An engineered extracellular vesicle and its use in nk cell expansion

Abstract

The application discloses an engineered extracellular vesicle, wherein the membrane surface of the extracellular vesicle displays an anti-CD16a nanobody. The application uses an engineered extracellular vesicle derived from a eukaryotic cell as an NK cell stimulating carrier, and does not need to use a blood tumor-derived living feeder cell such as K562, nor does it rely on membrane particles formed by crushing a K562 cell membrane. Thus, problems such as residual living feeder cells, insufficient inactivation, residual tumor-derived DNA or membrane components, and the like can be avoided, the safety risk and quality control difficulty in the preparation process of NK cells are reduced, the use of a tumor-derived living feeder cell is avoided, and the safety and regulatory risk are reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an engineered extracellular vesicle and its application in NK cell expansion. Background Technology

[0002] Natural killer (NK) cells are important effector cells of the innate immune system, capable of recognizing and killing tumor cells, virus-infected cells, and abnormally stressed cells without prior antigen sensitization. They have significant application value in tumor immunotherapy and the development of universal cell therapy products. Primary NK cells derived from peripheral blood have a good safety profile and well-defined functions, making them an important cell source for current adoptive NK cell therapy. However, the proportion of NK cells in peripheral blood is low, making it difficult to directly meet the cell quantity required for clinical reinfusion. Therefore, it is necessary to establish an efficient, stable, reproducible, and clinically-grade in vitro expansion system.

[0003] Existing NK cell expansion methods mainly include simple cytokine stimulation and feeder cell stimulation. When simply using soluble cytokines such as IL-2, IL-15, and IL-21 for stimulation, the system is relatively simple, but it lacks membrane-contact, multivalent aggregation, and spatially oriented activation signals, resulting in limited expansion efficiency, significant differences between donors, and insufficient functional maintenance after expansion. Engineered feeder cells based on K562 cells can significantly promote NK cell expansion by expressing molecules such as 4-1BBL, membrane-bound IL-15, and membrane-bound IL-21. However, K562 cells are hematologic malignancies, posing safety and regulatory risks such as residual live cells, inadequate inactivation, and residual host DNA or tumor-derived membrane components. Furthermore, the production system is complex, and batch-to-batch consistency and quality control are difficult.

[0004] To reduce the risks associated with live feeder cells, some technologies have attempted to use cell membrane granules, membrane vesicles, or plasma membrane granules derived from engineered cells such as K562 to replace intact feeder cells. Although such cell-free systems can retain membrane-bound stimuli to some extent, they are mostly derived from the fragmentation or disruption of tumor cell membranes and may still carry complex tumor-derived membrane components. Furthermore, they suffer from problems such as insufficient particle uniformity, impaired protein conformation, uncontrollable surface molecular orientation, and significant batch-to-batch variability.

[0005] Extracellular vesicles (EVs) are nanoscale membrane vesicles naturally released by cells, possessing excellent biocompatibility and surface molecular display potential. By expressing membrane-anchoring functional proteins in donor cells through genetic engineering, corresponding molecules can be loaded onto the surface of EVs, thereby constructing engineered extracellular vesicles with specific immunomodulatory functions. However, ordinary EVs usually lack the ability to target key activating receptors of NK cells. EVs carrying only a single cytokine or co-stimulatory molecule are also difficult to simultaneously satisfy the receptor activation, cytokine support, and co-stimulatory signals required for NK cell expansion.

[0006] CD16a is an important activation receptor on the surface of NK cells that mediates antibody-dependent cytotoxicity. Cross-linking and aggregation of CD16a can induce NK cell activation, degranulation, and enhanced cytotoxicity. Traditional anti-CD16 antibodies can be used for NK activation, but soluble antibodies are difficult to provide stable, spatially oriented membrane contact stimulation. Conventional antibodies have large molecular weights, which are not conducive to high-density display on the surface of nanoscale vesicles. VHH nanobodies have the characteristics of small molecular weight, good stability, and easy genetic engineering fusion expression, making them suitable as membrane-anchored CD16 activation modules on the surface of EVs.

[0007] Therefore, there is still a need in the field for a new in vitro expansion system for NK cells that can avoid the safety and regulatory risks of tumor-derived feeder cells such as K562, while overcoming problems such as insufficient efficiency of simple cytokine stimulation, complex composition of existing membrane particles, and insufficient activation capacity of ordinary EVs. Based on CD16 nanobodies, engineered extracellular vesicles are constructed, and CD16 aggregation activation signals, membrane-anchored cytokine signals, and co-stimulatory signals are synergistically displayed on their surface. This is expected to achieve efficient, stable, and controllable expansion of primary NK cells under conditions without live cell feeder layers. Summary of the Invention

[0008] The purpose of this invention is to solve the problems in the prior art by proposing an engineered extracellular vesicle and its application in NK cell expansion, which can solve the above problems.

[0009] To achieve the above objectives, the present invention proposes an engineered extracellular vesicle, wherein the surface of the extracellular vesicle membrane displays an anti-CD16a nanobody.

[0010] Preferably, the extracellular vesicles are derived from genetically engineered eukaryotic cells.

[0011] Preferably, the CD16a nanobody is expressed on the surface of an engineered eukaryotic cell membrane in a membrane-anchored form and is loaded onto the surface of the extracellular vesicle membrane during the extracellular vesicle formation process to form a membrane-anchored CD16a nanobody. Preferably, the membrane-anchored CD16a nanobody comprises, from the N-terminus to the C-terminus, a signal peptide, an anti-human CD16aVHH nanobody domain, a linker peptide, and a transmembrane anchoring region.

[0012] Preferably, the signal peptide is a signal peptide that can mediate protein secretion pathway expression; Preferably, the linker peptide is a flexible peptide rich in glycine and serine; Preferably, the transmembrane anchoring region is a transmembrane or membrane anchoring structure that enables the fusion protein to be located on the cell membrane and loaded onto the surface of the extracellular vesicle membrane.

[0013] Preferably, the signal peptide is selected from one or more of Igκ signal peptide, CD8α signal peptide, and IL-2 signal peptide; Preferably, the linker peptide is selected from one or more of GGGGS, (GGGGS)2, and (GGGGS)3; Preferably, the transmembrane anchoring region is selected from one or more of the following anchoring signals: CD8α, CD28, PDGFR, LAMP2B, CD63, CD81, and GPI.

[0014] Preferably, the membrane-anchored CD16a nanobody further includes an intracellular stabilizing region.

[0015] The CD16 nanobody can be stably positioned on the surface of engineered eukaryotic cell membranes and further enriched on the surface of extracellular vesicle membranes, enabling the extracellular vesicles to induce CD16a aggregation and activation in NK cells.

[0016] Preferably, the membrane-anchored CD16a nanobody structure contains the amino acid sequence shown in SEQ ID NO:3.

[0017] Preferably, the surface of the extracellular vesicle membrane also exhibits membrane-anchored IL-21 and / or membrane-anchored IL-15.

[0018] Both preferred membrane-anchored IL-21 and membrane-anchored IL-15 include: cytokine functional domains, linker peptides and / or stabilizing linker modules, and transmembrane anchoring regions.

[0019] The stabilizing linker is the CH2 / CH3 region in the IgG Fc domain; The linker peptide is a flexible peptide rich in glycine and serine, and the linker peptide is selected from one or more of GGGGS, (GGGGS)2, and (GGGGS)3.

[0020] The transmembrane anchoring region is selected from one or more of the following anchoring signals: CD8α, CD28, PDGFR, LAMP2B, CD63, CD81, and GPI.

[0021] Preferably, the membrane-anchored IL-21 includes an IL-21 functional domain, a linker region, and a transmembrane domain, and the membrane anchoring type contains the amino acid sequence shown in SEQ ID NO:4.

[0022] Preferably, the membrane-anchored IL-15 includes an IL-15 functional structural domain, an IL-15Rαsushi structural domain, a connection region, and a transmembrane structural domain.

[0023] Preferably, the membrane-anchored IL-15 contains the amino acid sequence shown in SEQ ID NO:5.

[0024] Preferably, the surface of the extracellular vesicle membrane also exhibits co-stimulatory molecules.

[0025] Preferably, the co-stimulatory molecule is CD86 and / or CD137L.

[0026] Preferably, CD86 provides a co-stimulatory signal associated with NK cell activation and contains the amino acid sequence shown in SEQ ID NO:6; CD137L promotes NK cell survival, proliferation and effector function maintenance through the CD137 pathway and contains the amino acid sequence shown in SEQ ID NO:7.

[0027] The present invention also provides an engineered eukaryotic cell for preparing the above-mentioned engineered extracellular vesicles.

[0028] The engineered eukaryotic cells are selected from one or more of the following: Chinese hamster ovary cells (CHO, CHO-K1, CHO-S, CHO-DG44); human embryonic kidney-derived cells (HEK293, HEK293T, HEK293F); and insect cells (Sf9, Sf21).

[0029] Preferably, the engineered eukaryotic cells are non-tumor-derived feeder cells, thereby avoiding safety and regulatory risks such as residual live cells, tumor-derived DNA, or membrane components that may arise from using tumor-derived cells such as K562 as feeder cells.

[0030] The engineered eukaryotic cells were obtained through transient transfection, stable transfection, viral vector transduction, site-directed genome integration, or other genetic engineering methods.

[0031] The engineered eukaryotic cells simultaneously or separately express the following molecules: Membrane-anchored CD16a nanobodies; Membrane-anchored IL-21 and / or IL-15; CD86 and / or CD137L.

[0032] The above molecules can be expressed by multiple expression vectors separately, or by the same polycistronic expression vector.

[0033] The expression vector may include CMV, EF1α, CAG, SV40, SFFV or other promoters suitable for eukaryotic cell expression.

[0034] Preferably, the engineered cells maintain a stable expression level of the above-mentioned molecules during continuous culture for 3–14 days, and the positive expression rate of the target protein on the cell membrane reaches 60–95%.

[0035] The present invention also provides a method for preparing the above-mentioned engineered extracellular vesicles, comprising the following steps: (1) Construct engineered eukaryotic cells expressing membrane-anchored CD16a nanobodies, membrane-anchored IL-21 and / or IL-15, CD86 and / or CD137L; (2) The engineered eukaryotic cells are cultured in a serum-free, low-serum, or low-vesicle background medium to secrete extracellular vesicles carrying the above-mentioned functional molecules. (3) Collect the supernatant from the culture of engineered eukaryotic cells; (4) Separate and purify the culture supernatant to obtain engineered extracellular vesicles; Preferably, the separation and purification methods in step (4) include low-speed centrifugation, filtration, ultrafiltration, tangential flow filtration, density gradient centrifugation, ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, or a combination thereof.

[0036] Preferably, the resulting engineered extracellular vesicles have a particle size mainly distributed between 30 and 300 nm. Preferably, the engineered extracellular vesicles have a particle size of 50–200 nm.

[0037] Preferably, the extracellular vesicles can express or carry extracellular vesicle markers such as CD63, CD81, CD9, and TSG101, and display CD16a nanobodies, membrane-anchored IL-21 and / or IL-15, CD86 and / or CD137L on their membrane surface.

[0038] The present invention also provides a method for expanding NK cells using the above-mentioned engineered extracellular vesicles, comprising the following steps: S1. Provide a starting cell population containing NK cells; S2. The initial cell population is seeded into NK cell expansion medium; S3. Add the above-mentioned engineered extracellular vesicles to the culture system; S4. Cultivate NK cells under suitable conditions to enable them to receive CD16a aggregation activation signals, membrane-anchored cytokine signals, and co-stimulatory signals; S5. Harvest the expanded NK cells.

