and antibody format will further investigate this TCR mimic antibody's potential as a tumor-agnostic therapeutic agent. Disclosures Klatt: MSKCC/EUREKA: Patents & Royalties: MSKCC AND EUREKA THERAPUETICS HAVE FILED A PATENT FOR THIS ANTIBODY/SCFV. Yang:Eureka Therapuetics: Current Employment, Current equity holder in private company, Patents & Royalties: MSKCC and Eureka have filed patent for this TCRm and ScvF. Liu:Eureka Therapue: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Dao:Eureka Therapeutics: Consultancy. Liu:Eureka Therapeutics: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Scheinberg:Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Arvenas: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Oncopep: Consultancy; Pfizer: Consultancy, Current equity holder in private company; Lantheus: Current equity holder in private company; Enscyse: Current equity holder in private company.

scholarly journals CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lena Andersch ◽  
Josefine Radke ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
Annika Winkler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
T Cell Therapy ◽  
Retinoblastoma Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

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 Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaojuan Shi ◽  
Daiqun Zhang ◽  
Feng Li ◽  
Zhen Zhang ◽  
Shumin Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inhibitory Effects ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractAsparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

2020 ◽  
Vol 8 (2) ◽  
pp. e000896
Author(s):  
Talia Velasco-Hernandez ◽  
Samanta Romina Zanetti ◽  
Heleia Roca-Ho ◽  
Francisco Gutierrez-Aguera ◽  
Paolo Petazzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
High Affinity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

scholarly journals Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 139 ◽  
Author(s):  
Jing Cui ◽  
Herui Wang ◽  
Rogelio Medina ◽  
Qi Zhang ◽  
Chen Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Therapeutic Potential ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

scholarly journals IMMU-11. LOCOREGIONAL DELIVERY OF TRANSIENT GD2 CAR T CELLS FOR SAFE AND EFFECTIVE TREATMENT OF DIPG

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii361-iii362
Author(s):  
Jessica Foster ◽  
Crystal Griffin ◽  
Allison Stern ◽  
Cameron Brimley ◽  
Tiffany Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Single Dose ◽  
T Cell ◽  
Indwelling Catheter ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Repeated Dosing

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor with a median survival of one year. Recently Mount et al (Nat Med 2018) discovered the disialoganglioside GD2 is present at high levels on the surface of DIPG and can be targeted using GD2-directed CAR T cells. However, permanently expressed CAR T cells created by lentiviral transduction resulted in a significant number of deaths from tumor swelling with uncontrolled T cell proliferation. We hypothesized that using mRNA to create transient GD2-directed CAR T cells delivered locally with repeated dosing would result in a safer yet equally effective way to treat DIPG using CAR T cell therapy. In vitro studies using mRNA GD2-directed CAR T cells resulted in robust tumor cytotoxicity and T cell degranulation across a panel of six DIPG cell lines. Using an orthotopic murine model of SU-DIPGXIIIP*, an extremely aggressive model, we delivered of a single dose of two million mRNA GD2-directed CAR T cells locoregionally to the pons via stereotactic injection. The mRNA GD2-directed CAR T cells resulted in no toxic deaths of the mice. In addition, a single dose of mRNA CAR T cells targeting GD2 prolonged survival of the mice by a median of six days (p<0.05). Ongoing studies using an indwelling catheter for repeated dosing of mRNA CAR T cells are currently underway and results expected at the time of presentation. This work will form of the basis of an mRNA CAR T cell trial targeting GD2 for patients with DIPG.

