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Published in final edited form as: Adv Biol (Weinh). 2021 Apr 19;5(7):e2000298. doi: 10.1002/adbi.202000298

NK-cell Biofactory as an Off-the-Shelf Cell-based Vector for Targeted in situ Synthesis of Engineered Proteins

Claire E Repellin 1, Marvin A Ssemadaali 1, Sherri Newmyer 1, Harikrishnan Radhakrishnan 1, Harold S Javitz 2, Parijat Bhatnagar 1,3,*
PMCID: PMC8275051  NIHMSID: NIHMS1702971  PMID: 33871182

Abstract

We have transformed the NK-92MI, a fast-growing cytolytic cell line with a track record of exerting clinical efficacy, into a vector for synthesizing calibrated amounts of desired engineered proteins at the disease site, i.e. NK-cell Biofactory. This provides an allogeneic option to our previously developed T-cell-based living vector that is limited by high manufacturing cost and product variability. The modularity of this pathway, which combines a “target” receptor with an “effector” function, enables reprogramming of the NK-cell Biofactory to target diseases with specific molecular biomarkers, such as cancer, viral infections or autoimmune disorders, and overcome barriers that may affect the advancement of NK-cell therapies.

Keywords: Cell engineering, CAR NK cells, CAR T cells, drug delivery, nuclear factor of activated T-cells response element

Graphical Abstract

graphic file with name nihms-1702971-f0005.jpg

NK-cell Biofactory, based on a clinically relevant NK cell line, is a living vector that secrete desired protein following recognition of a target cell while conserving the cytolytic activity. Its allogeneic nature, coupled with ease of manufacturing, transforms it into an off-the-shelf therapeutic option. The platform can be redirected for treating a broad range of diseases evading the immune system.

3.0. Introduction

The dynamic state of cell-based pathologies and inter-patient variability presents a challenge that, in the context of optimal dosing, requires continuous monitoring of the disease state and individual patient[1,2]. Current standard of care in drug delivery is to administer doses normalized to the body weight and surface area[3]. However, the disease burden is different for similar-sized patients[4,5]. If administered in excess, the therapeutic agents end up in the systemic circulation leading to morbidity. In the case of their suboptimal delivery, it leads to drug resistance. As such, options for many cell-based diseases (e.g. cancer, viral infections, autoimmune disorders) that evade the immune system are limited. To address this challenge, we recently reported on an antigen-specific T-cell Biofactory, which is a T-cell-based living vector that, upon interacting with the antigen-presenting target cell, synthesizes proportionate amounts of desired proteins in situ[6,7]. However, these and other T-cell therapies, such as T cells engineered with chimeric antigen receptors (CAR T cells), are primarily autologous in nature and present a pragmatic challenge in terms of manufacturing cost. Lymphodepleted patients have a low primary T cell count to draw from, and this number is further reduced when the cells are genetically modified. While the biology of primary T cells supports activation induced expansion, their scale-up for clinical use is non-trivial when starting with low cell count and suffers from debilitating source variability. This lack of availability of engineered T cell number required to observe the desired therapeutic effect continues to limit the translation of these treatments into the clinic. In addition, adverse events due to cytokine release syndrome, neurotoxicity, and graft-versus-host disease (GvHD) present other clinical barriers.