[0039] Preferably, the starting cell population may be derived from peripheral blood, umbilical cord blood, apheresis blood, bone marrow, or other samples containing NK cells.

[0040] Preferably, the starting cell population can be peripheral blood mononuclear cells or NK cell populations obtained by immunomagnetic bead sorting, flow cytometry sorting or other enrichment methods.

[0041] Preferably, the initial NK cell seeding density is 0.1 × 10⁻⁶. 6 –5×10 6 cells / mL.

[0042] Preferably, the initial NK cell seeding density is 0.2 × 10⁻⁶. 6 –2×10 6 cells / mL.

[0043] Preferably, the amount of engineered extracellular vesicles added is 0.1–50 μg / 10g based on total protein content. 6 NK cells, Preferably, the amount of engineered extracellular vesicles added is 1–20 μg / 10g based on total protein content. 6 NK cells; Preferably, the amount of engineered extracellular vesicles added is 1 × 10⁻⁶ particles. 8 –1×10 11 particles / 10 6 NK cells.

[0044] As a preferred option, low doses of soluble cytokines can also be added to the culture system to maintain NK cell survival and proliferation.

[0045] Preferably, the soluble cytokine is selected from IL-2, IL-7, IL-12, IL-18, or a combination thereof.

[0046] Preferably, the final concentration of IL-2 is 10–300 IU / mL.

[0047] Preferably, the NK cell culture period is 7–21 days. Preferably, the NK cell culture period is 10–14 days.

[0048] Preferably, fluid is replenished every 2–3 days during culture, and engineered extracellular vesicles are added according to cell density and stimulation intensity.

[0049] The beneficial effects of this invention are: (i) This invention uses extracellular vesicles derived from engineered eukaryotic cells as NK cell stimulation carriers, eliminating the need for live feeder cells from hematologic malignancies such as K562, and also avoiding reliance on membrane particles formed by K562 cell membrane disruption. This avoids problems such as residual live feeder cells, inadequate inactivation, and residual tumor-derived DNA or membrane components, reducing safety risks and quality control difficulties in NK cell preparation, and avoiding the use of tumor-derived live feeder cells, thus reducing safety and regulatory risks. (ii) The present invention displays CD16 nanobodies on the surface of engineered extracellular vesicles. The CD16a nanobodies can specifically bind to CD16a receptors on the surface of NK cells and are presented on the vesicle surface in a multivalent form, thereby inducing local aggregation of CD16a receptors and providing stable and targeted activation signals to NK cells. Compared with soluble anti-CD16 antibodies or simple cytokine stimulation, this method is closer to membrane contact activation, which is beneficial to improving the activation and expansion efficiency of NK cells. (III) The engineered extracellular vesicles described in this invention not only display CD16a nanobodies, but can also simultaneously carry membrane-anchored IL-21 and / or IL-15, CD86 and / or CD137L and other stimulating molecules; the above molecules are co-presented on the same vesicle surface, which can simultaneously provide CD16a aggregation activation signals, cytokine support signals and co-stimulatory signals, forming a multi-signal synergistic stimulation interface, thereby overcoming the defects of insufficient amplification efficiency, short signal duration and large differences between donors caused by simple soluble cytokine stimulation, and improving the NK cell amplification effect; (iv) The engineered extracellular vesicles of this invention promote NK cell proliferation while helping to maintain CD3 levels in expanded cells. - CD56 + NK cell phenotype, cell viability, CD16 expression, and expression of natural cytotoxic receptors; the expanded NK cells can maintain good tumor cell killing ability, degranulation ability, cytokine secretion ability, and CD16-mediated ADCC function, which is suitable for subsequent NK cell or CAR-NK cell preparation and helps maintain NK cell purity, viability, and effector function. (v) The engineered extracellular vesicles of the present invention are derived from genetically engineered eukaryotic cells, and their surface functional molecules can be regulated through expression vector design, engineered cell screening and production process control; the obtained vesicles can be quality controlled through detection of particle size, particle concentration, total protein content, EV markers, CD16a nanobodies, membrane-anchored cytokines, co-stimulatory molecules, host cell DNA, live cell residue, sterility, mycoplasma, endotoxins and virus safety-related tests, which facilitates the establishment of standardized and scalable preparation processes.

[0050] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0051] Figure 1 This is a graph showing the experimental results of Experiment 1; Figure 1 A is the initial screening result of phage ELISA for CD16a binding to VHH clone; Figure 1 B is the HPLC purity identification result of purified anti-CD16a-VHH-1; Figure 1 C is a diagram showing the dose-dependent binding results of anti-CD16a-VHH-1 to the extracellular region of recombinant human CD16a; Figure 1 D is a graph showing the purity and cell viability results of primary NK cells after enrichment. Figure 1 E is a graph showing the positive rate of binding of anti-CD16a-VHH-1 to CD16a on the surface of primary NK cells; Figure 1 F is a graph showing the expression results of primary NK cell activation markers induced by immobilized anti-CD16a-VHH-1; Figure 1 G is a graph showing the results of immobilized anti-CD16a-VHH-1 promoting the secretion of IFN-γ and TNF-α by primary NK cells; Figure 2 This is a graph showing the experimental results of Experiment 2; Figure 2 Figure A shows the positive expression rate of different membrane-anchored anti-CD16a VHH fusion proteins on the surface of HEK293T cells; Figure 2 B is a graph showing the NTA particle size range of different engineered extracellular vesicles; Figure 2 C is a graph showing the effect of different membrane anchoring structures on the loading of CD16a VHH onto the EV surface; Figure 3 This is a graph showing the experimental results of Experiment 3; Figure 3Figure A shows the positive expression rates of anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L on the surface of multi-signal engineered CHO-K1 cells. Figure 3 B is a graph showing the cell viability of multi-signal engineered CHO-K1 cells after 72 h of serum-free culture. Figure 3 C is the NTA particle size distribution of multi-signal engineered CHO-K1 cell-derived EVs; Figure 3 D is a graph showing the particle concentration results of multi-signal engineered CHO-K1 cell-derived EVs; Figure 3 E is a graph showing the surface detection results of EV markers and functional molecules in engineered EVs; Figure 4 This is a graph showing the results of Experiment 4; Figure 4 A is a graph showing the cell density of engineered CHO-K1 cells after 72 h of culture. Figure 4 B is a graph showing the cell viability of engineered CHO-K1 cells after 72 h of culture. Figure 4 C is a graph showing the NTA particle size parameters of CD16VHH / IL21 / CD86 / CD137L-EV; Figure 4 D is a graph showing the particle concentration results for Ctrl-EV and CD16VHH / IL21 / CD86 / CD137L-EV; Figure 4 E is the result of Western blot detection of EV markers; Figure 4 F is the image showing the results of transmission electron microscopy; Figure 4 G is a graph showing the positive rate of EV surface functional molecule loading; Figure 4 H is the result of MFI fold loading of functional molecules on EV surface; Figure 4 Figure I shows the dose-dependent binding results of EV surface anti-CD16a VHH and CD16a-Fc; Figure 4 J is the graph showing the total protein concentration of purified EV; Figure 4 K is the result of the EV particle / protein ratio; Figure 4 L is a graph showing the EV particle recovery rate; Figure 4 M is a graph showing the D50 particle size results for three batches of engineered EVs; Figure 4 N is a graph showing the particle concentration results for three batches of engineered EVs; Figure 4 O is a graph showing the total protein concentration results for three batches of engineered EVs; Figure 5 This is a graph showing the experimental results of Experiment 5; Figure 5 A represents the flow cytometry result of the purity of primary NK cells after sorting; Figure 5 B represents the expansion kinetics curves of primary NK cells under different treatment conditions; Figure 5 C represents the change in cell viability during primary NK cell culture under different treatment conditions; Figure 5 D represents the CD3 count of different treatment groups on day 14 of culture. - CD56 + Representative flow cytometry plot of NK cell proportion; Figure 5 E is a representative flow cytometry plot of NK cell functional phenotypes in different treatment groups on day 14 of culture; Figure 5 F represents the positive rates of NK cell activation and functional phenotypes in different treatment groups on day 14 of culture, including NKG2D, NKp30, NKp44, NKp46, CD69, CD25, NKG2A, PD-1, and TIM-3. Figure 5 G represents the fold increase in NK cells on day 14 in the CD16VHH / IL21 / CD86 / CD137L-EV group during the multi-donor replicate experiment. Figure 5 H represents the purity of CD3⁻CD56⁺ NK cells on day 14 in the CD16VHH / IL21 / CD86 / CD137L-EV group during multi-donor replicate experiments. Figure 6 This is a graph showing the experimental results of Experiment 6; Figure 6 A is a graph showing the NTA particle size parameters of engineered EVs with different functional molecule combinations; Figure 6 B is a graph showing the surface functional molecules of engineered EVs with different combinations of functional molecules; Figure 6 C is the result of the effect of engineered EVs with different functional molecular combinations on the expansion fold of primary NK cells on day 14; Figure 6 Figure D shows the effects of engineered EVs with different functional molecular combinations on the purity, viability, and CD16 retention rate of expanded NK cells. Figure 6E is a graph showing the K562 killing activity results after EVs were expanded into NK cells with different combinations of functional molecules. Figure 6 F is a graph showing the effect of engineered EVs with different functional molecular combinations on IFN-γ secretion in expanded NK cells; Figure 6 G is a graph showing the effect of engineered EVs with different functional molecular combinations on TNF-α secretion in expanded NK cells; Figure 7 This is a graph showing the results of Experiment 7; Figure 7 A is a graph showing the effects of different stimulation systems on the expansion kinetics of primary NK cells; Figure 7 B is a graph showing the cytotoxic activity of NK cells against K562 target cells after expansion under different stimulation systems; Figure 7 C is the graph showing the effect of different stimulation systems on the purity of expanded NK cells; Figure 7 D is a graph showing the effect of different stimulation systems on the CD16 positivity rate of expanded NK cells; Figure 7 E is a graph showing the IFN-γ secretion level of NK cells after expansion under different stimulation systems; Figure 7 F is a graph showing the TNF-α secretion level of NK cells after expansion under different stimulation systems; Figure 8 This is a graph showing the experimental results of Experiment 8; Figure 8 A is the graph showing the effect of EV protein addition dosage on the expansion curve of primary NK cells; Figure 8 Figure B shows the effect of EV protein addition dosage on the viability, purity, and CD16 positivity rate of expanded NK cells. Figure 8 C is the graph showing the effect of EV particle number and dosage on the expansion curve of primary NK cells; Figure 8 Figure D shows the effect of EV particle number and dosage on the viability, purity, and CD16 positivity rate of expanded NK cells. Figure 8 E is the graph showing the effect of EV supplementation method on the expansion curve of primary NK cells; Figure 8 F is the graph showing the effect of EV supplementation method on the viability, purity, and CD16 positivity rate of expanded NK cells; Figure 8 G is a graph showing the expansion curves of primary NK cells under different culture cycles; Figure 8H is a graph showing the results of NK cell viability, purity, and CD16 positivity rate at different culture cycles. Detailed Implementation

[0052] An engineered extracellular vesicle, wherein the surface of the extracellular vesicle membrane displays the following functional molecules: Anti-CD16 nanobody; Membrane-anchored IL-21 and / or membrane-anchored IL-15; CD86 and / or CD137L.

[0053] The anti-CD16a nanobody is a VHH nanobody that can specifically bind to human CD16a; the CD16a nanobody contains the amino acid sequence shown in SEQ ID NO:1.

