Designing Red Blood Cell-Derived Vesicle Natural Killer Cell Engagers for Agnostic Cancer Treatment 

1. Rationale

 

Immune cell engagers (ICEs) are an emerging feasible alternative to adoptive cell therapy that utilizes modified molecules to redirect immune effector cells (IECs), targeting specific tumour associated antigens (TAAs) on cancer cells. Conventional FDA-approved ICEs mainly consist of CD3+ T cell engagers (Shi, 2024), as the idea of T cell therapy is much more explored than any other adoptive cell therapies. However, despite its noteworthy performance, current T cell therapies face challenges like severe side effects (e.g., cytokine release syndrome (CRS), graft-versus-host disease (GvHD)), limited efficacy in solid tumours, manufacturing complexity, and risk of cancer relapse (Gershenson & Mello, 2022). 

Natural Killer (NK) cell therapy, introduced in 2005 (Karolinska Institutet, n.d.), brings an advantage over T cell therapy as an alternative functional type of immunotherapy. NK cells are cytotoxic lymphocytes that target diseased cells without prior priming, enabling “off-the-shelf” use and compatibility for patients ineligible for traditional T cell therapy. Early correlative research also highlights the integral trait of NK cell therapy in bypassing immunosuppressive tumor microenvironments (TMEs) (Lian et al., 2021), underscoring again the advantages of NK cell therapy against other IEC therapies. However, NK cell activation relies on multi-step signaling, and cancer cells can appear refractory to NK cell recognition by downregulating activational ligands like MHC class 1 molecules, reducing immunosurveillance, promoting metastasis, and rendering the adaptive immune system ineffective (Seliger & Koehl, 2022). Such resistance remains a key limitation for NK therapy. 

In recent years, specialized bispecific antibodies engineered to target NK cells and cancer cells have shown success in facilitating engagement and NK cytotoxicity. Yet, their clinical application is restrained by on-target, off-tumour toxicities due to their availability for binding of a singular activating ligand. Such bispecific antibodies are constrained to target a specific type of cancer, limiting their broader applications. 

One of the biggest challenges in immunotherapy is the complete eradication of solid tumors. Oncofetal chondroitin sulfate (ofCS) is a cancer-specific secondary modification to proteoglycans that is expressed in various solid tumors and metastasis but most commonly present on the placenta (Vidal-Calvo et al.). The malaria protein VAR2CSA binds to ofCS, enabling Plasmodium falciparum-infected erythrocytes to adhere to the placenta in order to avoid filtration by the spleen during merozoite invasion. Such adhesion was studied, and rVAR2 recombinant proteins were designed to target the ofCS glycoprotein on all cancers, functioning as a tumour-associated antigen (TAA), thereby maximizing the therapeutic index for ofCS-targeting therapies. 

In this project, we aim to address limitations in current NK cell therapy and immunotherapy through designing a biocompatible and histology-agnostic NK multifunctional engager platform, capable of preventing “off-target” cytotoxicity while ensuring cancer-specific targeting. Such a treatment will be delivered via red blood cell (RBC)-derived vesicles to ensure the biocompatible and biomimetic traits of the treatment to improve on current immunotherapy. RBC vesicles will be “armed” with specialized NK cell activating antibodies (anti-CD16, NKp46) and recombinant rVAR2 in vitro to conjugate and activate NK cells to cancer cells.

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2. Engineering Goal

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The goal of this project is to create multifunctional, biocompatible red blood cell (RBC)-derived vesicle NK cell engagers to stimulate NK cell activation and optimized antibody-dependent cytotoxicity towards ofCS+ cancer cells by “arming” biomimetic vesicles with NK activation antibodies (anti-CD16, NKp46) and rVAR2 recombinant protein at a 1:1:1 molar ratio. As aCD16 and NKp46 are both NK-activating antibodies, we hope to facilitate NK cell activation through our “armed” vesicles while targeting the ofCS protein on cancer cells. In doing so, we would potentially bridge the activated NK cell and targeted cancer cell together through the RBC-“armed” vesicles. 