In this work, to position the Cell Biofactory technology further along the path of clinical translation, we have exchanged the T-cell-based cellular chassis with the NK-92MI cell-line, i.e. antigen-specific NK-cell Biofactory (Figure 1).The artificial cell-signaling pathway, discussed previously in detail[6], was again utilized for generating the NK-cell Biofactory as a living vector (Figure 1A). The vector synthesizes engineered proteins in situ upon interacting with the antigen-presenting target cell. Briefly, a single plasmid comprising of three constant (Receptor, Actuator, Secretor) and two variable (Sensor, Effector) domains arranged in cis was generated (Figure 1B). As reported previously[6], the constant domains were optimally configured to provide functionality to the Cell Biofactory. They form part of the intracellular signaling pathway and include – a transmembrane molecule (Receptor) that mobilizes the calcium-dependent transcriptional machinery (Actuator) to upregulate the effector transgene fused to a signal peptide (Secretor) that assists in transporting it into the extracellular space. The variable domains are responsible for the broad applicability of this platform. They impart specificity to the Cell Biofactory against particular diseases and include – variable heavy-light (VH-VL) chain (Sensor, part of the transmembrane Receptor) to identify the antigen biomarker on the disease cell independent of the peptide – major histocompatibility complex; and the transgene (Effector). They are modular, i.e. the Sensor can be exchanged to reprogram the NK-cell Biofactory platform to target biomarkers specific to different cell-based diseases; and the Effector with a therapeutic transgene to neutralize the pathology that triggered the NK-cell Biofactory, essentially creating an off-the-shelf living vector, further enhanced by the innate cytolytic activity of the NK-92MI cell line.

Figure 1. Schematic illustration of the NK-cell Biofactory and plasmid constructs.

Figure 1.

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A) Fully assembled NK-cell Biofactory. B) Artificial cell-signaling pathway comprised of three constant domains (Receptor, Actuator, Secretor) and two variable domains (Sensor, Effector) arranged in cis. Detailed construction of the artificial cell-signaling pathway has been described previously (Ref:[6]).

4.0. Results

Figure 2 illustrates characterizations related to the NK-cell Biofactory function with specificity against the Folate-Receptor alpha (FRα) and mesothelin (MSLN) antigens that are overexpressed on multiple human cancers and whose expression on normal tissues is limited[8,9]. We used OVCAR3 human ovarian cancer cells, that endogenously overexpress FRα and MSLN, as target cells; and A2780cis human ovarian cancer cells, that lack the FRα and MSLN expression, as non-target negative controls. For the variable Sensor domain, we used VH-VL sequences from the anti-FRα antibody[10,11] (MORAb-003) and the anti-MSLN antibody[12,13] (MORAb-009) codon-optimized for human expression. The NanoLuc® (Nluc) (Promega) reporter enzyme was used to represent a variable Effector domain that can be replaced by a human or non-human protein or peptide to induce desired autocrine and paracrine effect. Figure 2A describes the role of constant domains in antigen-directed functional response of the NK-cell Biofactory. The details regarding the optimally configured artificial cell-signaling pathway in regard to the copies of nuclear factor of activated T-cell response element (NFAT-RE) (Actuator), intracellular co-stimulatory domains (Receptor), and signal peptides (Secretor) have been described earlier for the T-cell Biofactory[6]. In context of the NK-cell Biofactory, these constant domains were combined with FRα-specific-Sensor to demonstrate that while it is non-responsive to the FRαnegMSLNneg A2780cis non-target cells, it synthesizes and releases Effector protein upon engaging its target ligand on the surface of FRα+MSLN+ OVCAR3 target cells. The signal-to-noise ratio (S/N), as defined in the figure legend, quantitates specificity of the NK-cell Biofactory. The function of the Secretor domain for release of the Effector protein in the extracellular space was quantified by comparing the Nluc activity in the fully assembled NK-cell Biofactory to the control NK-cell Biofactory that lacked the Secretor domain. The presence of the Secretor domain robustly correlated with the accumulation of Nluc in the supernatant (p<0.0001) and its absence was associated with Nluc build up in the cell pellet (p<0.0001). Similarly, the function of the Receptor domain on regulating our artificial cell-signaling pathway and directing target-cell specificity (Sensor domain is a part of Receptor domain) was quantified by assessing the increased Nluc activity in the cell pellets of the two NK-cell Biofactory with versus without the Receptor domain (p<0.0001).

Figure 2. Engineered function of the NK-cell Biofactory.

Figure 2.