[0054] The CD16 nanobody is expressed on the surface of engineered eukaryotic cell membrane in a membrane-anchored form and is loaded onto the surface of extracellular vesicle membrane during the formation of extracellular vesicles, forming a membrane-anchored CD16 nanobody. The membrane-anchored CD16a nanobody includes, from the N-terminus to the C-terminus, a signal peptide, an anti-human CD16a VHH nanobody domain, a linker peptide, a transmembrane anchoring region, and an intracellular stabilizing region.

[0055] The signal peptide is selected from one or more of Igκ signal peptide, CD8α signal peptide, and IL-2 signal peptide; the linker peptide is selected from one or more of GGGGS, (GGGGS)2, and (GGGGS)3; and the transmembrane anchoring region is selected from one or more of CD8α, CD28, PDGFR, LAMP2B, CD63, CD81, and GPI anchoring signals.

[0056] The membrane-anchored CD16a nanobody structure contains the amino acid sequence shown in SEQ ID NO:3.

[0057] Both membrane-anchored IL-21 and membrane-anchored IL-15 include: cytokine functional domains, linker peptides and / or stabilizing linker modules, and transmembrane anchoring regions; the stabilizing linker module is the CH2 / CH3 segment in the IgG Fc domain.

[0058] The membrane-anchored IL-21 includes an IL-21 functional domain, a linker region, and a transmembrane domain, and the membrane anchoring type contains the amino acid sequence shown in SEQ ID NO:4; the membrane-anchored IL-15 includes an IL-15 functional domain, an IL-15Rαsushi domain, a linker region, and a transmembrane domain, and the membrane anchoring type IL-15 contains the amino acid sequence shown in SEQ ID NO:5.

[0059] CD86 contains the amino acid sequence shown in SEQ ID NO:6; CD137L contains the amino acid sequence shown in SEQ ID NO:7.

[0060] SEQ ID NO:6: MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYP EPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF SEQ ID NO:7: MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGSG A method for expanding NK cells using the above-mentioned engineered extracellular vesicles includes the following steps: S1. Provide a starting cell population containing NK cells; S2. The initial cell population is seeded into NK cell expansion medium; S3. Add the above-mentioned engineered extracellular vesicles to the culture system; S4. Cultivate NK cells under suitable conditions to enable them to receive CD16a aggregation activation signals, membrane-anchored cytokine signals, and co-stimulatory signals; S5. Harvest the expanded NK cells.

[0061] The initial cell population can be peripheral blood mononuclear cells, and the initial NK cell seeding density is 0.2 × 10⁻⁶. 6 –2×10 6 cells / mL.

[0062] The amount of engineered extracellular vesicles added is 0.1–50 μg / 10g based on total protein content. 6 NK cells, The amount of engineered extracellular vesicles added is 1 × 10⁻⁶ particles. 8 –1×10 11 particles / 10 6 NK cells.

[0063] Low doses of soluble cytokines were added to the culture system to maintain NK cell survival and proliferation.

[0064] The soluble cytokines are a combination of IL-2, IL-7, IL-12, and IL-18.

[0065] The final concentration of IL-2 is 10–300 IU / mL.

[0066] The NK cell culture period is 10–14 days.

[0067] During culture, fluid was replenished every 2–3 days, and engineered extracellular vesicles were added according to cell density and stimulation intensity.

[0068] Using the above method, NK cells can be expanded in vitro without the participation of feeder cells derived from live tumors, and the resulting NK cells have high CD3 content. - CD56 + It has a good ratio, good cell viability, and cytotoxic effects.

[0069] Experiment 1: Obtaining CD16a VHH nanobody sequence and identifying binding activity See Figure 1 This experiment screened, sequenced, and detected the binding activity of anti-human CD16a VHH nanobodies to obtain nanobodies sequences that can be used to construct membrane-anchored CD16 activation modules.

[0070] 1. Screening for CD16a-binding VHH clones Recombinant human CD16a extracellular region protein was used as the positive screening antigen, and recombinant human CD16b extracellular region protein, His tag protein and BSA were used as the negative screening or negative control antigens. The VHH library was displayed by phage display for screening.

[0071] Human CD16a extracellular protein was coated onto immunotubes or ELISA plates at a concentration of 5 μg / mL and incubated overnight at 4°C. The next day, the plates were blocked with 3% BSA for 1 h, and then VHH phage library was added and incubated at room temperature for 1 h. After washing with PBST, the bound phages were eluted and used to infect E. coli for amplification. Screening was performed in three rounds, with the antigen coating concentration decreasing to 5 μg / mL, 2 μg / mL and 0.5 μg / mL respectively, and the number of washes increasing to 10, 15 and 20 respectively.

[0072] After the third round of screening, 96 single clones were randomly selected for phage ELISA initial screening. The positive threshold for initial screening was 0.244, which was 3 times the average OD450 value of negative VHH to CD16a. The results showed that 7 clones had obvious binding signals to the extracellular region of human CD16a, and their OD450 values ​​were more than 3 times higher than those of the negative control.

[0073] After further excluding clones that clearly bind to CD16b, His tag protein, or BSA, a CD16a-VHH clone with good specificity was obtained and named anti-CD16a-VHH-1. The average OD450 value of this clone binding to CD16a was 1.422, while its binding signal to CD16b was about 6.2% of the binding signal to CD16a. The binding signals to His tag protein and BSA were both at low background levels, suggesting that anti-CD16a-VHH-1 has good initial screening binding selectivity for CD16a.

[0074] 2. Sequence identification of anti-CD16a-VHH-1 The anti-CD16a-VHH-1 phage clone plasmid was extracted and Sanger sequencing was performed. The sequencing results showed that the anti-CD16a-VHH-1 contained a complete VHH open reading frame and had a typical VHH frame region and complementarity-determining region structure.

[0075] The amino acid sequence of anti-CD16a-VHH-1 is shown in SEQ ID NO:1, and the nucleic acid sequence encoding it is shown in SEQ ID NO:2.

[0076] in: SEQ ID NO:1: Anti-CD16a-VHH-1 amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGSIFSFAMSWVRQAPGKGLEWVSRIGSDDRVTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQTDLRDWTVREYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWVRQAPGKGLEWVSSITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNPWPVAAPRSGTYWGQGTLVTVSS SEQ ID NO:2: Nucleic acid sequence encoding anti-CD16a-VHH-1 GAGGTCCAACTGGTGGAGAGCGGTGGGGGATTGGTGCAGCCTGGGGGGTCCCTGCGCCTGAGCTGCGCAGCTTCAGGAAGCATCTTTAGTTTTGCCATGAGTTGGGTGCGGCAAGCCCCCGGGAAAGGGTTGGAATGGGTGTCAAGAATTGGTTCAGACGACAGGGTGACCTACGCCGATAGTGTGAAGGGC CGGTTCACAATCTCCAGAGACAATTCCAAGAACACCTTGTATCTGCAGATGAACTCCCTGCGAGCTGAGGACACAGCAGTGTACTATTGCGCAAAACAGACCGACCTGCGCGACTGGACAGTCAGAGAATATTGGGGCCAAGGCACCCTTGTGACGGTGTCCAGCGGCGGAGGCGGGAGCGGCGGGGGGGGA AGCGGAGGCGGCGGCTCTGAGGTACAGCTCGTCGAAAGCGGAGGGGGCTTGGTCCAGCCAGGGGGAAGCCTCCGGCTGTCATGCGCGGCAAGTGGCCTGACCTTCAGTTCCTACAATATGGGCTGGGTGAGGCAGGCTCCAGGCAAGGGACTGGAGTGGGTCTCATCTATTACTTGGAGTGGCAGGGATACA TTTTACGCAGACTCTGTTAAAGGTCGGTTCACTATCTCCCGAGACACAGCAAGAACACATTGTACCTGCAGATGAACTCTCTGAGAGCCGAGGATACCGCGGTCTACTATTGTGCGAAAAATCCCTGGCCGGTCGCCGCACCACGCTCTGGGACATATTGGGGTCAAGGGACCCTCGTGACTGTGTCCAGC 3. Recombinant Expression and Purification of Anti-CD16a-VHH-1 The anti-CD16a-VHH-1 coding sequence was cloned into the pET series expression vector, which had a 6×His tag at its C-terminus; the recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and positive clones were selected for expansion culture; when the OD600 of the bacterial culture reached 0.6–0.8, IPTG was added to a final concentration of 0.5 mmol / L, and expression was induced at 20℃ for 16 h.

[0077] After induction, bacterial cells were collected, sonicated, and the supernatant was collected by centrifugation. The anti-CD16a-VHH-1 protein was obtained by Ni-NTA affinity chromatography. HPLC analysis showed that the purified protein had a single main peak at about 28 kDa, which was consistent with the theoretical molecular weight. The analysis showed that the protein purity was >95% and the protein concentration was 1.43 mg / mL.

[0078] 4. ELISA detection of the binding between anti-CD16a-VHH-1 and CD16a. Recombinant human CD16a extracellular protein was coated onto an ELISA plate at a concentration of 2 μg / mL and incubated overnight at 4°C. After blocking with 3% BSA, serially diluted anti-CD16a-VHH-1 protein was added at concentrations ranging from 0.01 to 1000 nmol / L and incubated at room temperature for 1 h. After washing, HRP-labeled anti-His antibody was added, and TMB was used for color development. The absorbance was read at 450 nm.

[0079] The results showed that anti-CD16aVHH-1 could bind to the extracellular region of human CD16a in a dose-dependent manner; after fitting the CD16a binding curve with four parameters, its EC50 for binding to CD16a was 3.17 nmol / L; no obvious binding signal was observed in the unrelated VHH control within the same concentration range, and no obvious non-specific binding was also observed in the BSA-coated group.

[0080] At a concentration of 1000 nmol / L, the binding signal of anti-CD16a-VHH-1 to the extracellular region of CD16b was 7.3% of the binding signal of CD16a, suggesting that it has good binding selectivity for CD16a.

[0081] 5. Binding of anti-CD16a-VHH-1 to CD16a on the surface of primary NK cells. Peripheral blood was collected from healthy donors, and PBMCs were separated using Ficoll density gradient centrifugation. Primary NK cells were then enriched using an NK cell negative sorting kit. Flow cytometry analysis showed that after enrichment, CD3+ was significantly increased. - CD56 + The proportion of NK cells was 95.4%, and the cell viability was 96.9%; CD3 + The proportion of T cells was at a low level, and no obvious T cell enrichment was observed.

[0082] Anti-CD16a-VHH-1 was fluorescently labeled, or a tagged anti-CD16a-VHH-1 was combined with a fluorescent secondary antibody for detection; 1×10 6 One NK cell was incubated with anti-CD16a-VHH-1 to a final concentration of 1 μg / mL at 4°C for 30 min; after washing, the binding of anti-CD16a-VHH-1 to NK cells was detected by flow cytometry.

[0083] The results showed that anti-CD16a-VHH-1 could significantly bind to CD3. - CD56 + NK cell population; the positive rate of anti-CD16a-VHH-1 staining was 75.0%, while the positive rate of staining in the negative VHH control group was 2.87%; the average fluorescence intensity of the anti-CD16a-VHH-1 treatment group was 26.1 times that of the negative VHH control; after competitive blocking with commercial anti-CD16 antibody, the positive rate of anti-CD16a-VHH-1 staining decreased to 14.4%, and the binding signal after background correction decreased by 80.8%, indicating that the binding signal of anti-CD16a-VHH-1 is related to the CD16a receptor on the surface of NK cells.

