Engineered Cell-Membrane-Coated Nanoparticles Directly Present Tumor Antigens to Promote Anticancer Immunity

Abstract

The recent success of immunotherapies has highlighted the power of leveraging the immune system in the fight against cancer. In order for most immune-based therapies to succeed, T cell subsets with the correct tumor-targeting specificities must be mobilized. When such specificities are lacking, providing the immune system with tumor antigen material for processing and presentation is a common strategy for stimulating antigen-specific T cell populations. While straightforward in principle, experience has shown that manipulation of the antigen presentation process can be incredibly complex, necessitating sophisticated strategies that are difficult to translate. Herein, the design of a biomimetic nanoparticle platform is reported that can be used to directly stimulate T cells without the need for professional antigen-presenting cells. The nanoparticles are fabricated using a cell membrane coating derived from cancer cells engineered to express a co-stimulatory marker. Combined with the peptide epitopes naturally presented on the membrane surface, the final formulation contains the necessary signals to promote tumor antigen-specific immune responses, priming T cells that can be used to control tumor growth. The reported approach represents an emerging strategy that can be used to develop multiantigenic, personalized cancer immunotherapies.

Keywords: anticancer vaccines; artificial antigen presentation; cell-membrane-coated nanoparticles; genetic engineering; immunotherapy.

Figure 1.

Schematic of engineered cell membrane-coated nanoparticles for direct antigen presentation. a) Wild-type cancer cells, which naturally present their own antigens via MHC-I, are engineered to express CD80, a co-stimulatory signal. The plasma membrane from these cells is then derived and coated onto polymeric nanoparticle cores. b) The resulting antigen-presenting nanoparticles (AP-NPs) can directly stimulate tumor antigen-specific T cells through engagement of the cognate T cell receptor (TCR) and CD28. Upon activation, the T cells are capable of controlling tumor growth by killing cancer cells that express the same antigens.

Figure 2.

Characterization and biological activity of engineered cancer cells capable of direct antigen presentation. a) Western blot probing for OVA on B16-CD80/OVA cells and control cells. b,c) Expression of CD80 (b) and presentation of an MHC-I-restricted OVA peptide (Kb-SIINFEKL, c) by B16-CD80/OVA cells and control cells. d) Co-expression of CD80 and Kb-SIINFEKL by B16-CD80/OVA cells and control cells. e-h) Expression of CD69 (e,f) and CD25 (g,h) by OT-I CD8⁺ T cells incubated with B16-CD80/OVA cells and control cells for 24 h (n = 3, mean + SD). i,j) Frequency of memory phenotypes CD44^highCD62L^high (i) and CD44^highCD62L^low (j) among OT-I CD8⁺ T cells incubated with B16-CD80/OVA cells and control cells for 24 h (n = 3, mean + SD). k,l) Secretion of IL-2 (k) and IFNγ (l) by OT-I CD8⁺ T cells incubated with B16-CD80/OVA cells and control cells for 24 h (n = 3, mean + SD). m) Expression of CD69 by CD8⁺ T cells in a population of pmel-1 splenocytes incubated with B16- OVA or B16-CD80/OVA cells for 2 days (n = 3, mean + SD). **p < 0.01, ****p < 0.0001 (compared to B16-CD80/OVA); one-way ANOVA.

Figure 3.

Fabrication and characterization of engineered antigen-presenting nanoparticles. a) Size of [CD80/OVA]NPs at different membrane to core weight ratios when suspended in water or PBS (n = 3; mean + SD). b,c) Hydrodynamic diameter (b) and surface zeta potential (c) of bare PLGA cores, B16-CD80/OVA membrane vesicles, and [CD80/OVA]NPs (n = 3; mean + SD). d,e) Transmission electron microscope images of [CD80/OVA]NPs immediately after synthesis (d) and after 1 week of storage (e). Scale bars = 100 nm. f) Size of [CD80/OVA]NPs over 2 weeks (n = 3; mean ± SD). g,h) Relative binding of antibodies against Kb-SIINFEKL (g) and CD80 (h) to [WT]NPs, [OVA]NPs, and [CD80/OVA]NPs (n = 3; mean + SD).

Figure 4.

Biological activity of engineered antigen-presenting nanoparticles. a-d) Expression of CD69 (a,b) and CD25 (c,d) by OT-I CD8⁺ T cells incubated with [CD80/OVA]NPs and control nanoparticles for 3 days (n = 3, mean + SD). e,f) Frequency of memory phenotypes CD44^highCD62L^high (e) and CD44^highCD62L^low (f) among OT-I CD8⁺ T cells incubated with [CD80/OVA]NPs and control nanoparticles for 3 days (n = 3, mean + SD). g,h) Secretion of IL-2 (g) and IFNγ (h) by OT-I CD8⁺ T cells incubated with [CD80/OVA]NPs and control nanoparticles for 3 days (n = 3, mean + SD). i,j) Expression of CD69 (i) and CD25 (j) by CD8⁺ T cells in a population of OT-I splenocytes after 3 days of incubation with [CD80/OVA]NPs either freshly made or stored for 1 week (n = 3, mean + SD). k,l) Fluorescent signal dilution of CD8⁺ T cells in a population of OT-I splenocytes labeled with CellTrace Violet after incubation with [CD80/OVA]NPs and control nanoparticles (k) or [CD80/OVA]NPs at various concentrations (l) for 3 days. m) Fold expansion of CD8⁺ T cells in a population of OT-I splenocytes after incubation with [CD80/OVA]NPs and control nanoparticles for 4 days (n = 3, mean + SD). n) Cell killing by OT-I CD8⁺ cells activated by [CD80/OVA]NPs for 3 days and then incubated with B16-OVA or B16-WT cells at various effector to target (E:T) ratios for 18 h (n = 3, mean ± SD). o) Expression of CD69 by CD8⁺ T cells in a population of pmel-1 splenocytes incubated with [OVA]NPs or [CD80/OVA]NPs for 3 days (n = 3, mean + SD). **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to [CD80/OVA]NP); one-way ANOVA. ##p < 0.01, ####p < 0.0001; Student’s t-test.

Figure 5.

In vivo delivery and activity of engineered antigen-presenting nanoparticles. a) Immunofluorescence images of draining lymph node sections taken from OT-I mice at different periods after administration of dye-labeled [CD80/OVA]NPs. Red: [CD80/OVA]NPs, green: CD8⁺ cells; scale bar = 250 μm. b) Expression of CD69 by OT-I CD8⁺ T cells in the draining lymph nodes 3 days after administration of [CD80/OVA]NPs or control nanoparticles into C57BL/6 mice adoptively transferred with OT-I splenocytes (n = 4, mean + SD). c) Secretion of IFNγ by draining lymph node cells 4 days after administration of [CD80/OVA]NPs or control nanoparticles into C57BL/6 mice adoptively transferred with OT-I splenocytes (n = 3, mean + SD). ***p < 0.001, ****p < 0.0001 (compared to [CD80/OVA]NP); one-way ANOVA.

Figure 6.

In vivo prophylactic and therapeutic efficacy. a) Experimental timeline for prophylactic efficacy study. b-d) Average tumor sizes (b), individual tumor growth kinetics (c), and survival (d) over time for the prophylactic efficacy study (n = 6; mean ± SEM). e) Experimental timeline for therapeutic efficacy study. f-h) Average tumor sizes (f), individual tumor growth kinetics (g), and survival (h) over time for the therapeutic efficacy study (n = 6; mean ± SEM). ** p < 0.01, *** p < 0.001 (compared to [CD80/OVA]NP in survival plot); log-rank test.