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A) The NK-cell Biofactory synthesizes and secretes Effector proteins upon stimulation by antigen-presenting target cells. The presence of the Secretor domain significantly correlated with the accumulation of Nluc in the supernatant (p<0.0001), while its absence led to Nluc retention in the cell pellet (p<0.0001). Also, the significant difference between the Nluc activity in the cell pellets of the two NK-cell Biofactory (with and without Receptor) (p<0.0001) shows the function of the Receptor domain on regulating the intracellular signaling and directing the specificity (Sensor domain is a part of Receptor domain). B) The Nluc activity was observed within 2 h (p<0.0001) when stimulated by the target (FRα+MSLN+ OVCAR3) versus non-target (FRαnegMSLNneg A2780cis) cells C) The Effector protein expression in the NK-cell Biofactory was the function of the number of target cells. D) The Tukey box-and-whisker plot shows the Nluc activity in the two NK-cell Biofactory (MSLN-specific or FRα-specific when stimulated by the target (FRα+MSLN+ OVCAR3) versus non-target (FRαnegMSLNneg A2780cis) cells. (A-D) Unless otherwise mentioned, the data was collected at NK-cell Biofactory = 12,500 and target/non-target cells = 2,500. The FRα- (A, D) or MSLN-specific (B-D) NK-cell Biofactory were generated per Fig. 1 with FRα- or MSLN-specific Sensor-Receptor but without the Secretor. Nluc activity for all observations was measured using n = 4. The error bars represent 1 standard deviation (SD) above and below the mean and can also be considered as one half-width of an 68% confidence interval for that mean. See Methods section for details on statistical analysis.

Figure 2B shows the kinetics of Effector synthesis from the MSLN-specific NK-cell Biofactory. Nluc activity reporting Effector synthesis increased as early as 2 h (S/N ~2 @ 2 h; S/N ~3.3 @ 6 h, p<0.0001) when MSLN-specific NK-cell Biofactory were stimulated by OVCAR3 (target) cells at Effector-Cell –to– Target-Cell ratio (E:T) = 5:1, compared to A2780cis (non-target) cells. The activity stabilized within 24 h and continued until at least 96 h. Figure 2C shows Effector protein activity from the MSLN-specific NK-cell Biofactory as a function of the target cell mass. While target cell-induced Nluc activity in the NK-cell Biofactory stimulated at low target/non-target cell count was not detectable, it increased exponentially with increasing target cell count and was statistically significantly different from when stimulated by the non-target cells at E:T ≤ 5:1 (p<0.05). To show that the NK-cell Biofactory, similar to the T-cell Biofactory[6], can be redirected to different target antigens, we exchanged the Sensor domain from MLSN-specific single chain variable fragment (scFv)[12,13] to FRα-specific scFv[10,11] sequence. We observed that the Nluc activity in the FRα-specific NK-cell Biofactory, when stimulated by the same number OVCAR3 target cells over a period of 24 h, is significantly upregulated compared to MSLN-specific NK-cell Biofactory (E:T = 5:1) (Figure 2D). This is similar to FRα-specificity and MSLN-specificity observed previously in the T-cell Biofactory[6], and is probably due to higher expression of FRα on OVCAR3 cells, compared to MSLN expression[14]. Alternatively, it could also be due to higher integration of the FRα-specific artificial cell-signaling pathway in the NK-cell Biofactory, compared to the integration of MSLN-specific artificial cell-signaling pathway. Additional validations at 24, 48, and 72 h with different E:T are shown in Figure S1.

Figure 3 reports the cytolytic function of MSLN-specific and FRα-specific NK-cell Biofactory (Effector-Cell) against FRα+MSLN+ OVCAR3 (target) and FRαnegMSLNneg A2780cis (non-target) cells. For this, the target and non-target cells were engineered to express Luc2® (firefly luciferase) (Promega), a 60.6-kDa, ATP-dependent bioluminescent reporter for in vitro and in vivo cell viability. Normalized Luc2 activity from the target and non-target cells, as indicated on the y-axis, is the readout for the NK-cell Biofactory cytolytic function[14].

Figure 3. Innate cytolytic function the NK-cell Biofactory.

Figure 3.