[0084] 6. Detection of NK cell activation by immobilized anti-CD16a-VHH-1 Anti-CD16a-VHH-1 was coated onto a 96-well plate at 5 μg / mL and incubated overnight at 4°C. The plate was washed with PBS the next day and blocked with 1% BSA for 1 h. 1×10⁻⁶ μg / mL of anti-CD16a-VHH-1 was added to each well. 5 Primary NK cells were cultured and IL-2 was added to a final concentration of 100 IU / mL. An uncoated VHH-coated group and an uncoated group were set up as controls. After 24 h of culture, the expression of CD69, CD25 and CD107a in NK cells was detected.

[0085] The results showed that, compared with the unrelated VHH control group, the anti-CD16a-VHH-1 coated group could significantly induce NK cell activation; the positive rates of CD69, CD25, and CD107a in NK cells of the unrelated VHH control group were 22.3%, 13.1%, and 10.4%, respectively; while the positive rates of CD69, CD25, and CD107a in NK cells of the anti-CD16a-VHH-1 coated group increased to 66.0%, 44.1%, and 32.6%, respectively.

[0086] Further analysis of cytokine levels in the culture supernatant revealed that the IFN-γ level in the unrelated VHH control group was 238.0 pg / mL, and the TNF-α level was 71.3 pg / mL. In the anti-CD16a-VHH-1 coated group, the IFN-γ level increased to 1035.2 pg / mL, and the TNF-α level increased to 329.0 pg / mL. In the uncoated group, the IFN-γ and TNF-α levels were 160.5 pg / mL and 49.0 pg / mL, respectively.

[0087] The results showed that anti-CD16a-VHH-1 can not only specifically bind to human CD16a, but also induce NK cell activation under immobilized or multivalent presentation conditions. Therefore, anti-CD16a-VHH-1 is suitable as a CD16a-binding activation module on the surface of engineered extracellular vesicles for subsequent construction of engineered extracellular vesicle stimulation systems based on CD16 nanobodies.

[0088] Experiment 2: Design and expression of membrane-anchored anti-CD16a VHH fusion protein See Figure 2 In this experiment, the anti-CD16a-VHH-1 obtained in Experiment 1 was designed to be membrane anchored, and the effects of different membrane anchoring structures on anti-CD16a-VHH cell membrane expression and extracellular vesicle loading were compared, so as to determine the anti-CD16a-VHH membrane display structure suitable for constructing engineered extracellular vesicles.

[0089] 1. Structural design of membrane-anchored anti-CD16a VHH fusion protein Using the anti-CD16a-VHH-1 obtained in Experiment 1 as the antigen-binding domain, a membrane-anchored anti-CD16a VHH fusion protein was designed. The fusion protein includes, from the N-terminus to the C-terminus, an Igκ signal peptide, an anti-CD16a-VHH-1 nanobody domain, a linker peptide, and a transmembrane anchoring region.

[0090] Among them, the Igκ signal peptide is used to mediate the entry of the fusion protein into the secretion pathway; the anti-CD16a-VHH-1 amino acid sequence is shown in SEQ ID NO:1; the linker peptide is (GGGGS)3; the transmembrane anchoring regions are selected from the PDGFR transmembrane region, the LAMP2B membrane localization structure and the CD63 membrane localization structure, respectively.

[0091] Three fusion proteins were constructed and named as follows: CD16VHH-PDGFR-TM; CD16VHH-LAMP2B-TM; CD16VHH-CD63-TM.

[0092] Each fusion protein was synthesized after mammalian cell codon optimization, and a Kozak sequence was added to the 5' end; after optimization, they were cloned into eukaryotic expression vectors driven by the CMV promoter.

[0093] 2. Expression vector construction and sequencing validation The coding sequences of the three fusion proteins were inserted into the pcDNA3.1 expression vector by restriction endonuclease ligation or homologous recombination. The recombinant plasmids were transformed into E. coli DH5α competent cells, and single clones were selected for amplification and plasmid extraction.

[0094] Enzyme digestion analysis revealed that each recombinant plasmid contained the target fragment of the theoretical size.

[0095] Further Sanger sequencing revealed that the inserted fragments in all three expression vectors were complete, with correct reading frames, and no base mutations or frameshift mutations were found.

[0096] The obtained expression vectors were named as follows: pcDNA-CD16VHH-PDGFR-TM; pcDNA-CD16VHH-LAMP2B-TM; pcDNA-CD16VHH-CD63-TM.

[0097] 3. Transient transfection of engineered cells HEK293T cells were selected for preliminary expression validation; 24 h before transfection, HEK293T cells were seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 5 This method involves using individual cells to achieve a cell confluency of 70%–80% during transfection.

[0098] Add 2 μg of recombinant plasmid to each well and transfect using liposome transfection reagent; after adding cells to the transfection system, incubate in a 37℃, 5% CO2 incubator; replace with fresh complete culture medium 6 h after transfection; collect cells 48 h after transfection for membrane surface expression detection.

[0099] An empty vector transfection group was set up as a negative control.

[0100] 4. Flow cytometry detection of anti-CD16a VHH membrane surface expression 48 h after transfection, HEK293T cells from each group were collected and washed twice with PBS; 1×10⁶ cells were collected from each group. 6 Cells were incubated with recombinant human CD16a-Fc protein to a final concentration of 2 μg / mL at 4°C for 30 min. After washing, fluorescently labeled anti-human Fc secondary antibody was added and incubated at 4°C in the dark for 30 min. After washing, the binding signal between CD16a-Fc and anti-CD16a VHH on the cell surface was detected by flow cytometry.

[0101] The results showed that all three membrane-anchored anti-CD16a VHH fusion proteins were expressed on the surface of HEK293T cells. Among them, the CD16VHH-CD63TM group and the CD16VHH-LAMP2B group had higher expression levels, with CD16a-Fc binding positivity rates of 80.6% and 68.8%, respectively, and average fluorescence intensities of 38.9 and 33.2 times that of the negative control group, respectively. No obvious CD16a-Fc binding signal was detected in the empty vector control group.

[0102] 5. Preliminary detection of anti-CD16a VHH loading in extracellular vesicles To preliminarily compare the effects of different membrane anchoring structures on the loading of CD16a VHH into extracellular vesicles, culture supernatants of HEK293T cells from each transfection group were collected. The culture supernatants were centrifuged at 300×g for 10 min to remove cells, centrifuged at 2000×g for 20 min to remove cell debris, and centrifuged at 10000×g for 30 min to remove large particulate impurities. The supernatants were then concentrated using a 100 kDa ultrafiltration tube. The concentrated samples were then subjected to size exclusion chromatography to collect the extracellular vesicle enriched components.

[0103] The size of vesicles in each group was detected by NTA, and the results showed that the size of extracellular vesicles in each group was mainly concentrated in the range of 113–176 nm. Further, the functional display of anti-CD16a VHH on the surface of extracellular vesicles was detected by flow cytometry using CD16a-Fc. The results showed that CD16VHH-LAMP2B-EV and CD16VHH-CD63-EV could significantly bind CD16a-Fc, and their fluorescence intensities were 48.4 times and 49.2 times that of the negative EV control, respectively, suggesting that anti-CD16a VHH can be loaded on the surface of extracellular vesicles in a binding active form.

[0104] Experiment 3: Construction and Identification of Multi-Signal Engineered Eukaryotic Cells See Figure 3 Based on the membrane-anchored anti-CD16a VHH structure determined in Experiment 2, this experiment further constructed engineered eukaryotic cells that simultaneously express anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L, for subsequent preparation of engineered extracellular vesicles with NK cell expansion activity.

[0105] 1. Design of multi-signal expression molecules CHO-K1 cells were selected as donor cells for engineered extracellular vesicles. The following four membrane-associated stimulating molecules were constructed: (1) Membrane-anchored anti-CD16a VHH fusion protein: The CD16VHH-LAMP2B structure obtained in Experiment 2 was used. The fusion protein consists of an Igκ signal peptide, anti-CD16a-VHH-1, (GGGGS)3 linker peptide and LAMP2B membrane localization structure from the N-terminus to the C-terminus. (2) Membrane-anchored IL-21 fusion protein: from N-terminus to C-terminus are Igκ signal peptide, human IL-21 functional domain, (GGGGS)2 linker peptide and CD8α transmembrane region; (3) CD86: The full-length coding sequence of human CD86 is adopted, and its natural signal peptide, extracellular region, transmembrane region and intracellular short tail region are retained; (4) CD137L: The full-length coding sequence of human CD137L is used, preserving its natural membrane localization structure and receptor binding domain.

[0106] 2. Expression vector construction and plasmid validation The above four gene sequences were all optimized for mammalian cell codons and cloned into the pcDNA3.1 expression vector respectively; the recombinant plasmids were transformed into Escherichia coli DH5α competent cells, and single clones were selected for expansion culture and plasmids were extracted.

[0107] Restriction enzyme digestion confirmed that all four expression vectors contained the target fragments of the theoretical size. Further Sanger sequencing confirmed that the insertion sequences of CD16VHH-LAMP2B, mbIL21, CD86, and CD137L were complete, the reading frames were correct, and no frameshift mutations or premature stop codons were found.

[0108] 3. CHO-K1 cell co-transfection 24 hours before transfection, CHO-K1 cells were seeded into 6-well plates at a density of 4 × 10⁶ cells per well. 5 This method involves using individual cells to achieve a cell confluency of 70%–80% during transfection.

[0109] pcDNA-CD16VHH-LAMP2B, pcDNA-mbIL21, pcDNA-CD86, and pcDNA-CD137L were mixed at a mass ratio of 2:1:1:1. 2.5 μg of the mixed plasmid was added to each well, and transfection was performed using liposome transfection reagent. After 6 h of transfection, the medium was replaced with fresh complete medium, and the culture was continued at 37 °C and 5% CO2.

[0110] An empty vector transfection group was set up as a negative control, and groups transfected with CD16VHH-LAMP2B, mbIL21, CD86, or CD137L individually were set up as single-molecule expression controls.

[0111] 4. Detection of cell surface expression of multiple signaling molecules 48 h after transfection, CHO-K1 cells from each group were collected and washed twice with PBS; 1×10⁶ cells were taken from each group. 6 Each cell was stained for detection.

[0112] Surface expression of anti-CD16a VHH was detected by adding fluorescently labeled anti-human Fc secondary antibody after binding to human CD16a-Fc protein; membrane-anchored IL-21 was detected by anti-IL-21 fluorescent antibody; CD86 and CD137L were detected by anti-CD86 fluorescent antibody and anti-CD137L fluorescent antibody, respectively.

[0113] Flow cytometry results showed that after co-transfection with multiple plasmids, anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L could be detected simultaneously on the surface of CHO-K1 cells. The positive rates of each molecule were as follows: Anti-CD16a VHH: 74.6%; Membrane-anchored IL-21: 62.9%; CD86: 68.6%; CD137L: 60.2%.

[0114] Among them, anti-CD16a VHH + IL-21 + CD86 + CD137L + The proportion of four positive cells was 43.8%.

[0115] No significant positive signals of the above molecules were detected in the empty vector control group; expression of the corresponding molecules was detected in all single-molecule transfection groups, indicating that each expression vector can be effectively expressed in CHO-K1 cells; all four target molecules in the co-transfection group showed significant positive expression; these results indicate that CHO-K1 cells can simultaneously express anti-CD16a VHH, membrane-anchored IL-21, CD86 and CD137L, providing a cellular basis for the preparation of multi-signal engineered extracellular vesicles.

