Bioengineered Nanocage from HBc Protein for Combination Cancer

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A Bioengineered Nanocage from HBc Protein for Combination Cancer Immunotherapy Wenjun Shan, Haiping Zheng, Guofeng Fu, Chenfeng liu, Zizhen li, Yuhan Ye, jie zhao, Dan Xu, Liping Sun, Xin wang, Xiao Lei Chen, Shengli Bi, Lei Ren, and Guo Fu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04722 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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A Bioengineered Nanocage from HBc Protein for Combination Cancer Immunotherapy Wenjun Shan,† Haiping Zheng,‡ Guofeng Fu,‡ Chenfeng Liu,‡ Zizhen Li,∥ Yuhan Ye,# Jie Zhao,† Dan Xu,† Liping Sun,† Xin Wang,∥ Xiao Lei Chen,‡ Shengli Bi,⊥ Lei Ren,*† and Guo Fu*‡§

†Department

of Biomaterials, Key Laboratory of Biomedical Engineering of Fujian Province,

State Key Lab of Physical Chemistry of Solid Surface, College of Materials, Xiamen University, Xiamen, Fujian 361005, P. R. China. ‡State

Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling

Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. ∥National

Institute of Diagnostics and Vaccine Development in Infectious Diseases, School

of Public Health, Xiamen University, Xiamen, Fujian 361102, P. R. China. #Zhongshan ⊥Chinese

Hospital, Xiamen University, Xiamen, Fujian 361005, P. R. China.

Center for Disease Control & Prevention Institute for Viral Disease Control &

Prevention, Beijing 102206, P. R. China. §Cancer

Research Center of Xiamen University, Xiamen, Fujian 361102, China.

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ABSTRACT Protein nanocages are promising multifunctional platforms for nanomedicine owing to the ability to decorate their surfaces with multiple functionalities through genetic and/or chemical modification to achieve desired properties for therapeutic and diagnostic purposes. Here, we describe a model antigen (OVA peptide), was conjugated to the surface of a naturally occurring hepatitis B core protein nanocage (HBc NC) by genetic modification. The engineered OVA-HBc nanocages (OVA-HBc NCs), displaying high density repetitive array of epitopes in a limited space by self-assembling into symmetrical structure, can not only induce bone marrow derived dendritic cells (BMDC) maturation effectively, but also be enriched in the draining lymph nodes. Naïve C57BL/6 mice immunized with OVA-HBc NCs are able to generate significant and specific cytotoxic T lymphocyte (CTL) responses. Moreover, OVA-HBc NCs as a robust nanovaccine can trigger preventive antitumor immunity and significantly delay tumor growth. When combined with a low-dose chemotherapy drug (paclitaxel), OVA-HBc NCs could specifically inhibit progression of established tumor. Our findings support HBc-based nanocages with modularity and scalability are an attractive nanoplatform for combination cancer immunotherapy.

KEYWORDS Protein nanocage, Hepatitis B core protein, Antigen delivery, Vaccination, Cancer immuotherapy

Chemotherapy and radiotherapy are widely used in clinicals as the golden standard for cancer treatment.1. However, a severe side-effect is that both therapies can not only kill cancerous cells but also normal cells due to lack of target specificity.2 In contrast, immunotherapy, including vaccines and immune checkpoint blockade, can induce a durable 2 ACS Paragon Plus Environment

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population of highly potent, tumor-specific T cells that lyse tumor cells and eradicate cancers.3-5 Although initially thought inappropriate to combine chemotherapy with immunotherapy due to the immunosuppressive effects commonly associated with chemotherapy, it is now accepted that low dose chemotherapy can reverse the immunosuppressive status within tumor microenvironment,6-8 and enhance the anti-tumor effects of immunotherapy.9 The induction of a strong tumor-specific cytotoxic T cell (CTL) response is a prerequisite for peptide vaccine based immunotherapy.10 However, in most cases, the neoantigens which can elicit anti-tumor CTL response may vary from patient to patient significantly, even among those patients with a similar diagnosis.11 The recent innovation in cancer exome sequencing has partially solved the problem by allowing the identification of patient-specific neoantigens, and thus promoted the development of personalized peptide-based cancer vaccines.12 Unfortunately, the therapeutic effect of peptide-based vaccines has been poor in some clinical trials partially due to their low immunogenicity and partially due to the immunosuppression mechanism within the tumor.13 Moreover, the preparation of peptide-based vaccines derived from tumor neoantigen can be very expensive and time consuming, which will not only increase the financial burden of patients but more importantly lose the precious time of those patients.14 Nanoparticle-based vaccine is an emerging new branch of cancer immunotherapy, and recent progress has been focusing on improving the co-delivery of antigen and adjuvant to evoke a productive immune response. However, at present, only a few nano-platforms have both adjustable size, shape and good biocompatibility.15 Naturally derived protein nanocage is a highly symmetrical structure, formed by the self-assembly of multi-repetitive and defined protein. Meanwhile, the outer and inner surface of protein nanocages can be modified, and its internal cavity can be used as a template to store and release small molecule cargo.16 The 3 ACS Paragon Plus Environment