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3. Materials and Methods

 

a. RBC-vesicle generation

Whole blood will be collected in EDTA tubes and processed within 24 hours of collection. RBCs will be isolated from human peripheral blood using a Ficoll-based method, followed by centrifugation to obtain a purified RBC pellet. The isolated RBCs are then harvested to be lysed progressively with hypotonic solution (1:3 PBS 1x with ddH2O) to release hemoglobin. The resulting RBC ghosts will be washed with cold phosphate-buffered saline (PBS) to ensure complete hemoglobin removal. This state of the vesicles is termed micro-erythrocytes (MERs), which will then be washed repeatedly to better obtain RBC ghosts for vesicle production. Aliquots will then be sonicated at 60% amplitude with a 1-second on, 1-second off cycle, performed in triplicates, to disrupt the ghost membranes into nanoscale vesicles (NERs) using ultrasonic energy. The resulting NERs will then be characterized using a zetasizer to obtain zeta potential and particle mobility through Dynamic Light Scattering (DLS).

 

b. Labeling with 6 Azidohexanoic Acid NHS ester Click Chemistry Reagent and Density Gradient Centrifugation

To adjoin aCD16, NKp46, and rVAR2 recombinant protein to the RBC vesicle membrane, the NHS-DBCO click chemistry linkage is used for stable conjugation. The bifunctional linker enables conjugation of molecules by using a linker with two reactive groups. The NHS ester reacts selectively with primary amines present on the RBC-vesicle membrane. The DBCO group undergoes a strain-promoted alkyne-azide cycloaddition (SPAAC) with azide-functionalized antibodies. RBC-vesicles are labeled with NHS, and DBCO is conjugated to antibodies separately. Vesicles and 6 azido hexanoic acid NHS (Vector Labs) will be incubated for 2 hours at room temperature to enable conjugation. Excess linker is removed using OptiPrep density gradient centrifugation, where samples are separated in layers through the difference in density of the materials and PBS. A 50% OptiPrep (Sigma) bed will be layered at the bottom of Eppendorf tubes to prevent aggregation of NHS-labeled vesicles when spun for 50 minutes at 20,000 G. Pelleted labeled vesicles are then collected and stored for later characterization. 

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Fig 1 - Density gradient centrifuge process 

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Protein quantification will be performed using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), which measures Cu2+ reduction Cu1+ from by peptide bonds, producing a purple color proportional to protein content. Results of the BCA assay will be identified on a spectrophotometer at a wavelength of 562 nm and calculated using a standard curve. Flow cytometry will be used to confirm NHS incorporation on NERs. Since NERs are likely to be in the nanoscale and too small for direct detection by the cytometer, they will be conjugated to 4-μm latex beads (Thermo Fisher Scientific) for better visibility. A fluorescent secondary tag (DBCO-488) will be labeled to BEADS-NER-NHS for detection. Excess beads and DBCO-488 will be removed through a multiple washing process. A fluorescent shift analyzed through the 488 channel (FL1-FITC) between the controlled and NHS-stained groups will be an indicator of successful labeling. 

 

c. Labeling Proteins (aCD16, NKp46, rVAR2 recombinant protein) with DBCO-PEG4-NHS for Click Chemistry 
DBCO-PEG4-NHS (Vector Labs) and protein (aCD16 (Thermo Fisher Scientific), NKp46 (Ottawa Hospital Research Institute), and rVAR2 (Vancouver Prostate Centre))will be conjugated at a 3:1 molar ratio. Pre-labeling conditions will be recorded to detect the wavelengths before the addition of DBCO to establish baseline wavelengths. The absorbance rate is to be measured at 280 (protein) and 309 nm (DBCO-PEG4-NHS). The reaction mixture (protein + DBCO-PEG4-NHS) will be incubated at room temperature for 1 hour with shaking at 600 rpm to facilitate conjugation. To filter out non-reactive reagents, a desalting column (Thermo Fisher Scientific) will be prepared according to the manufacturer’s instructions to isolate conjugated protein-DBCO molecules from residual DBCO linker. The post-labeling absorbance value will be measured at 280 nm and 309 nm after labeling to calculate the degree of labeling. This process is to be repeated for all three proteins. 

