scholarly journals Importance of T, NK, CAR T and CAR NK Cell Metabolic Fitness for Effective Anti-Cancer Therapy: A Continuous Learning Process Allowing the Optimization of T, NK and CAR-Based Anti-Cancer Therapies

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 183
Author(s):  
Adrien Krug ◽  
Adriana Martinez-Turtos ◽  
Els Verhoeyen
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Building Blocks ◽  
Cancer Therapies ◽  
Cell Therapies ◽  
Anti Cancer ◽  
Car T ◽  
Metabolic Fitness

Chimeric antigen receptor (CAR) T and CAR NK cell therapies opened new avenues for cancer treatment. Although original successes of CAR T and CAR NK cells for the treatment of hematological malignancies were extraordinary, several obstacles have since been revealed, in particular their use for the treatment of solid cancers. The tumor microenvironment (TME) is competing for nutrients with T and NK cells and their CAR-expressing counterparts, paralyzing their metabolic effective and active states. Consequently, this can lead to alterations in their anti-tumoral capacity and persistence in vivo. High glucose uptake and the depletion of key amino acids by the TME can deprive T and NK cells of energy and building blocks, which turns them into a state of anergy, where they are unable to exert cytotoxic activity against cancer cells. This is especially true in the context of an immune-suppressive TME. In order to re-invigorate the T, NK, CAR T and CAR NK cell-mediated antitumor response, the field is now attempting to understand how metabolic pathways might change T and NK responses and functions, as well as those from their CAR-expressing partners. This revealed ways to metabolically rewire these cells by using metabolic enhancers or optimizing pre-infusion in vitro cultures of these cells. Importantly, next-generation CAR T and CAR NK products might include in the future the necessary metabolic requirements by improving their design, manufacturing process and other parameters. This will allow the overcoming of current limitations due to their interaction with the suppressive TME. In a clinical setting, this might improve their anti-cancer effector activity in synergy with immunotherapies. In this review, we discuss how the tumor cells and TME interfere with T and NK cell metabolic requirements. This may potentially lead to therapeutic approaches that enhance the metabolic fitness of CAR T and CAR NK cells, with the objective to improve their anti-cancer capacity.

scholarly journals A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4809-4809
Author(s):  
Alexander G Allen ◽  
Rithu Pattali ◽  
Kaitlyn M Izzo ◽  
Jared A Getgano ◽  
Kevin M Wasko ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Constitutive Expression ◽  
Publicly Traded ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T ◽  
Membrane Bound

Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

scholarly journals FcεRIγ-negative NK cells persist in vivo and enhance efficacy of therapeutic monoclonal antibodies in multiple myeloma

2021 ◽  
Vol 5 (15) ◽  
pp. 3021-3031
Author(s):  
Austin B. Bigley ◽  
Shanae Spade ◽  
Nadia H. Agha ◽  
Sujit Biswas ◽  
Suni Tang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
Nk Cells ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Central Component ◽  
Clinical Issue ◽  
Cell Therapies

Abstract Monoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (∼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.

scholarly journals Enhancing Human NK Cell Function and Specificity for Cancer Immunotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2044-2044
Author(s):  
Pomeroy Emily ◽  
Hunzeker John ◽  
Kluesner Mitchell ◽  
Crosby Margaret ◽  
Laura Bendzick ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Peripheral Blood ◽  
High Efficiency ◽  
Cell Function ◽  
Nk Cell ◽  
Negative Regulator ◽  
Car T

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.

scholarly journals Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

2021 ◽  
Vol 12 ◽  
Author(s):  
Hemant K. Mishra ◽  
Kate J. Dixon ◽  
Nabendu Pore ◽  
Martin Felices ◽  
Jeffrey S. Miller ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Feedback System ◽  
Cytotoxic Lymphocytes ◽  
Cell Therapies ◽  
Surface Receptors

Natural killer (NK) cells are innate cytotoxic lymphocytes that can recognize assorted determinants on tumor cells and rapidly kill these cells. Due to their anti-tumor effector functions and potential for allogeneic use, various NK cell platforms are being examined for adoptive cell therapies. However, their limited in vivo persistence is a current challenge. Cytokine-mediated activation of these cells is under extensive investigation and interleukin-15 (IL-15) is a particular focus since it drives their activation and proliferation. IL-15 efficacy though is limited in part by its induction of regulatory checkpoints. A disintegrin and metalloproteinase-17 (ADAM17) is broadly expressed by leukocytes, including NK cells, and it plays a central role in cleaving cell surface receptors, a process that regulates cell activation and cell-cell interactions. We report that ADAM17 blockade with a monoclonal antibody markedly increased human NK cell proliferation by IL-15 both in vitro and in a xenograft mouse model. Blocking ADAM17 resulted in a significant increase in surface levels of the homing receptor CD62L on proliferating NK cells. We show that NK cell proliferation in vivo by IL-15 and the augmentation of this process upon blocking ADAM17 are dependent on CD62L. Hence, our findings reveal for the first time that ADAM17 activation in NK cells by IL-15 limits their proliferation, presumably functioning as a feedback system, and that its substrate CD62L has a key role in this process in vivo. ADAM17 blockade in combination with IL-15 may provide a new approach to improve NK cell persistence and function in cancer patients.