[0116] 5. Preliminary detection of the ability of engineered cells to release extracellular vesicles Stable CHO-CD16VHH / IL21 / CD86 / CD137L cells were seeded in serum-free medium at an initial density of 5 × 10⁶ cells / year. 5 The cell count was 97.3% at 72 h.

[0117] After pretreatment by centrifugation at 300×g for 10 min, 2000×g for 20 min, and 10000×g for 30 min, the culture supernatant was concentrated using a 100 kDa ultrafiltration tube, and the extracellular vesicle enriched components were collected by size exclusion chromatography. NTA detection showed that the obtained particles were mainly distributed in the range of 112–281 nm, with a D50 of 184.3 nm and a particle concentration of 1.22×10¹¹ particles / mL.

[0118] Flow cytometry analysis showed that extracellular vesicle markers such as CD63 and CD81 could be detected in the extracellular vesicle samples, as well as signals of anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L. In extracellular vesicles derived from the empty vector CHO-K1, only extracellular vesicle markers were detected, and the above-mentioned stimulating molecular signals were not detected.

[0119] This experiment successfully constructed multi-signal engineered CHO-K1 cells that simultaneously express anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L. These engineered cells can stably express a variety of NK cell stimulation-related molecules and release extracellular vesicles carrying these molecules on their surface. The resulting engineered cells provide a stable source of donor cells for the subsequent preparation of engineered extracellular vesicles based on CD16 nanobodies and for conducting in vitro NK cell expansion experiments.

[0120] Experiment 4: Preparation, purification and characterization of engineered extracellular vesicles See Figure 4 In this experiment, engineered extracellular vesicles were prepared using CHO-CD16VHH / IL21 / CD86 / CD137L engineered cells constructed in Experiment 3. The particle size distribution, particle concentration, vesicle markers, functional molecule loading, and safety indicators were detected to confirm that the obtained extracellular vesicles can be used as cell-free stimulation carriers for subsequent NK cell expansion.

[0121] 1. Engineered CHO cell culture Engineered CHO-K1 cells expressing CD16VHH / IL21 / CD86 / CD137L, obtained in Experiment 3, were collected during the logarithmic growth phase. Cells were resuspended in serum-free CHO cell culture medium, and the cell density was adjusted to 5 × 10⁶ cells / year. 5 cells / mL, seeded in T175 cell culture flasks or shake flasks.

[0122] Cells were cultured at 37°C and 5% CO2 for 72 h; the culture medium was not changed during the culture period; at the end of the culture, the cell density was ~1.7 × 10⁻⁶. 6 The cell count / mL was >95%; empty vector CHO-K1 cells were cultured under the same conditions as a negative extracellular vesicle control.

[0123] 2. Collection and pretreatment of culture supernatant After 72 h of culture, the culture supernatant of engineered CHO cells was collected. To remove intact cells, cell debris, and large particulate impurities, the culture supernatant was subjected to the following treatments at 4°C: Centrifuge at 300×g for 10 min to remove intact cells; Centrifuge at 2000×g for 20 min to remove cell debris and apoptotic bodies; Centrifuge at 10000×g for 30 min to remove large particles and organelle debris; The solution was filtered through a 0.22μm low-protein adsorption membrane to obtain a clear supernatant.

[0124] After the above pretreatment, no obvious cell residue or visible precipitation was observed; the supernatant after pretreatment was used for subsequent extracellular vesicle concentration and purification.

[0125] 3. Extracellular vesicle concentration and purification The pretreated culture supernatant was added to an ultrafiltration device with a molecular weight cutoff of 100 kDa and concentrated by ultrafiltration at 4°C to reduce the sample volume to 1 / 30–1 / 50 of the original volume.

[0126] The concentrated sample was purified by size exclusion chromatography column equilibrated with PBS; the extracellular vesicle enrichment eluent was collected using sterile PBS as the mobile phase; based on the protein absorption peak and particle detection results, the extracellular vesicle enrichment was combined and concentrated again to the target volume using a 100 kDa ultrafiltration tube.

[0127] The purified engineered extracellular vesicles were named CD16VHH / IL21 / CD86 / CD137L-EV(co-expression); the extracellular vesicles derived from the empty vector CHO-K1 were named MOCK-EV.

[0128] 4. Particle size distribution and particle concentration detection The particle size distribution and particle concentration of CD16VHH / IL21 / CD86 / CD137L-EV were analyzed using a nanoparticle tracking analyzer. The results showed that CD16VHH / IL21 / CD86 / CD137L-EV exhibited a typical nanoscale particle distribution, with particle sizes mainly concentrated between 123.1 and 435.8 nm, and an average particle size of 284.6 nm; the particle concentration was 1.03 × 10⁻⁶. 11 particles / mL; the particle size distribution of MOCK-EV is basically the same as that of engineered EV.

[0129] The above results indicate that the expression of engineered stimulating molecules did not significantly alter the overall particle size characteristics of extracellular vesicles derived from CHO cells.

[0130] 5. Observation using transmission electron microscopy The purified CD16VHH / IL21 / CD86 / CD137L-EV sample was dropped onto a copper grid, allowed to stand for adsorption, and then negatively stained with phosphotungstic acid or uranium acetate. After drying, the sample was observed using a transmission electron microscope.

[0131] The results showed that the obtained extracellular vesicles were round or cup-shaped membranous vesicles with clear particle boundaries and particle size that was basically consistent with the NTA detection results; no large amount of cell debris or irregular aggregates were observed.

[0132] 6. Detection of extracellular vesicle markers Extracellular vesicle markers were detected by Western blot. CD16VHH / IL21 / CD86 / CD137L-EV and MOCK-EV samples were taken, and 10 μg of total protein was added to each well. After SDS-PAGE electrophoresis and membrane transfer, anti-CD63, anti-CD81, anti-CD9 and anti-TSG101 antibodies were used for detection.

[0133] The results showed that positive bands for CD63, CD81, CD9 and TSG101 could be detected in CD16VHH / IL21 / CD86 / CD137L-EV; the above extracellular vesicle markers could also be detected in Ctrl-EV; indicating that the obtained samples had good extracellular vesicle characteristics and low endoplasmic reticulum contamination.

[0134] 7. Detection of functional molecular loading on EV surface Flow cytometry was used to detect surface functional molecules of CD16VHH / IL21 / CD86 / CD137L-EV using microsphere-conjugated microspheres. Human CD16a-Fc protein, anti-IL-21 antibody, anti-CD86 antibody, and anti-CD137L antibody were added to the sample for flow cytometry detection. Results showed that the positive rates for CD16a-Fc binding in CD16VHH / IL21 / CD86 / CD137L-EV conjugated microspheres were 84.9%, IL-21 72.8%, CD86 78.1%, and CD137L 67.1%, respectively; the corresponding MFI folds were 20.6, 13.1, 16.3, and 11.2, respectively. No obvious positive signal was observed in Ctrl-EV conjugated microspheres.

[0135] The results indicate that anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L can all be loaded into engineered extracellular vesicles and displayed on the EV surface in a detectable form.

[0136] 8. CD16a binding activity detection To confirm that the anti-CD16a VHH on the surface of EVs still has CD16a binding ability, CD16VHH / IL21 / CD86 / CD137L-EV was coupled to latex microspheres, and different concentrations of human CD16a-Fc protein were added, at concentrations of 0.1, 0.3, 1, 3 and 10 μg / mL, respectively. After incubation, fluorescently labeled anti-human Fc secondary antibody was added, and the binding signal was detected by flow cytometry.

[0137] The results showed that CD16VHH / IL21 / CD86 / CD137L-EV could bind to human CD16a-Fc in a dose-dependent manner; the fluorescence intensity gradually increased with increasing CD16a-Fc concentration and tended to plateau in the range of 3–10 μg / mL; no obvious binding signal was shown in the Ctrl-EV group and the unrelated Fc protein treatment group.

[0138] This result indicates that anti-CD16a VHH retains CD16a binding activity after being loaded onto the surface of extracellular vesicles.

[0139] 9. Detection of total protein, particulate protein ratio and recovery rate The total protein concentration of the purified EV samples was determined using the BCA method; the protein concentration of CD16VHH / IL21 / CD86 / CD137L-EV was 0.065 mg / mL, and the protein concentration of Ctrl-EV was 0.049 mg / mL.

[0140] The particle-to-protein ratio was calculated based on NTA particle concentration; the particle-to-protein ratio of CD16VHH / IL21 / CD86 / CD137L-EV was 2.07 × 10⁻⁶. 10 The particle-to-protein ratio for Ctrl-EV is 1.70 × 10⁻⁶. 10 The particle / μg protein recovery rate of engineered EVs after ultrafiltration and size exclusion chromatography was 62.7%.

[0141] The results indicate that the process described in this experiment can obtain engineered extracellular vesicle samples with high concentrations and relatively stable particulate proteins.

[0142] 11. Batch consistency testing Three batches of CD16VHH / IL21 / CD86 / CD137L-EV were prepared consecutively using the same process and named EV-B1, EV-B2 and EV-B3, respectively. The particle size, particle concentration, total protein concentration and surface functional molecule expression of each batch were detected.

[0143] The results showed that the D50 values ​​of the three batches of EVs were 112.2 nm, 115.3 nm, and 113.1 nm, respectively; the particle concentrations were 13.1 × 10⁻⁶. 11 13.8×10 11 and 13.3×10 11 The coefficient of variation for the CD16a-Fc binding signal was 2.0%; the coefficients of variation for the surface detection signals of IL-21, CD86, and CD137L were 2.3%, 1.9%, and 2.7%, respectively.

[0144] The results show that the preparation process has good reproducibility and can stably obtain engineered extracellular vesicles with surface-carrying anti-CD16a VHH, membrane-anchored IL-21, CD86 and CD137L.

[0145] 12. Conclusion This experiment successfully established a method for the preparation and purification of engineered extracellular vesicles (EVs) with CD16VHH / IL21 / CD86 / CD137L. The obtained EVs exhibited typical extracellular vesicle particle size, morphology, and marker expression, and stably displayed anti-CD16aVHH, membrane-anchored IL-21, CD86, and CD137L on their surface. The anti-CD16aVHH on the EV surface retained CD16a binding activity, and no live cell residues were detected in the samples. The results of host cell DNA, endotoxin, and microbial contamination detection met the requirements for subsequent NK cell amplification experiments. This engineered EV can serve as a cell-free, multi-signal synergistic NK cell amplification stimulation vector.

[0146] Experiment 5: Engineered extracellular vesicles promote the in vitro expansion of primary NK cells. See Figure 5 In this experiment, CD16VHH / IL21 / CD86 / CD137L-EV prepared in Experiment 4 was used to stimulate primary NK cells, and its effects on in vitro expansion, cell viability and NK cell purity were detected to verify the role of the engineered extracellular vesicles as a cell-free stimulation carrier in promoting NK cell expansion.

[0147] 1. Isolation of primary NK cells Peripheral blood (50 mL) was collected from healthy donors, diluted with an equal volume of PBS, and peripheral blood mononuclear cells were separated using Ficoll density gradient centrifugation. Centrifugation conditions were 400 × g for 30 min at room temperature without brakes. The mononuclear cell layer was collected and washed twice with PBS. Primary NK cells were enriched using an NK cell negative sorting kit. Samples were then analyzed by flow cytometry. Results showed that the obtained cells contained CD3+. - CD56 + The proportion of NK cells was 96.9%; the sorted NK cells were used for subsequent expansion experiments.