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All experiments included both NK-cell Biofactory [(i) MSLN-specific Sensor-Receptor and (ii) FRα-specific Sensor-Receptor] stimulated by either FRα+MSLN+Luc2–2A-E2Crimson+ OVCAR3 (target) cells or FRαnegMSLNnegLuc2–2A-E2Crimson+ A2780cis (non-target) cells. Both NK-cell Biofactory were generated per Fig. 1 with FRα- or MSLN-specific Sensor-Receptor but without the Secretor. Luc2 activity in target or non-target cells was assessed as a surrogate biomarker for live cells. In all experiments, 0.5% Tween-20 was used as positive control for cell killing. A) The cytolytic activity at 6 h as a function of E:T was fit using a four-parameter logistic model. B) The cytolytic activity at E:T of 0.94:1 as a function of time was fit using a four-parameter logistic model. C) Tukey box-and-whisker plots show comparison between the cytolytic activity at 6 h, E:T of 3.75:1 and 7.5:1 were used. D) Tukey box-and-whisker plots show comparison between the cytolytic activity at 24 h, E:T of 0.94:1 and 1.87:1 were used. (A-D) All data were collected at target/non-target cells = 2,500. Luc2 activity for all observations was measured using n = 3. The error bars represent 1 SD above and below the mean and can also be considered as one half-width of an 68% confidence interval for that mean. See Methods section for details on statistical analysis.

Figure 3A (i-ii) shows the cytolytic activity of (i) MSLN-specific and (ii) FRα-specific NK-cell Biofactory at 6 h co-culture with OVCAR3 (target) cells, which was statistically significantly higher (Figure 3A (i) p<0.05 at all E:T ≥ 0.94:1; Figure 3A (ii) p<0.05 at all E:T ≥ 3.75:1) compared to that against A2780cis (non-target) cells. A parameter for defining the target-specific cytolytic efficiency, η(E:T)50, was determined as the E:T at which Luc2 activity in target or non-target cells was 50% of the difference between the maximum and minimum values of their respective normalized Luc2 activities, when co-cultured with the NK-cell Biofactory for 6 h. The η(E:T)50 of non-target A2780cis cells was higher (~6.5-fold for MSLN-specific NK-cell Biofactory and ~2.5-fold for FRα-specific NK-cell Biofactory) than that of the target OVCAR3 cells.

Figure 3B (i-ii) shows the cytolytic effect of the two NK-cell Biofactory as a function of time and at E:T = 0.94:1. OVCAR3 (target) cell killing was again statistically significantly higher (Figure 3B (i-ii) p<0.05 at all co-culture periods) compared to that of A2780cis (non-target) cells. Time50 was determined as the duration of stimulation at which Luc2 activity in target or non-target cells was 50% of the difference between the maximum and minimum values of their respective normalized Luc2 activities, when co-cultured with the NK-cell Biofactory at E:T = 0.94:1. The difference between the means became more pronounced at 24 h and diminished again over longer durations, which allowed for the cumulative increase in non-specific cytolytic activity. This effect is further emphasized in Figure S2.

Figures 3C (i-ii) and 3D (i-ii) assess the cytolytic function of the two NK-cell Biofactory at different E:T at 6 and 24 h of co-culture with OVCAR3 (target) cells and A2780cis (non-target) cells, respectively. A lower E:T demonstrated sufficient differential cytolytic effect at longer co-culture periods, an effect that has also been computationally calculated[15]. Therefore, E:T along with the duration over which the target-specific cytolytic effect is observed, should be carefully balanced when designing in vitro assays. Additional validations with a larger E:T range are shown in Figure S2(A-C) [(A): 24 h; (B): 48 h; (C); 72 h].