scholarly journals Engineered Memory-like NK Cars Targeting a Neoepitope Derived from Intracellular NPM1c Exhibit Potent Activity and Specificity Against Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Han Dong ◽  
Guozhu Xie ◽  
Yong Liang ◽  
James Dongjoo Ham ◽  
Juliana Vergara ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Tumor Resistance ◽  
Cell Therapies ◽  
Car T

Introduction: Acute myeloid leukemia (AML) continues to be a major therapeutic challenge. There is an emerging need to develop less toxic and more effective targeted therapies. Natural Killer (NK) cells possess many of the key attributes critical for effective cancer therapies- "born to kill" but without apparent risk of graft versus host disease, cytokine release syndrome, or neurotoxicity. Furthermore, their intrinsic propensity to target myeloid blasts makes them particularly attractive for AML. Despite promising clinical results in blood cancer, the development of NK cell-based therapy remains challenging mostly due to NK cells' short lifespan, inadequate proliferation and lack of specific tumor targeting. Here, we utilized a new approach to arm NK cells for adoptive immunotherapy based on innate cell memory. Chimeric antigen receptors (CARs) significantly enhance anti-tumor specificity and activity of immune effector cells. Our innovative CAR-NK cells target a tumor-specific neoepitope in AML and harness potent function pathways in their design to enhance efficacy and minimize toxicity. Methods: 1. Mutated NPM1c as a CAR Target in AML. Most CAR-T cell therapies target tumor-associated antigens (TAAs), which could lead to on-target/off-tumor toxicity as well as tumor resistance. One way to overcome these drawbacks is to target tumor-specific oncogenic driver mutations. The four-nucleotide duplication in nucleophosmin, referred to as NPM1c, is a driver oncogene mutation in about 35% of AML. The mutation creates a neoepitope that is presented by the most common HLA-A2 allele. Using yeast surface display, we have isolated a human single-chain variable fragment (scFv) that specifically binds to the NPM1c epitope-HLA-A2 complex, but not HLA-A2 alone or HLA-A2 loaded with control peptides. 2. Cytokine-Induced Memory-Like (CIML) NK Cells as a CAR Platform. CIML NK cells can provide a unique platform for development of NK cell CARs based on the favorable safety profile, increased proliferation, prolonged persistence and enhanced anti-leukemia function that we have observed in pre-clinical models (Romee et al, Blood 2012) and in patients (Romee et al, Science Trans Med 2016) treated with un-modified CIML NK cells. 3. Efficient Gene Editing in Primary NK Cells. We have overcome the transduction block in primary human and mouse NK cells by utilizing an unconventional pseudotyped lentivirus based on a unique protein with high expression on CIML NK cells. Results: 1. Engineered CAR-T cells with the isolated scFv exhibit potent cytotoxicity both in vitro and in vivo against NPM1c+HLA-A2+ leukemia cells (OCI-AML3) and primary AML blasts, but not NPM1c-HLA-A2+ leukemia cells (OCI-AML2) or HLA-A2- tumor cells (PC-3). 2. The in vivo anti-leukemia efficacy of anti-NPM1c CAR-T cells was however transient (overall survival extended from 28 to 42 days, median survival extended from 21 to 37 days, compared with the control mice adoptively transferred with untraduced T cells), with unneglectable toxicity. 3. Utilizing an unconventional pseudotyped lentivirus to transduce CIML NK cells from healthy donor blood (n = 5 donors), we have successfully generated anti-NPM1c CAR-NK cells with high transduction efficiency (using MOI = 10: transduction rate mean 48%, range 32% to 65%; compared with 2%, range 0.8% to 4.5% for the conventional approach with VSVG pseudotyped lentivirus). 4. Harnessing key cytokine pathways in the CAR design substantially promoted CAR-NK cell survival (indicated by the enhanced cell viability from 29.7% to 75.2%) and proliferation (marked by the increased levels of ki-67 from 60.2% to 94.5%). 5. Anti-NPM1c CAR significantly promoted anti-tumor function (represented by CD107a, IFN-gamma) and tumor-specific killing (measured by annexin V and 7-AAD) of CIML NK cells against AML with NPM1c oncogene (OCI-AML3). 6. Dual-armed CIML NK cells with CAR and cytokine signaling exhibited optimal specificity and sustainability against AML targets. Conclusion: These results demonstrate that the innovative CAR-CIML NK cells could be developed as an efficient cellular immunotherapy for treating NPM1c+HLA-A2+ AML with potentially reduced on-target/off-tumor toxicity and tumor resistance. Our study should drive novel conception and design of CAR-NK cell therapies against myeloid malignancies in the clinic. Figure Disclosures Ritz: Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