[0148] 2. Engineered EV processing grouping The sorted primary NK cells were resuspended in serum-free NK cell expansion medium and the cell density was adjusted to 2 × 10⁶ cells / year. 5 cells / mL; add 1 mL of cell suspension to each well and seed in a 24-well plate.

[0149] The experiment was set up with the following 4 groups: Control group 1: Only IL-2 was added, with a final concentration of 300 IU / mL; Control group 2: Ctrl-EV and IL-2 were added, with Ctrl-EV added at a concentration of 10 μg EV protein / 10 6 NK cells, with a final IL-2 concentration of 300 IU / mL; Experimental group 1: CD16VHH-EV and IL-2 were added, with CD16VHH-EV added at a concentration of 10 μg EV protein / 10 6 NK cells, with a final IL-2 concentration of 300 IU / mL; Experimental group 2: CD16VHH / IL21 / CD86 / CD137L-EV and IL-2 were added, with EV added at a rate of 30 μg EV protein / 10 6 NK cells, with a final IL-2 concentration of 100 IU / mL.

[0150] Among them, Ctrl-EV is the extracellular vesicle derived from the empty vector CHO-K1 cells described in Experiment 4; CD16VHH-EV is an engineered extracellular vesicle that only exhibits anti-CD16a VHH; and CD16VHH / IL21 / CD86 / CD137L-EV is a multi-signal engineered extracellular vesicle prepared in Experiment 4.

[0151] Each group has 3 duplicate holes.

[0152] 3. NK cell culture and EV supplementation Cells from each group were cultured in a 37°C, 5% CO2 incubator. Cell status was observed every 2–3 days during culture, and cell density and viability were measured.

[0153] When cell density exceeds 1×10 6 When the cell density reaches 0.5 × 10⁶ cells / mL, fresh serum-free NK cell expansion medium is added to maintain the cell density at 0.5 × 10⁶ cells / mL. 6 –1.0×10 6 cells / mL; each time the culture volume is replenished, the corresponding amount of EV is added according to the newly added culture volume to keep the effective stimulation concentration of EV consistent; IL-2 is added at a final concentration of 300 IU / mL.

[0154] The culture period was 14 days; samples were taken on days 0, 3, 7, 10 and 14 to detect cell number, cell viability and phenotype.

[0155] 4. NK cell expansion fold detection The total number of cells and the number of viable cells in each group were determined using an automated cell counter combined with trypan blue staining.

[0156] The results showed that on day 7 of culture, NK cells in all groups expanded to some extent, but the cell proliferation rate in the CD16VHH / IL21 / CD86 / CD137L-EV group was significantly higher than that in the other groups. On day 14 of culture, the fold increases in each group were as follows: Control group 1: Amplified 251.0 times; Control group 2: Amplified 284.7 times; Experimental group 1: Amplification 590.7-fold; Experimental group 2: Amplified 1654.0 times.

[0157] Among them, the amplification fold of experimental group 2 was significantly higher than that of the IL-2 control group, the Ctrl-EV control group, and the CD16VHH-EV group; this result indicates that engineered EVs that simultaneously exhibit anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L can significantly promote the in vitro expansion of primary NK cells.

[0158] 5. Cell viability detection Cell viability was assessed in each group on days 0, 7, and 14 using trypan blue expulsion assay or flow cytometry with live / dead cell dyes. Results showed that cell viability in each group on day 14 was as follows: Control group 1: 89.4%; Control group 2: 88.7%; Experimental group 1: 92.1%; Experimental group 2: 94.7%.

[0159] Experimental group 2 promoted cell proliferation without causing significant cell death, and the cell viability remained at a high level; no abnormal cell aggregation, significant increase in debris, or abnormal cell morphology were observed during the culture process.

[0160] 6. NK cell purity detection On day 14 of culture, cells from each group were collected and analyzed by flow cytometry using anti-CD3 and anti-CD56 antibodies; 1×10⁶ cells were collected from each group. 6 Cells were stained at 4°C in the dark for 30 minutes, washed, and then analyzed.

[0161] The results showed that CD3 in each group - CD56 + The proportions of NK cells are as follows: Control group 1: 96.4%; Control group 2: 97.1%; Experimental group 1: 97.1%; Experimental group 2: 96.3%.

[0162] CD3 in experimental group 2 - CD56 +The proportion of NK cells remained above 96.3%, and no obvious abnormal expansion of T cells or other non-NK cells was observed. This result indicates that CD16VHH / IL21 / CD86 / CD137L-EV can maintain high NK cell purity while promoting NK cell expansion.

[0163] 7. Detection of NK cell activation and functional phenotype On day 14 of culture, the functional phenotypes of NK cells in each group were detected by flow cytometry; the indicators detected included CD16, NKG2D, NKp30, NKp44, NKp46, CD69, CD25, NKG2A, PD-1 and TIM-3.

[0164] The NK cells obtained from experimental group 2 maintained high CD16 expression, with a CD16 positivity rate of 82.9% and an average fluorescence intensity of 7083. Compared with the control group, the expression levels of NKG2D, NKp30, and NKp46 in experimental group 2 remained high, at 90.5%, 75.7%, and 85.9%, respectively. The expression of CD69 and CD25 was moderately increased, at 57.6% and 45.0%, respectively. Meanwhile, the PD-1 and TIM-3 in experimental group 2 were 8.4% and 14.1%, respectively, without significant abnormal increases, indicating that the NK cells were in an effectively activated state.

[0165] Meanwhile, no significant abnormal increases in PD-1 and TIM-3 were observed in experimental group 2, indicating that stimulation with CD16VHH / IL21 / CD86 / CD137L-EV did not lead to a significant increase in exhaustion-like phenotypes.

[0166] 8. Multiple donors repeatedly validated To evaluate the consistency of amplification effects among different donors, the above amplification experiments were repeated using primary NK cells from three healthy donors; the culture conditions, EV addition amount, and detection methods were the same as described above.

[0167] The results of testing on day 14 showed that the NK cell expansion folds in the CD16VHH / IL21 / CD86 / CD137L-EV groups of the three donors were 1608.0-fold, 1689.0-fold, and 1665.0-fold, respectively, with an average expansion fold of 1654.0 ± 41.6-fold and a coefficient of variation of 2.5%; the corresponding CD3... - CD56 + The proportions of NK cells were 94.1%, 95.0%, and 94.6%, respectively, and the average purity of NK cells was 94.6 ± 0.5%. The results indicate that the engineered EV of this invention can promote the expansion of primary NK cells from different donors and has good reproducibility.

[0168] 9. Conclusion The results of this experiment indicate that CD16VHH / IL21 / CD86 / CD137L-EV can promote the in vitro expansion of primary NK cells without the participation of live feeder cells. Compared with the IL-2 group alone, the Ctrl-EV group, and the CD16VHH-EV group alone, the multi-signal engineered EV group significantly increased the fold expansion of NK cells while maintaining high cell viability and CD3+. - CD56 + NK cell ratio; the resulting NK cells retain functional phenotypes such as CD16, NKG2D and natural cytotoxic receptors, and are suitable for subsequent functional testing and NK or CAR-NK cell preparation.

[0169] Experiment 6: Engineered extracellular vesicles promote NK cell expansion through multi-signal synergistic effect. See Figure 6 This experiment compares the effects of engineered extracellular vesicles with different functional molecular combinations on the expansion of primary NK cells, in order to verify that anti-CD16a VHH, membrane-anchored IL-21, CD86 and CD137L have a synergistic stimulatory effect when co-presented on the same extracellular vesicle surface.

[0170] 1. Preparation of EVs with different functional molecule combinations Following the methods in Experiments 3 and 4, the following engineered extracellular vesicles were prepared: Ctrl-EV: Empty vector CHO cell-derived EV; CD16VHH-EV: EV with surface resistance to CD16a VHH; IL21-EV: EV of surface-displaying film-anchored IL-21; CD86 / CD137L-EV: Surface display of the EV versions of CD86 and CD137L; CD16VHH / IL21-EV: The surfaces together exhibit resistance to CD16a VHH and membrane-anchored IL-21 in EV form; CD16VHH / IL21 / CD86 / CD137L-EV: The surface together exhibits resistance to CD16a VHH, membrane-anchored IL-21, CD86 and CD137L EV.

[0171] All EVs were prepared and purified using the same process. NTA assay showed that all EVs exhibited a nanoscale particle distribution, with particle sizes mainly concentrated between 124.5 and 432.4 nm. Among them, the average particle size of CD16VHH / IL21 / CD86 / CD137L-EV was 283.6 nm, and the D50 was 279.8 nm. The average particle sizes of Ctrl-EV, CD16VHH-EV, IL21-EV, CD86 / CD137L-EV, and CD16VHH / IL21-EV were 281.6 nm, 285.7 nm, 283.8 nm, 288.6 nm, and 282.2 nm, respectively. These results indicate that the engineered modification of different functional molecule combinations did not significantly alter the overall nanoparticle distribution characteristics of CHO cell-derived EVs. After BCA assay, all EVs were used for subsequent NK cell expansion experiments with the same protein content.

[0172] The expression of molecules on the surface of each EV group was detected by EV-microsphere coupled flow cytometry. The results showed that the corresponding functional molecule signals could be detected in each engineered EV, but no positive signals for anti-CD16a VHH, IL-21, CD86 or CD137L were detected in Ctrl-EV.

[0173] 2. Primary NK cell expansion experiment Peripheral blood was collected from healthy donors, and PBMCs were separated using Ficoll density gradient centrifugation. Primary NK cells were obtained using an NK cell negative sorting kit.

[0174] NK cells were seeded in 24-well plates at an initial density of 2 × 10⁶ cells / well. 5 cells / mL; IL-2 was added to each group to a final concentration of 300 IU / mL, and EV was added separately at a concentration of 10 μg EV protein / 10 6 NK cells; culture period is 14 days, with fresh culture medium, IL-2 and corresponding EV added every 2-3 days.

[0175] The experimental groups are as follows:

[0176] 3. Results of NK cell expansion On day 14 of culture, the number of live NK cells in each group was measured, and the NK cell expansion fold was calculated.

[0177] On day 14 of culture, the fold expansion of NK cells in each group was as follows: G1: IL-2, 251.0 times; G2: Ctrl-EV+IL-2, 284.0 times; G3: CD16VHH-EV+IL-2, 583.3 times; G4: IL21-EV+IL-2, 713.7 times; G5: CD86 / CD137L-EV+IL-2, 439.7 times; G6: CD16VHH / IL21-EV+IL-2, 1129.0 times; G7: CD16VHH / IL21 / CD86 / CD137L-EV+IL-2, 1654.0-fold increase; the results showed that CD16VHH-EV, IL21-EV and CD86 / CD137L-EV could all increase the fold increase of NK cells to some extent, but the promoting effect of a single signal or a single class of signal was limited; the amplification effect of CD16VHH / IL21-EV was higher than that of CD16VHH-EV and IL21-EV, indicating that there is a synergistic trend between CD16a aggregation activation signal and membrane anchoring IL-21 signal.

[0178] The four-signal group CD16VHH / IL21 / CD86 / CD137L-EV showed the highest fold increase, significantly higher than CD16VHH / IL21-EV, CD16VHH-EV, IL21-EV, and CD86 / CD137L-EV, suggesting that the CD86 / CD137L co-stimulatory signal further enhanced the promoting effect of CD16a activation signal and IL-21 proliferation signal on NK cell expansion.

[0179] 4. Comparison of NK cell purity, viability, and CD16 retention rate On day 14 of culture, CD3 counts in each group were measured by flow cytometry. - CD56 + NK cell proportion, cell viability, and CD16 expression.