The NK-92MI is a clinically relevant cell line and the cells are irradiated at 10 Gy before human infusion[16]. This stops further proliferation and renders them non-oncogenic. Up to 10 billion NK cells/m2 have been safely infused in humans with no severe side effects[17]. However, the resulting DNA damage also has the potential to disrupt the Actuator and Effector sequences presenting a risk for the NK-cell Biofactory to be non-functional. Figure 4 demonstrates that the integrity of the artificial cell-signaling pathway and ultimately that of the NK-cell Biofactory is preserved after exposure to 15 Gy radiation, thereby making it safe for clinical use. Figure 4A shows the kinetics of Effector synthesis from the FRα-specific NK-cell Biofactory. Nluc activity reporting Effector synthesis increased within 5 h (S/N ~3 @ 5 h, p<0.05) when stimulated by OVCAR3 (target) cells compared to A2780cis (non-target) cells (E:T = 5:1), and increased linearly until at least 72 h. Figure 4B shows the Effector protein activity from the irradiated FRα-specific NK-cell Biofactory as a function of the target cell mass. Target cell-induced Nluc activity by the NK-cell Biofactory was statistically elevated when stimulated by OVCAR3 (target) cells compared to A2780cis (non-target) cells at all cell counts (p<0.05). The minor increase in Nluc activity seen with non-target A2780cis cells is the result of non-specific stimulation of the NK-cell Biofactory, a characteristic of immune cell signaling also reported earlier[6]. Figure 4C shows that the Effector protein activity from the same irradiated FRα-specific NK-cell Biofactory increases as a function of their cell number with a constant target/non-target cell mass. The Nluc activity was statistically elevated when stimulated by OVCAR3 (target) cells compared to A2780cis (non-target) cells at all cell counts with E:T ≥ 0.625:1 (p<0.01). The dose 10 Gy has also been found to be acceptable in clinical trials in terms of their cytolytic efficacy[17]. We conducted the cytolytic assay with the FRα-specific NK-cell Biofactory irradiated at 15 Gy and the results are shown in Figure 4D. The irradiated NK-cell Biofactory was co-cultured for 6 h with OVCAR3 (target) cells, resulting in a statistically significant increase of target-specific (OVCAR3) cytolysis (p<0.05 at E:T = 100:1) compared to that against A2780cis (non-target) cells. Of note, this effect is lower than that of non-irradiated NK-cell Biofactory (Figure 3A).

Figure 4. Engineered and innate functions of the irradiated NK-cell Biofactory.

Figure 4.

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The integrity of the artificial-cell-signaling pathway in FRα-specific NK-cell Biofactory (generated per Fig. 1 with FRα-specific Sensor-Receptor but without the Secretor) is preserved when irradiated at 15 Gy as measured by its engineered and innate functions. A) The Nluc activity was observed within 5 h (p<0.05) when stimulated by the target (FRα+MSLN+ OVCAR3) versus non-target (FRαnegMSLNneg A2780cis) cells. B) The Effector protein expression in the NK-cell Biofactory was proportionate to the number of target cells. C) The Effector protein expression in the NK-cell Biofactory was the function of the number of effector cells. D) The cytolytic activity at 6 h as a function of E:T was fit using a four-parameter logistic model. Luc2 activity in target (FRα+MSLN+Luc2–2A-E2Crimson+ OVCAR3) or non-target (FRαnegMSLNnegLuc2–2A-E2Crimson+ A2780cis) cells was assessed as a surrogate biomarker for live cells; 0.5% Tween-20 was used as positive control for cell killing. (A-D) Unless otherwise mentioned, the data was collected at NK-cell Biofactory = 12,500 and target/non-target cells = 2,500. The error bars represent 1 SD above and below the mean and can also be considered as one half-width of an 68% confidence interval for that mean. (A-C) n = 3, (D) n = 4. See Methods section for details on statistical analysis.

5.0. Discussion

Despite recent approvals by the U.S. Food and Drug Administration (FDA)[18] for cell therapies, the full impact of T-cell-based life-saving drugs has been limited. Primarily due to the autologous nature of adoptive cell therapies, which contributes to high manufacturing cost, product variability, and potential for adverse events; we recognize the possibility of a clinical obstacle that may emerge during the translation of our previously reported primary T-cell Biofactory[6,7]. Therefore, we have utilized the NK-92MI cell line as a cellular chassis, which allowed us to generate and test NK-cell Biofactory in parallel to the T-cell Biofactory[6,7,14]. With a track record for exerting anti-tumor effect in the clinic[17,19–21] and doubling time of ~ 24 h [19,21], NK-92MI is a strong candidate offering the potential for off-the-shelf allogeneic option[22].