scholarly journals Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

2021 ◽  
Vol 8 (6) ◽  
pp. 110
Author(s):  
Nathalie Meijerink ◽  
Jean E. de Oliveira ◽  
Daphne A. van Haarlem ◽  
Guilherme Hosotani ◽  
David M. Lamot ◽  
...  
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Relative Abundance ◽  
Nk Cell ◽  
Feed Additives ◽  
Microbiota Composition ◽  
In Ovo ◽  
Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals 114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A126-A126
Author(s):  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Immune Cell ◽  
Nk Cell ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

scholarly journals 150 GAIA-102: a new class off-the-shelf allogeneic NK-like cells that can eliminate solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A163-A163
Author(s):  
Yui Harada ◽  
Yoshikazu Yonemitsu
Keyword(s):  
Nk Cells ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Adoptive Immunotherapy ◽  
Nk Cell ◽  
Peritoneal Dissemination ◽  
Checkpoint Inhibitors ◽  
Antitumor Effects ◽  
Car T

BackgroundCancer immunotherapy has been established as a new therapeutic category since the recent success of immune checkpoint inhibitors and a type of adoptive immunotherapy, namely chimeric antigen receptor-modified T cells (CAR-T). Although CAR-T demonstrated impressive clinical results, serious adverse effects (cytokine storm and on-target off-tumor toxicity) and undefined efficacy on solid tumors are important issues to be solved. We’ve developed a cutting-edge, simple, and feeder-free method to generate highly activated and expanded human NK cells from peripheral blood (US9404083, PCT/JP2019/012744, PCT/JP2020/012386), and have been conducting further investigation why our new type of NK cells, named as GAIA-102, are so effective to kill malignant cells.MethodsCryopreserved PBMCs purchased from vendors were mixed and processed by using LOVO and CliniMACS® Prodigy (automated/closed systems). CD3+ and CD34+ cells were depleted, and the cells were cultured with high concentration of hIL-2 and 5% UltraGRO® for 14 days in our original closed system. Then, we confirmed the expression of surface markers, CD107a mobilization and cell-mediated cytotoxicity against various tumor cells and normal cells with or without monoclonal antibody drugs in vitro and antitumor effects against peritoneal dissemination model using SKOV3 in vivo.ResultsImportantly, we’ve found that our GAIA-102 exhibited CD3-/CD56bright/CD57- immature phenotype that could kill various tumor cells efficiently from various origins, including Raji cells that was highly resistant to NK cell killing. More importantly, massive accumulation, retention, infiltration and sphere destruction by GAIA-102 were affected neither by myeloid-derived suppressor cells nor regulatory T-lymphocytes. GAIA-102 was also effective in vivo to murine model of peritoneal dissemination of human ovarian cancer; thus, these findings indicate that GAIA-102 has a potential to be an ‘upward compatible’ modality over CAR-T strategy, and would be a new and promising candidate for adoptive immunotherapy against solid tumors.ConclusionsWe now just started GMP/GCTP production of this new and powerful NK cells and first-in-human clinical trials in use of GAIA-102 will be initiated on 2021.Ethics ApprovalThe animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Kyushu University (approval nos. A30-234-0 and A30-359-0).

Prevascularized, Co-Culture Model for Breast Cancer Drug Development

2012 ◽  
Author(s):  
Lauren Marshall ◽  
Isabel Löwstedt ◽  
Paul Gatenholm ◽  
Joel Berry
Keyword(s):  
Breast Cancer ◽  
Drug Development ◽  
Three Dimensional ◽  
Human Tumor ◽  
3D Models ◽  
Cancer Drug ◽  
Cancer Therapies ◽  
Anti Cancer

The objective of this study was to create 3D engineered tissue models to accelerate identification of safe and efficacious breast cancer drug therapies. It is expected that this platform will dramatically reduce the time and costs associated with development and regulatory approval of anti-cancer therapies, currently a multi-billion dollar endeavor [1]. Existing two-dimensional (2D) in vitro and in vivo animal studies required for identification of effective cancer therapies account for much of the high costs of anti-cancer medications and health insurance premiums borne by patients, many of whom cannot afford it. An emerging paradigm in pharmaceutical drug development is the use of three-dimensional (3D) cell/biomaterial models that will accurately screen novel therapeutic compounds, repurpose existing compounds and terminate ineffective ones. In particular, identification of effective chemotherapies for breast cancer are anticipated to occur more quickly in 3D in vitro models than 2D in vitro environments and in vivo animal models, neither of which accurately mimic natural human tumor environments [2]. Moreover, these 3D models can be multi-cellular and designed with extracellular matrix (ECM) function and mechanical properties similar to that of natural in vivo cancer environments [3].

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