[0180]

[0181] The results showed that group G7 achieved the highest amplification fold while also having low CD3... - CD56 + The proportion of NK cells remained above 94.7%, the cell viability remained above 94.8%, and the CD16 positivity rate remained above 83.5%. These results indicate that the four-signal EV not only promotes the expansion of NK cell numbers, but also maintains NK cell purity, viability, and CD16-related functional phenotypes.

[0182] 5. Comparison of NK cell functional activities On day 14 of culture, K562 cells were used as target cells to detect the killing function of expanded NK cells in each group; the effector-target ratio was set to E:T=5:1, and the killing rate was detected by LDH release method after 4 h of co-culture.

[0183] The K562 killing assay showed that the G7 group had the highest NK cell killing rate, at 81.2%. Further analysis of cytokines in the culture supernatant revealed that the G7 group had an IFN-γ secretion level of 3011.7 pg / mL and an TNF-α secretion level of 705.0 pg / mL, both higher than other groups.

[0184] These results indicate that the synergistic effect of the four-signal EVs is not only reflected in the number of NK cells expanded, but also in the maintenance and enhancement of the effector function of NK cells after expansion.

[0185] 7. Conclusion The results of this experiment show that when anti-CD16a VHH, membrane-anchored IL-21, CD86, and CD137L are co-presented on the surface of engineered extracellular vesicles, a significant multi-signal synergistic effect can be produced. Compared with anti-CD16a VHH alone, IL-21 alone, the CD86 / CD137L combination, or the CD16VHH / IL21 dual-signal EV, CD16VHH / IL21 / CD86 / CD137L-EV can more significantly promote the expansion of primary NK cells and maintain higher NK cell purity, cell viability, CD16 expression, and cytotoxic effects. These results demonstrate that the technical effect of the engineered extracellular vesicles of this invention originates from the synergistic co-presentation of CD16a aggregation activation signals, membrane-anchored cytokine signals, and co-stimulatory signals on the same vesicle surface.

[0186] Experiment 7: Comparison of the engineered vesicles of this invention with the K562 feeder layer / membrane particle system See Figure 7 This experiment compares the roles of CD16VHH / IL21 / CD86 / CD137L-EV and K562 engineered feeder cells and K562-derived membrane particles in the expansion of primary NK cells to evaluate the advantages of the system of this invention in terms of expansion efficiency, functional maintenance and safety.

[0187] 1. Preparation of experimental materials The following stimulation systems were used for comparison: G1: IL-2 control group, with a final IL-2 concentration of 100 IU / mL; G2: K562 feeder group, using engineered K562 cells expressing membrane-anchored IL-21 and CD137L, which were inactivated by γ-ray irradiation before use; G3: K562 membrane particle group, obtained from engineered K562 cells through cell disruption, membrane component separation and particle granulation, quantified according to total protein content; G4: The EV group of this invention uses CD16VHH / IL21 / CD86 / CD137L-EV prepared in Experiment 4.

[0188] The initial ratio of NK cells to K562 feeder cells in group G2 was set at 2:1; the amount of K562 membrane particles added to group G3 was 10 μg protein / 10 μg. 6 NK cells; G4 group: EV addition amount of this invention was 10 μg EV protein / 10 6 NK cells.

[0189] 2. Primary NK cell culture Peripheral blood was collected from healthy donors, and PBMCs were separated by Ficoll density gradient centrifugation. Primary NK cells were then enriched using an NK cell negative sorting kit. After sorting, CD3+ cells were analyzed. - CD56 + NK cell proportion >95%, cell viability >95%.

[0190] Adjust NK cells to 2×10 5 Cells / mL were seeded into 24-well plates, 1 mL per well; IL-2 was added to each group to a final concentration of 100 IU / mL, and corresponding stimulating materials were added; the culture period was 14 days, and fresh culture medium, IL-2 and corresponding stimulating materials were added every 2-3 days.

[0191] 3. Comparison of NK cell expansion efficiency On day 14 of culture, the number of viable NK cells in each group was measured and the fold increase was calculated. The fold increase of NK cells in each group on day 14 of culture is as follows: G1: IL-2, control group, amplified 238.0-fold; G2: K562 feeder group, amplified 1777.7 times; G3: K562 membrane particle group, amplified 1182.3 times; G4: The EV group of this invention, amplified 1649.7 times.

[0192] The results showed that the EV group of the present invention could significantly promote the expansion of primary NK cells without the participation of K562 live feeder cells, and the expansion effect was significantly better than that of the IL-2 control group, and reached or exceeded that of the K562 membrane particle group.

[0193] 4. Comparison of NK cell purity, viability, and CD16 expression On day 14 of culture, CD3 was detected by flow cytometry. - CD56 + NK cell percentage, cell viability, and CD16 expression.

[0194]

[0195] The results showed that the EV group of this invention promoted NK cell expansion while also reducing CD3... - CD56 +The proportion of NK cells remained above 97.4%, the cell viability remained above 97.2%, and CD16 expression remained high, suggesting that it is beneficial to maintain the functional phenotype of NK cells.

[0196] 5. Comparison of cytotoxic killing activities On day 14 of culture, K562 cells were used as target cells to detect the killing function of NK cells in each group; the effector-target ratio was E:T=5:1, and the killing rate was detected after 4 h of co-culture; the results showed that the NK cells obtained by the EV group of the present invention had strong K562 killing activity, indicating that it not only promotes the expansion of NK cell numbers, but also maintains or enhances the effector function of NK cells.

[0197] 7. Conclusion The results of this experiment show that the CD16VHH / IL21 / CD86 / CD137L-EV of this invention can effectively promote the expansion of primary NK cells and maintain high NK cell purity, viability, CD16 expression, and cytotoxic function without the participation of K562 live feeder cells and K562 tumor-derived membrane particles. Compared with K562 feeder cells and K562 membrane particles, the EV of this invention has a clear source, controllable composition, and no risk of live cell stimulation, making it more suitable for clinical-grade NK cell preparation.

[0198] Experiment 8: Optimization of the dosage and supplementation method of engineered extracellular vesicles See Figure 8 In this experiment, CD16VHH / IL21 / CD86 / CD137L-EV prepared in Experiment 4 was used to optimize the dosage, supplementation method and culture period of primary NK cell expansion in order to determine the optimal conditions for in vitro expansion of NK cells.

[0199] 1. Preparation of primary NK cells Peripheral blood was collected from healthy donors, and PBMCs were separated by Ficoll density gradient centrifugation. Primary NK cells were then enriched using an NK cell negative sorting kit. After sorting, CD3+ cells were analyzed. - CD56 + NK cell proportion >95%, cell viability >95%.

[0200] NK cells were resuspended in serum-free NK cell expansion medium, and the cell density was adjusted to 2 × 10⁶ cells / day. 5 Cells / mL were seeded into 24-well plates, 1 mL per well; IL-2 was added to each group to a final concentration of 100 IU / mL.

[0201] 2. EV Dosage Optimization Different dosages of CD16VHH / IL21 / CD86 / CD137L-EV were set according to the total protein content: G1: IL-2 control group, without added EV; G2: 1μg EV protein / 10 6 NK cells; G3: 5μg EV protein / 10 6 NK cells; G4: 10μg EV protein / 10 6 NK cells; G5: 20μg EV protein / 10 6 NK cells; G6: 50μg EV protein / 10 6 NK cells.

[0202] Each group was cultured for 14 days, with fresh culture medium, IL-2, and corresponding doses of EV added every 2–3 days; on day 14, NK cell expansion, viability, and CD3 count were measured. - CD56 + Proportion.

[0203] The results are as follows:

[0204] The results showed that as the EV addition dose increased from 1 μg / 10 6 NK cells increased to 10–20 μg / 10 6 NK cells, the NK cell expansion rate gradually increased; when the EV dose was increased to 50 μg / 10 6 When NK cells were used, the expansion fold did not increase significantly further, and some samples showed a trend of slowed cell proliferation or decreased viability.

[0205] Considering factors such as fold expansion, cell viability, and NK cell purity, the preferred dosage for CD16VHH / IL21 / CD86 / CD137L-EV is 5–20 μg EV protein / 10 6 NK cells, more preferably 10 μg EV protein / 10 6 NK cells.

[0206] 3. EV Particle Count and Dosage Validation To further determine the EV usage range based on particle count, the following dosages were set for CD16VHH / IL21 / CD86 / CD137L-EV according to particle count: G1: 1×10 8 particles / 10 6 NK cells; G2: 1×10 9 particles / 10 6 NK cells; G3: 1×10 10 particles / 10 6 NK cells; G4: 1×10 11 particles / 10 6 NK cells.

[0207] The cultivation conditions are the same as before, and the cultivation period is 14 days.

[0208] The results are as follows:

[0209] The results showed that 1×10 9 –1×10 10 particles / 10 6 Good expansion and cell viability can be obtained within the NK cell range; when the granule number is further increased to 1×10 11 particles / 10 6 When NK cells were used, the expansion effect tended to plateau; this result is consistent with the dose optimization results based on EV protein content.

[0210] 4. Comparison of EV replenishment methods In determining 10 μg EV protein / 10 6 After determining the optimal dose of NK cells, the effects of different supplementation methods on NK cell expansion were compared.

[0211] The experimental groups are as follows: G1: Add all EVs at once on Day 0; G2: Add EV twice, on day 0 and day 7; G3: EV is added in divided doses on days 0, 3, 6, 9, and 12 along with the rehydration solution; G4: Add half the amount of EV on day 0, and then add the remaining EV each time you refill the solution.

[0212] The total amount of EV added in each group was the same, which was 10 μg EV protein / 10 6 The equivalent total number of NK cells; the culture period is 14 days.

[0213] The results are as follows:

[0214] The results showed that the amplification effect of the group that was supplemented with EV in multiple doses was better than that of the group that was supplemented once on day 0. Among them, the G3 group, which was supplemented with EV in multiple doses every 2–3 days with fluid replenishment, had the highest amplification fold and maintained good cell viability and NK cell purity. This result indicates that maintaining the intensity of engineered EV stimulation continuously is beneficial to the continuous proliferation of NK cells.

[0215] 5. Optimization of cultivation cycle The preferred conditions were adopted, namely, the amount of CD16VHH / IL21 / CD86 / CD137L-EV added was 10 μg EV protein / 10 6 NK cells were cultured, and EVs were added every 2–3 days with replenishment fluid to compare the NK cell expansion effects under different culture cycles.

[0216] Samples were taken and tested on days 7, 10, 14 and 21.

[0217]

[0218] The results showed that NK cells expanded rapidly from Day 7 to Day 14, and the expansion rate, cell viability and NK cell purity were all in a good state at Day 14. After extending the culture to Day 21, the expansion rate of some samples decreased and there was a trend of decreased CD16 expression or decreased cell viability.

[0219] 6. Conclusion The results of this experiment indicate that CD16VHH / IL21 / CD86 / CD137L-EV has a dose-dependent promoting effect on the expansion of primary NK cells. Within an appropriate dose range, it can significantly increase the fold increase of NK cells while maintaining high cell viability and CD3+. - CD56 + The proportion; the fractionated addition method is better than the one-time addition method, indicating that continuous provision of EV surface anti-CD16a VHH, membrane-anchored IL-21, CD86 and CD137L signals is beneficial to the continuous expansion of NK cells; considering all indicators, 10 μg EV protein / 10 6 The optimal NK cell expansion conditions of this invention are: NK cells are added in portions every 2–3 days and cultured for 10–14 days.