Tumor cells develop resistance to NK cells by reduced expression of NK-cell activating ligands on their surfaces[23,24]. Primary NK cells and NK cell lines have been engineered with CARs (CAR NK cells) to overcome this resistance[25,26] and improve killing of solid tumors[22,27–30]. While the CAR architecture employed in CAR NK cells was derived from the ones used in CAR T cell therapies, comparatively more effective designs that draw from NK cell activation domains have now surfaced[31] and further offer the potential to improve the artificial cell-signaling pathway employed in generating NK-cell Biofactory. No evidence of GvHD was observed in 17 different clinical studies with allogeneic NK cells[32], further emphasizing that NK-cell Biofactory could be an off-the-shelf cell-based living vector. Although the relatively short half-life of primary NK cells (1–2 weeks in vivo) compared to that of T cells remains a concern, the decreased longevity provides the benefit of preventing continued targeting of healthy cells. Genetic manipulation and expansion of primary NK cells are more challenging compared to primary T cells and is undoubtedly a barrier[33]; however the use of the NK-92MI cell line engineered in these studies offers an alternative means to generate NK cell-based immunotherapy. The use of induced pluripotent stem (iPS) cells as starting materials also offers another means to mitigate challenges with primary NK cell therapies[31,34].

Beyond the advantages that the use of NK cell offers, the significance of the NK-cell Biofactory platform stems from its modularity. Antigen-specificity, as expected, overcomes tumor resistance and directs the cytolytic function exclusively towards different antigen-presenting target cells; and the artificial cell-signaling pathway introduces a new capability to serve as a vector— for producing calibrated amounts of protein-based therapeutics and inducing desired autocrine and paracrine signaling, upon engaging the target antigen. The technology presents the capability of focused synthesis of the desired biologics at the target site and offers the hope to extend treatment duration for better outcome by limiting systemic toxicity. We have shown that the integrity of the NK-cell Biofactory artificial cell-signaling pathway and cytolytic activity are conserved following irradiation, although lower than that of non-irradiated NK-cell Biofactory, further emphasizing its potential for safe use in the clinic.

Successful implementation of the artificial cell-signaling pathway in an NK-cell line presented in this work, in addition to T-cell line[6] and primary T cells[7] extend the options for selecting a cellular chassis based on the advantages they offer. This work additionally raises curiosity for investigating other cell types, e.g. primary NK cells, iPS cells, mesenchymal stem cells, TALL-104, K562, as cellular chassis, which may offer alternative therapeutic benefits. Overall, challenges still remain but continuous improvements in cell manufacturing and emerging strategies in cellular therapies offer a promise of advancing the primary cell-based and the cell-line based Biofactory into a clinical technology.

Supplementary Material

adbi.202000298_SupportingInformation_RawData
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6.0. Acknowledgements

Research reported in this publication was supported in part by the National Institute of Biomedical Imaging and Bioengineering (DP2EB024245: NIH Director’s New Innovator Award Program (https://commonfund.nih.gov/newinnovator) and the National Cancer Institute (R21CA236640, R33CA247739) of the National Institutes of Health (NIH); and the Defense Advanced Research Projects Agency (DARPA) (D19AP00024: DARPA Young Faculty Award (https://www.darpa.mil/work-with-us/for-universities/young-faculty-award). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or DARPA. Authors thank Didier Trono (Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland) for providing the lentivirus packaging plasmids.

Footnotes

8.0

Additional information

Competing financial interests. The authors declare no competing financial interests.

9.0

Experimental section. See Supporting Information.

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Supplementary Materials

adbi.202000298_SupportingInformation_RawData
NIHMS1702971-supplement-adbi_202000298_SupportingInformation_RawData.xlsx (66.1KB, xlsx)
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