T- & NK-cell Engineering

Accelerating Development & Commercialization of Enhanced Potency T- & NK-Cell Therapies


Whether exploring your first immunotherapy or your next-generation follow-on therapy, turn to MaxCyte’s fully scalable, cGMP-compliant delivery platform to develop and commercialize enhanced potency immunotherapies such as CAR-T cells. Accelerate timelines for IND applications, clinical studies and commercialization by safely, efficiently and more cost-effectively engineering primary immune cells that display increased therapeutic potential using MaxCyte’s non-viral delivery platform.

  • Clinically proven
  • Non-viral, inherently safe with a clear regulatory pathway – cGMP compliant and FDA Master File
  • Seamless scalability from bench to commercial patient treatment
  • Produces therapies with enhanced potency
  • Computer-controlled, closed environment for robust manufacturing
  • Unmatched primary & stem cell transfection efficiencies



Case Study: mRNA CAR Delivery in Expanded T- & NK-cells


MaxCyte’s universal delivery platform is ideal for automated loading of CAR (chimeric antigen receptor) mRNA into primary immune cells. It results in high T- and NK-cell viability and transfection efficiencies with expression of the encoded CAR over multiple days following mRNA loading and enhanced anti-tumor activities.


CAR Expression in Primary T Cells Following mRNA Delivery


CAR expression as measured by mean fluorescent intensity (MFI) at different time points after electroporation with CD19-BBz mRNA in anti-CD3 and CD28 stimulated peripheral blood T cells (open histograms). Non-electroporated T cells were used as negative control (filled histogram).

mRNA CAR-T Cells Display Antigen-specific Cell Killing



Peripheral blood lymphocytes (PBLs) were electroporated with CD19-BBz or no mRNA (Mock). mRNA CAR+ T cells specifically kill CD19 targets. A flow-based CTL assay was conducted on the indicated day post electroporation with K562-CD19 as the target cells. Figure adapted from Hum Gene Ther. 22(12): 1575–1586, 2011.

In Vivo Trafficking and Proliferation of mRNA CAR-T Cells



Specific trafficking and proliferation of RNA CARs in tumor-bearing mice. (Left) NOD/SCID/γc–/– (NSG) mice were injected IV with 106 Nalm-6 cells followed 7 days later with 5×106 T cells 4 hr after electroporation with the indicated mRNA constructs. The T cells had been stably transfected with a lentiviral construct to express firefly luciferase, and mice were imaged for bioluminescence. The graph indicates average of individual total photon flux±the standard error for each of the indicated groups (n=8). (Right) CD19 RNA CARs exhibit increasing bioluminescence signal and anatomic distribution consistent with migration to sites of disease and CAR T-cell proliferation. Photon density heat maps on day 3 post injection suggest that mock T cells or T cells expressing RNA CARs with irrelevant specificity against mesothelin pool passively in the spleen (left flank on heat map) and do not increase in photon density, indicating a lack of proliferation. Note that the 5×106 cells produce a p/sec/cm2 flux of ∼2×107, equivalent among all groups immediately after injection. Saline-treated mice represent the background autoluminescence of 5×105 p/sec/cm2. Figure adapted from Hum Gene Ther. 22(12): 1575–1586, 2011.

In Vivo Efficacy of mRNA CAR-T Cells



Dose splitting impacts efficacy of mRNA CAR T cells. This is a composite survival of 4 independent experiments (with 3 different donors, n=20) using 106 Nalm-6 on day 0 and either a single dose 2.5×107 on day 7, or split infusions on days 7, 14, and 28 with interval CTX at 60 mg/kg IP×1 24 hr before CTL doses 2 and 3. Survival is significantly improved by the 20–5–5 schedule despite the total dose of T cells being equivalent (p=0.00103). The only long-term survivors are observed with this dose schedule. Figure adapted from Hum Gene Ther., 24(8), 717-727, 2013.