 

d. Labeling Proteins (aCD16, NKp46, rVAR2 recombinant protein) with DBCO-PEG4-NHS for Click Chemistry 
NERs-NHS will be conjugated with proteins (aCD16, NKp46, and rVAR2 recombinant protein) at equimolar ratios (1:1:1). The extent of NERs-NHS molecules available for protein attachment will be quantified using a BCA assay, performed according to standard protocols. DBCO-functionalized proteins will be incubated with NERs-NHS for 2 hours on ice on a shaking plate to promote conjugation via SPAAC. Following incubation, the NER-NHS-DBCO-protein mixture will be layered on top of a discontinuous OptiPrep density gradient (10% and 50% w/v) and ultracentrifuged for 2 hours at 100,000 G at 4°C. This step allows separation between “armed” NERs, which sediment through the 10% layer but are retained above the 50% layer due to their density, from residual supernatants, which remain above the 10% interface. The purified NER-proteins will be collected from the 10-50% interface and sterilized through a 0.22 μm sterilization spin cup. 
 

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Fig 2 - Density gradient ultracentrifugation process

 

Subsequent characterization involves a BCA assay to quantify conjugated protein content and DLS using a zetasizer to assess NER size stability post-conjugation. Flow cytometry will be run to validate the presence of all proteins being successfully conjugated to NERs. Flow cytometry will be used to validate successful conjugation of all proteins, detected through the pre-existing fluorescence of aCD16 and NKp46 clones and Alexa-labeled rVAR2, obviating the need for additional fluorescent secondaries conjugation. Data acquired on the flow cytometry will be further analyzed using FlowJo software (v10.8). Fluorescence shifts are detected by switching to the protein-corresponded channels (e.g., FITC for aCD16, PE for NKp36, Alexa Fluor 647 for rVAR2 TAA), with shifts in fluorescence intensity depicting successful protein incorporation onto NERs. 

 

e. Cytotoxicity Assessment of “Armed” NER-Activated NK-92 Cells Against K562 Lymphoma Cells through Lactate Dehydrogenase Release (LDH) Assay
To evaluate the cytotoxic performance of NK-92 cells activated via “armed” NERs against human K562 lymphoma cells, a lactate dehydrogenase (LDH) release assay (Abcam), which tests the quantity of LDH released during cell death, will be optimized in a 96-well plate format. NK-92 cells will be seeded at 5 x 104 cells/well in RPMI 1640 medium and with 10% FBS and 200 U/ml IL-2 to be pre-incubated with “armed” NERs for 2 hours at 37oC to allow antibody-facilitated NK cell activation via aCD16 and NKp46. K562 cells will be seeded at 2 x 104 cells/well, either alone for spontaneous LDH release (S) or co-cultured with activated NK-92 cells at a 5:1 effector to target (E:T) ratio for 4 hours at 37oC in 5% CO2. Supernatants, collected at 50 µL, will be transferred to a separate assay plate, mixed with 50 µL LDH reagents, incubated for 1 hour in the dark, and interrupted with 50 µL M acetic acid. The LDH reagent reacts with the release of LDH, producing formazan, a purple dye that will be measured as optical density (OD) at 490 nm by a SpectraMax M5 plate reader. The total LDH release (T) will be determined by lysing K562 cells with 10 µL 1% Triton X-100 (v/v) for 3 minutes. 