scholarly journals 126 Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A135-A135
Author(s):  
Hee Jun Lee ◽  
Cody Cullen ◽  
John Murad ◽  
Jason Yang ◽  
Wen-Chung Chang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Models ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundWhile chimeric antigen receptor (CAR) T cell therapy has shown impressive clinical efficacy for hematological malignancies,1 efficacy remains limited for solid tumors due in large part to the immunosuppressive tumor microenvironment.2 Tumor-associated glycoprotein 72 (TAG72) is an aberrantly glycosylated protein overexpressed on ovarian cancer3 and is an exciting target for CAR T cell immunotherapy. Our lab previously developed a second-generation TAG72 CAR T cell product and showed its potency against TAG72-expressing ovarian tumor cells both in vitro and in preclinical mouse models.4 We report here further modification of our TAG72 CAR T cells, with incorporation of interleukin-12 (IL-12) and interleukin-15 (IL-15), and evaluate the therapeutic benefits in peritoneal ovarian tumor models.MethodsIn this preclinical study, we build upon our earlier work with in vitro and in vivo evaluation of 9 different second-generation TAG72 CAR constructs varying in single-chain variable fragment, extracellular spacer, transmembrane, and intracellular co-stimulatory domains. We then engineer CAR T cells with two types of cytokines – IL-12 and IL-15 – and put these engineered cells against challenging in vivo tumor models.ResultsThrough in vitro and in vivo studies, we identify the most optimal construct with which we aim to evaluate in a phase 1 clinical trial targeting TAG72-positive ovarian cancer in 2021. Despite thorough optimizations to the CAR backbone, CAR T cells can be additionally engineered for improved anti-tumor response. Therefore, we further engineered CAR T cells with IL-12 or IL-15 production that greatly improves the effectiveness of TAG72-CAR T cells in difficult-to-treat in vivo tumor models. We observed that modification of CAR T cells with IL-15 displayed toxicity when regionally delivered in vivo, yet introduction of IL-12 not only demonstrated safe and superior therapeutic responses, but also allowed the regional administration of CAR T cells to address systemic disease. We are now expanding these findings by evaluating these therapies using syngeneic immunocompetent mouse tumor models.ConclusionsThe tumor microenvironment (TME) harbors various factors that thwart the killing of tumor cells by CAR T cells. Thus, CAR T cells will likely require further engineering to overcome this barrier. We show that amplifying cytokine pathways is one way to overcome the TME and improve the efficacy of CAR T cell therapy for solid tumors.ReferencesMaude SL, Teachey DT, Porter DL, Grupp SA. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood 2015 Jun 25;125(26):4017–23.Priceman SJ, Forman SJ, Brown CE. Smart CARs engineered for cancer immunotherapy. Curr Opin Oncol 2015 Nov;27(6):466–74.Chauhan SC, Vinayek N, Maher DM, Bell MC, Dunham KA, Koch MD, Lio Y, Jaggi M. Combined Staining of TAG-72, MUC1, and CA125 Improves Labeling Sensitivity in Ovarian Cancer: Antigens for Multi-targeted Antibody-guided Therapy. J Histochem Cytochem 2007 Aug;55(8):867–75.Murad JP, Kozlowska AK, Lee HJ, Ramamurthy M, Chang WC, Yazaki P, Colcher D, Shively J, Cristea M, Forman SJ, Priceman SJ. Effective Targeting of TAG72+ Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol 2018 Nov 19;9:2268.

scholarly journals 111 Armored CAR T cells secreting 4–1BB ligand crosslinked to PD-1 checkpoint inhibitor for enhanced solid tumor efficacy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Zachary Dunn ◽  
Yun Qu ◽  
Melanie MacMullan ◽  
Xianhui Chen ◽  
Gunce Cinay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Solid Tumor ◽  
Single Chain ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 scFv, as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality.1 We have since matured the platform to create a superior cellular product – CAR T cells secreting single-chain trimeric 4-1BB ligand crosslinked to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocytes proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder anti-tumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand.MethodsWe hypothesized that crosslinking the current anti-PD-1 scFv with 4-1BB ligand would provide additional benefits to CAR T cells and is potentially of translational value in the management of tumors resistant to PD-1 blockade due to lack of T cell function. Therefore, we engineered CAR T cells to secrete a novel immunomodulatory fusion protein consisting of anti-PD-1 scFv crosslinked to a single-chain format of trimeric 4-1BB ligand, in which three extracellular domain units of 41BBL are connected with polypeptide linkers. The CAR T cells were then characterized in vitro and subcutaneous tumor models.ResultsIn vitro and in vivo, CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy and persistence of αPD1-41BBL secreting CAR T cells.ConclusionsGiven the significantly better therapeutic efficacy of αPD1-41BBL expressing T cells over αPD1 expressing T cells, we believe that it is of high translational value to adopt secretion of αPD1-41BBL fusion protein to improve CAR T cell solid tumor efficacy, especially given the large number of patients that are PD1/PD-L1 therapy resistant.ReferencesLi S, Siriwon N, Zhang X, Yang S, Jin T, He F, et al. Enhanced cancer immunotherapy by chimeric antigen receptor–modified T cells engineered to secrete checkpoint inhibitors. Clin Cancer Res 2017;23(22):6982–92.

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