[0220] 7. Alternative Solutions Without departing from the overall technical concept of this invention, those skilled in the art can make appropriate substitutions or adjustments to the source cells of engineered extracellular vesicles, the structure of CD16 nanobody, the membrane anchoring method, the composition of cytokines and co-stimulatory molecules, the vesicle preparation process, and the NK cell expansion conditions; as long as the above-mentioned alternative solutions can achieve CD16-mediated NK cell activation, cytokine support, and synergistic presentation of co-stimulatory signals, they should all be regarded as equivalent implementations of the technical solution of this invention.

[0221] (1) Alternatives to engineered cell sources The present invention preferably uses CHO-K1 cells as the source cells for engineered extracellular vesicles, but the present invention is not limited thereto; the engineered eukaryotic cells may also be selected from CHO-S, CHO-DG44, HEK293, HEK293T, HEK293F, Expi293, BHK, Vero, PER.C6, insect cells, or other eukaryotic cells that can stably express exogenous membrane proteins and secrete extracellular vesicles.

[0222] The engineered cells do not use feeder cells derived from blood tumors, such as K562, to reduce the risk of residual tumor-derived live cells, tumor-derived DNA, or complex membrane components.

[0223] (2) Alternatives to CD16 nanobodies The present invention preferably uses anti-human CD16a VHH nanobody anti-CD16a-VHH-1, but the present invention is not limited to this specific clone; the CD16 nanobody may be a VHH that specifically binds to human CD16a, a single-domain antibody, a camelid nanobody, a humanized VHH, an affinity-matured VHH or its antigen-binding functional variant.

[0224] The CD16 nanobody may contain an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:1 and still retains CD16a binding activity; it may also contain a CDR sequence that is the same as or has undergone conservative substitution as the CDR sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0225] SEQ ID NO:3: MALPVTALLLPLALLLHAARPEQKLISEEDLGGGGSEVQLVESGGGLVQPGESLTLSCVVAGSIFSFAMSWYRQAPGKERELVARIGSDDRVTYADSVKGRFTISRDNIKRTAGLQMNSLKPEDTAVYYCNAQTDLRDWTVREYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGG ELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT SEQ ID NO:4: MWLQSLLLLGTVACSISQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSMALIVLGGVAGLLLFIGLGIFF SEQ ID NO:5: MWLQSLLLLGTVACSISMRISKPHLRSISIQLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSMALIVLGGVAGLLLFIGLGIFF In addition to VHH, the CD16 binding module can also be replaced with anti-CD16a scFv, Fab, single-chain antibody fragments, affinity molecules, DARPins, single-domain engineered proteins, or other binding domains that can bind to CD16a and induce its aggregation.

[0226] (3) Alternatives to membrane anchoring structures The anti-CD16a VHH of the present invention can be anchored to the surface of engineered cells and their extracellular vesicles via the transmembrane region of LAMP2B, CD63 or CD8α, but the present invention is not limited thereto; the membrane anchoring structure may also be selected from CD28, PDGFR, CD81, CD9, Lamp1, GPI anchoring signal, transmembrane region of viral envelope protein or other structures that can promote the localization of fusion protein to the cell membrane and extracellular vesicle membrane.

[0227] The structural sequence of the anti-CD16a VHH fusion protein can also be adjusted to signal peptide-anti-CD16a VHH-linker peptide-transmembrane region, or signal peptide-dimerization domain-anti-CD16a VHH-linker peptide-transmembrane region; the linker peptide can be GGGGS, (GGGGS)2, (GGGGS)3, EAAAK, IgG hinge, or other linker sequences that can maintain the spatial freedom of the antigen-binding domain.

[0228] (4) Alternatives to membrane-anchored cytokines The present invention preferably uses membrane-anchored IL-21, but membrane-anchored IL-15 can also be used; the present invention may also use IL-15 / IL-15Rαsushi complex structure, membrane-anchored IL-2, IL-7, IL-12, IL-18, IL-21 or their functional fragments, mutants, fusion proteins or combinations thereof.

[0229] Among them, membrane-anchored IL-15 may include the IL-15 functional domain and the IL-15Rαsushi domain to enhance the stable presentation of IL-15; membrane-anchored IL-21 may include the IL-21 functional domain, a flexible junction region and a transmembrane region to provide signals for NK cell proliferation and function maintenance; the above cytokines may be used alone or in combination on the surface of the same engineered extracellular vesicle.

[0230] (5) Alternatives to co-stimulatory molecules The present invention preferably uses CD86 and CD137L as co-stimulatory molecules, but the present invention is not limited thereto; CD86 can be replaced by CD80, ICOSL, B7-H3, B7-H6 or other B7 family molecules; CD137L can be replaced by CD40L, OX40L, GITRL, CD70, LIGHT or functional fragments thereof.

[0231] In this invention, the co-stimulatory molecule is co-presented with CD16 nanobody, membrane-anchored IL-21 and / or IL-15; the preferred embodiment is a combination of anti-CD16a VHH, membrane-anchored IL-21, CD86 and CD137L; alternative embodiments may be a combination of anti-CD16a VHH, membrane-anchored IL-15, CD80 and CD137L, or a combination of anti-CD16a VHH, membrane-anchored IL-21, CD86 and OX40L.

[0232] (6) Alternatives to multi-signal representation This invention can use multiple expression vectors to co-transfect engineered cells, or use a single polycistronic expression vector to express multiple functional molecules; the polycistronic expression system may include IRES, 2A peptide sequence, dual promoter or multipromoter expression cassette.

[0233] The expression vector may be a plasmid vector, lentiviral vector, adenovirus vector, adeno-associated virus vector, transposon vector, or site-directed integration vector; preferably, a non-replicating expression system with low viral safety risk or that has undergone adequate viral safety evaluation is used; the promoter may be selected from CMV, EF1α, CAG, SV40, PGK, SFFV, or other promoters suitable for eukaryotic cell expression.

[0234] (7) Alternative solutions for extracellular vesicle preparation processes This invention uses culture supernatant collection, fractionation centrifugation, ultrafiltration concentration, and size exclusion chromatography to prepare engineered extracellular vesicles, but this invention is not limited to this; the extracellular vesicles can be prepared and purified by differential centrifugation, ultracentrifugation, density gradient centrifugation, tangential flow filtration, ultrafiltration, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer precipitation, microfluidic separation, or combinations thereof.

[0235] The culture system can be adherent culture, suspension culture, cell factory culture, shake flask culture, wave bioreactor culture, or stirred tank bioreactor culture; the culture medium can be serum-free culture medium, animal-free culture medium, or low vesicle background culture medium; the obtained extracellular vesicles can be exosomes, microvesicles, small extracellular vesicles, or mixtures thereof, preferably with a particle size mainly distributed in the range of 30–300 nm, more preferably 50–200 nm.

[0236] (8) Alternative solutions for NK cell sources and expansion conditions The present invention preferably uses primary NK cells derived from peripheral blood, but the present invention is not limited thereto; the NK cells may be derived from peripheral blood, umbilical cord blood, apheresis blood, bone marrow, placental tissue, NK cells derived from induced pluripotent stem cell differentiation, NK cells derived from hematopoietic stem cell differentiation, or NK cell lines.

[0237] The starting cells can be PBMCs or NK cells enriched by immunomagnetic beads, flow cytometry, or other methods; the initial seeding density can be 0.1 × 10⁻⁶ cells / year. 6 –5×10 6 cells / mL, preferably 0.2 × 10⁻⁶. 6 –2×10 6 cells / mL; the amount of engineered extracellular vesicles added can be 0.1–50 μg EV protein / 10 6 NK cells, or 1×10 8 –1×10 11 particles / 10 6 NK cells; the culture period can be 7–21 days, preferably 10–14 days.

[0238] IL-2, IL-15, IL-21, IL-12, IL-18 or combinations thereof can be added to the culture system to maintain NK cell survival and expansion; the extracellular vesicles can be added all at once on day 0 of culture, or added in several times each time the fluid is replenished.

[0239] The above embodiments are illustrative of the present invention and are not intended to limit the present invention. Any simple modifications to the present invention are within the scope of protection of the present invention.

Claims

1. An engineered extracellular vesicle, characterized in that: The surface of the extracellular vesicle membrane displays anti-CD16a nanobodies.

2. The engineered extracellular vesicles as described in claim 1, characterized in that: The extracellular vesicles are derived from genetically engineered eukaryotic cells.

3. The engineered extracellular vesicles as described in claim 1, characterized in that: The anti-CD16a nanobody is a VHH nanobody that can specifically bind to human CD16a; the CD16a nanobody contains the amino acid sequence shown in SEQ ID NO:1 or contains an amino acid sequence that has more than 80% sequence identity with SEQ ID NO:1 and still retains the activity of specifically binding to human CD16a.

4. The engineered extracellular vesicles as described in claim 3, characterized in that: The CD16a nanobody is expressed on the surface of engineered eukaryotic cell membrane in a membrane-anchored form and is loaded onto the surface of extracellular vesicle membrane during the extracellular vesicle formation process to form a membrane-anchored CD16a nanobody. The membrane-anchored CD16a nanobody includes, from the N-terminus to the C-terminus, a signal peptide, an anti-human CD16a VHH nanobody domain, a linker peptide, and a transmembrane anchoring region.

5. The engineered extracellular vesicles as described in claim 4, characterized in that: The signal peptide is selected from one or more of Igκ signal peptide, CD8α signal peptide, and IL-2 signal peptide; the linker peptide is selected from one or more of GGGGS, (GGGGS)2, and (GGGGS)3; and the transmembrane anchoring region is selected from one or more of CD8α, CD28, PDGFR, LAMP2B, CD63, CD81, and GPI anchoring signals.

6. The engineered extracellular vesicles as described in claim 4, characterized in that: The membrane-anchored anti-CD16a nanobody contains the amino acid sequence shown in SEQ ID NO:

3.

7. The engineered extracellular vesicles as described in claim 1, characterized in that: The extracellular vesicle membrane surface also displays membrane-anchored IL-21 and / or membrane-anchored IL-15, both of which include: cytokine functional domains, linker peptides and / or stabilizing linker modules, and transmembrane anchoring regions; the stabilizing linker module is the CH2 / CH3 segment in the IgG Fc domain.

8. The engineered extracellular vesicles as described in claim 7, characterized in that: The membrane-anchored IL-21 includes an IL-21 functional domain, a linker region, and a transmembrane domain, and the membrane anchoring type contains the amino acid sequence shown in SEQ ID NO:4; the membrane-anchored IL-15 includes an IL-15 functional domain, an IL-15Rαsushi domain, a linker region, and a transmembrane domain, and the membrane anchoring type IL-15 contains the amino acid sequence shown in SEQ ID NO:

5.

9. The engineered extracellular vesicles as described in claim 1, characterized in that: The surface of the extracellular vesicle membrane also displays co-stimulatory molecules, namely CD86 and / or CD137L; CD86 contains the amino acid sequence shown in SEQ ID NO:6; CD137L contains the amino acid sequence shown in SEQ ID NO:

7.

10. A method for in vitro expansion of NK cells, characterized in that, Includes the following steps: S1. Provide a starting cell population containing NK cells; S2. The initial cell population is seeded into NK cell expansion medium; S3. Add the engineered extracellular vesicles as described in any one of claims 1 to 9 to the culture system; S4. Cultivate NK cells under suitable conditions to allow them to receive CD16a aggregation activation signals, membrane-anchored cytokine signals, and / or co-stimulatory signals. S5. Harvest the expanded NK cells.

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