 

4. RISK AND SAFETY 

The Laboratory Safety and Biosafety Certificate is received before the start of the project. All experimental procedures will be conducted under institutional biosafety and chemical safety guidelines. Supervision is provided at all times by research associate/safety rep. Personal protective equipment (PPE) is provided and worn at all times in the lab to minimize risks. All procedures involving CL2 containment, such as cell lines and human peripheral blood, will be performed in the CL2 tissue culture lab. CL2 liquid waste is treated with bleach, and CL2 dry waste is disposed of in bio-tubs sent for incineration. ​

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5. Data Analysis ​

​The cytotoxicity of NER-activated NK-92 against K562 will be calculated in the following formula: 

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​where: 
OD490LDHr (LDH release) = OD490 from K526 cells killed by NKp46 with or without NERs

OD490S (spontaneous, controls) = OD490 from K562 alone 
OD490NK (NK-92 background) = OD490 from NK-92 with or without NERs

OD490T (Total) = OD490 from K562 fully lysed with 1% Triton X-100 

Conditions will be tested in quadruplicate, with data analyzed using SoftMax Pro v7.1, comparing NER-activated NK-92 cells (E:T+NER) to unstimulated controls (E:T) to assess NERs’ enhancement of NK cell-mediated specific cytotoxicity. 

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6. Bibliography

Abel, Alex M., et al. “Natural Killer Cells: Development, Maturation, and Clinical Utilization.” Frontiers in Immunology, vol. 9, 13 Aug. 2018, https://doi.org/10.3389/fimmu.2018.01869. Accessed 19 Feb. 2025. 

---. “Natural Killer Cells: Development, Maturation, and Clinical Utilization.” Frontiers in Immunology, vol. 9, 13 Aug. 2018, https://doi.org/10.3389/fimmu.2018.01869. Accessed 19 Feb. 2025. 

Ansari, Azim, et al. “Nanovesicles Based Drug Targeting to Control Tumor Growth and Metastasis.” Advances in Cancer Biology - Metastasis, vol. 7, July 2023, p. 100083, https://doi.org/10.1016/j.adcanc.2022.100083. Accessed 19 Feb. 2025. 

Au, Kin Man, et al. “Abstract 1723: Trispecific Natural Killer Cell Nanoengagers for Targeted Chemoimmunotherapy.” Cancer Research, vol. 80, no. 16_Supplement, 15 Aug. 2020, pp. 1723–1723, https://doi.org/10.1158/1538-7445.am2020-1723. Accessed 19 Feb. 2025. 

Cao, Wendy, et al. “Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions.” Bioconjugate Chemistry, vol. 34, no. 3, 14 Feb. 2023, pp. 510–517, https://doi.org/10.1021/acs.bioconjchem.2c00576. Accessed 19 Feb. 2025. 

Gauthier, Laurent, et al. “Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity.” Cell, vol. 177, no. 7, June 2019, pp. 1701-1713.e16, https://doi.org/10.1016/j.cell.2019.04.041. Accessed 19 Feb. 2025. 

Jiang, Dandan, et al. “Driving Natural Killer Cell-Based Cancer Immunotherapy for Cancer Treatment: An Arduous Journey to Promising Ground.” Biomedicine & Pharmacotherapy, vol. 165, Sept. 2023, p. 115004,  https://doi.org/10.1016/j.biopha.2023.115004. Accessed 19 Feb. 2025. 

Khazamipour, Nastaran, et al. “Oncofetal Chondroitin Sulfate: A Putative Therapeutic Target in Adult and Pediatric Solid Tumors.” Cells, vol. 9, no. 4, 28 Mar. 2020, p. 818, https://doi.org/10.3390/cells9040818. Accessed 19 Feb. 2025. 

 

---. “Oncofetal Chondroitin Sulfate: A Putative Therapeutic Target in Adult and Pediatric Solid Tumors.” Cells, vol. 9, no. 4, 28 Mar. 2020, p. 818, https://doi.org/10.3390/cells9040818. Accessed 19 Feb. 2025. 