α-Mesothelin CAR Progression to Human POC Studies Targeting Solid Tumors

Cancer Immunol Res., 2(2), 112-120, 2014 & J Clin Oncol 33, 2015 (suppl; ASCO 2015 abstr 3007)

Cell Engineering:
• α-Mesothelin CAR mRNA loaded into ex vivo expanded T-cells using the MaxCyte GT® for manufacturing of all product lots
• Final Product: ~98% CD3+ T-cells, ~95% cell viability, and ~95% cells expressed CAR molecule


Clinical Observations:
• Product infusions were without any overt evidence of ‘on-target off-tumor’ toxicity against normal tissues or evidence of cytokine release syndrome (CRS)
• α-Mesothelin CAR T-cells persisted transiently 5 – 7 days in peripheral blood after i.v. infusion & observed to traffic to primary & metastatic tumor sites
• α-Mesothelin CAR T-cells elicited anti-tumor immune responses


Additional mRNA-CAR Products in Human POC Studies
MaxCyte and its collaborators are currently evaluating the safety and biological activity of CAR mRNA engineered T-cells and NK-cells in multiple human clinical trials targeting validated tumor-associated antigens in multiple cancer indications.


Case Study: Enhanced NK-cell Cytotoxicity via CAR Expression

Anti-CD19-BBZ Expression Following mRNA Transfection of Expanded NK-cells



NK-cells were isolated from peripheral blood mononuclear cells and expanded by coculture with K562-mb15-41BBL cells for 6–12 days. NK were then harvested and transfected with mRNA encoding the anti-CD19-BBZ chimeric antigen receptor (receptor that binds CD19 and delivers CD3z and 4–1BB activation signals). Left). FACS analysis for anti-CD19-BBZ expression following NK cell transfection. Right). Post electroporation viability and anti-CD19-BB-z expression of NK cells transfected ± mRNA encoding anti-CD19-BB-z. Results were obtained from two different donors and performed 10 times in total. Figure adapted from Cancer Gene Therapy, 1-8, 2009.

Antigen-specific Cytotoxicity of Engineered NK-cells



Specific killing of OP-1 cells (CD19+ human B-lineage ALL cell line) by expanded NK cells that were not electroporated (control), electroporated with no mRNA or electroporated with mRNA-antiCD19-BBZ. Cell killing analyzed by using the Calcein-AM method. Figure adapted from Cancer Gene Therapy, 1-8, 2009.

Case Study: Augmented ADCC & Lymph Node Trafficking via NK-cell Engineering


Enhanced Migration Toward CCL19 of Expanded NK-cells Transfected with CCR7 mRNA

Ex vivo-expanded NK-cells were electroporated with mRNA encoding CCR7. Left) Representative example of CCR7 expression on NK-cells 8 hours after electroporation with CCR7 mRNA (CCR7-EP) compared to non-electroporated (non-EP) NK cells. Right) Trans-well migration of non-EP and CCR7-EP NK-cells against a gradient of CCL19 (a ligand for CCR7). Error bars, SEM. A paired t-test was used in for analysis. Figures adapted from Frontiers in Immunology, Vol 7, 22, 2016.

Enhanced ADCC Capacity of NK-cells Engineered to Express CD16


Ex vivo-expanded NK-cells were electroporated with mRNA coding for the high-affinity Fc receptor CD16-158V. Left) Representative example and average cell surface expression of CD16 on NK-cells 24 h after electroporation with CD16 mRNA (CD16-EP) compared to non-electroporated (non-EP) NK cells (n = 7). Right) Kinetics of NK-cell rituximab-induced ADCC following electroporation (n = 3).

Key Scientific Publications


  • Efficient mRNA-based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-induced ADCC against Lymphoma and Targets NK cell Migration Towards the Lymph Node Associated Chemokine CCL19. Frontiers in Immunology, Vol 7, 22, 2016.
  • Clinical Scale Zinc Finger Nuclease-Mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma. Molecular Therapy, Vol. 23(8): 1380–1390, 2015.
  • Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol Res., 2(2), 112-120, 2014.
  • Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia. Hum Gene Ther., 24(8), 717-727, 2013.
  • Treatment of Advanced Leukemia in Mice with mRNA Engineered T Cells. Hum Gene Ther. 22(12): 1575–1586, 2011.
  • Multiple Injections of Electroporated Autologous T Cells Expressing a Chimeric Antigen Receptor Mediate Regression of Human Disseminated Tumor. Cancer Research, Volume 70(22), 2010.
  • Expression of Chimeric Antigen Receptors in Natural Killer Cells with a Regulatory-compliant Non-viral Method. Cancer Gene Therapy, 1-8, 2009.


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