Murugan, Dhanashree, et al. “Nanoparticle Enhancement of Natural Killer (NK) Cell-Based Immunotherapy.” Cancers, vol. 14, no. 21, 4 Nov. 2022, p. 5438, https://doi.org/10.3390/cancers14215438. Accessed 19 Feb. 2025. 
 

Pickens, Chad J., et al. “Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide–Alkyne Cycloaddition.” Bioconjugate Chemistry, vol. 29, no. 3, 29 Dec. 2017, pp. 686–701, https://doi.org/10.1021/acs.bioconjchem.7b00633. Accessed 19 Feb. 2025. 
 

Salanti, Ali, et al. “Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.” Cancer Cell, vol. 28, no. 4, Oct. 2015, pp. 500–514, https://doi.org/10.1016/j.ccell.2015.09.003. Accessed 19 Feb. 2025. 
 

Seiler, Roland, et al. “An Oncofetal Glycosaminoglycan Modification Provides Therapeutic Access to Cisplatin-Resistant Bladder Cancer.” European Urology, vol. 72, no. 1, July 2017, pp. 142–150, https://doi.org/10.1016/j.eururo.2017.03.021. Accessed 19 Feb. 2025. 
 

Singh, Ajit, et al. “Overcoming the Challenges Associated with CD3+ T-Cell Redirection in Cancer.” British Journal of Cancer, vol. 124, no. 6, 19 Jan. 2021, pp. 1037–1048, https://doi.org/10.1038/s41416-020-01225-5. Accessed 19 Feb. 2025. 
 

Topham, Nicola J., and Eric W. Hewitt. “Natural Killer Cell Cytotoxicity: How Do They Pull the Trigger?” Immunology, vol. 128, no. 1, 4 Aug. 2009, pp. 7–15, https://doi.org/10.1111/j.1365-2567.2009.03123.x. Accessed 19 Feb. 2025.

 

van der Horst, Hilma J., et al. “Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies.” Cancers, vol. 12, no. 10, 19 Oct. 2020, p. 3041, https://doi.org/10.3390/cancers12103041. Accessed 19 Feb. 2025. 
 

Vidal-Calvo, Elena Ethel, et al. “Tumor-Agnostic Cancer Therapy Using Antibodies Targeting Oncofetal Chondroitin Sulfate.” Nature Communications, vol. 15, no. 1, 30 Aug. 2024, https://doi.org/10.1038/s41467-024-51781-0. Accessed 19 Feb. 2025. 
 

Wang, Chris Kedong, et al. “Internalization and Trafficking of CSPG-Bound Recombinant VAR2CSA Lectins in Cancer Cells.” Scientific Reports, vol. 12, no. 1, 23 Feb. 2022, https://doi.org/10.1038/s41598-022-07025-6. Accessed 19 Feb. 2025. 
 

Yang, Xiao-mei, et al. “Nanobody-Based Bispecific T-Cell Engager (Nb-BiTE): A New Platform for Enhanced T-Cell Immunotherapy.” Signal Transduction and Targeted Therapy, vol. 8, no. 1, 4 Sept. 2023, https://doi.org/10.1038/s41392-023-01523-3. Accessed 19 Feb. 2025. 
 

Ye, Qian-Ni, et al. “Orchestrating NK and T Cells via Tri-Specific Nano-Antibodies for Synergistic Antitumor Immunity.” Nature Communications, vol. 15, no. 1, 23 July 2024, https://doi.org/10.1038/s41467-024-50474-y. Accessed 19 Feb. 2025. 
 

Zhang, Minchuan, et al. “Natural Killer Cell Engagers (NKCEs): A New Frontier in Cancer Immunotherapy.” Frontiers in Immunology, vol. 14, 9 Aug. 2023, 
https://doi.org/10.3389/fimmu.2023.1207276. Accessed 19 Feb. 2025.