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main advantage of protein nanocages is the spatially controlable display of functional groups at well-defined locations through genetic or chemical modifications.17 Moreover, protein nanocages themselves possess potent adjuvant activity and can enhance the immunogenicity of antigenic peptides/proteins, which otherwise could be of poor immunogenicity and unable to induce an effective immune response.18 Thus owing to these features, protein cages hold tremendous promise as a carrier candidate in nanomedicine. Several commercial nanocage-based vaccines, which are derived from hepatitis B virus and human papillomavirus, have been approved by U.S. Food and Drug Administration.19 Among these, hepatitis B core protein (HBc) is the most promising model for displaying candidate antigens, due to their excellent safety profiles and strong immunogenicity.20,

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remarkable character of HBc-based nanocages (HBc NCs) is that it can accommodate a large amount of foreign peptides meanwhile self-assemble into corrected folding in Escherichia coli expression system, a unique feature not shared by other nanocages. Herein we designed a HBc NCs-based vaccine displaying the OVA254-267 peptide with a high-density on the surface of the nanocages (i.e. OVA-HBc NCs), and evaluated its efficacy to induce anti-tumor immune responses (Scheme 1). The model antigen, OVA254-267 peptide (SIINFEKL), is biochemically well-defined and can be recognized in the context of MHC-I H2-Kb allele by the T cells derived from OT-I TCR transgenic mice. This model system had been extensively used to study antigen-specific cytotoxic T cell response, thus faciliating comparing our results to others.22 We measured various aspects of the T cell responses induced by OVA-HBc NCs both in vitro and in vivo, and then evaluated the immunotherapy efficacy of OVA-HBc NCs in a murine melanoma model. We found that OVA-HBc NCs can induce a potent anti-tumor immune response either used alone or jointly with low dose chemotherapy.


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Given that peptide-based vaccines have had only limited success in certain clinical trials presumably due to the immunosuppression mechanism in tumors, we wondered whether HBc NCs could be used as a surrogate antigen nanocarrier to trigger cytotoxic T cell response in tumors. For this purpose, we genetically inserted OVA257-264 (SIINFEKL) peptide together with flanking linkers into the surface-exposed major immunogenic loop region of HBc-183 protein and generated OVA-HBc chimeric protein. (Figure 1A). Thereafter we performed Monte Carlo ( MC ) simulations to mimic the 3D structure of OVA-HBc chimeric protein formed nanocages, OVA-HBc NCs. We particularly focused on the surface-exposed major immunodominant loop region and explored the conformational structure of the inserted OVA257-264 peptide. The computer simulation showed that the inserted OVA257-264 peptide motif, like a spike, could be exposed to the outer surface of the nanocage with a high density. Meanwhile the inserted glycine–serine-rich linkers could reduce the interference of other amino acid residues on the OVA257-264 epitope (Figure 1B). To experimentally characterize OVA-HBc NCs, we expessed OVA-HBc protein in E. coli and purified the resultant OVAHBc NCs by gel filtration chromatography. We determined the molecular weight of OVAHBc monomer by MALDI-TOF mass spectrometry and SDS-PAGE analyses. While the SDS-PAGE showed a single band around 25 kDa (Figure S1), the accurate molecular weight was found to be 24.53 kDa by MALDI-TOF, well matched to the protein sequence (Figure S2). Morphologically, OVA-HBc NCs showed a monodispersed spherical shape by transmission electron microscopy (TEM) observation (Figure 1C). The hydrodynamic diameter of OVA-HBc NCs appeared to be 34.3 ± 1.6 nm by Dynamic Light Scattering (DLS) analysis (Figure 1D). Thus introduction of OVA257-264 peptide into the major immunodominant loop region of HBc seems neither inhibit OVA-HBc expression nor interfere with OVA-HBc self-assembly into nanocages. The Zeta potential of OVA-HBc NCs was -20.8 ± 5.2 mV (Figure S3) which was usually stable in suspension, as the surface charge prevents aggregation of the particles. To test the stability of the OVA-HBc NCs under 5 ACS Paragon Plus Environment

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physiological conditions, a size distribution experiment was conducted in DMEM medium. As shown in Figure S4, the distribution of OVA-HBc NCs was stable in serum-free DMEM medium for 48 h. We also evaluated the reactivity of OVA-HBc to mouse anti-HBc monoclonal antibody, which reportedly bind the major immunodominant loop region of HBc-183 protein. As shown in Figure 1E whereas anti-HBc antibody could effectively bind to HBc-183 protein, it does not bind OVA-HBc chimeric protein, indicating that the insertion of OVA257-264 could abrogate the risk of pre-existed anti-HBc antibody neutralizing OVA-HBc formed nanocages if applied to patients with chronic hepatitis B. Dendritic cells (DCs) play an essential role in inducing adaptive immune responses.23 In the scenario of T cell-mediated cancer immunity, DCs can capture tumor-associated antigens and migrate to the draining lymph nodes where they can prime and activate T cells.24 As illustrated in Figure S5, OVA-HBc NCs could be effectively phagocytosed by DC 2.4 cells and bone marrow-derived DCs (BMDCs) in a time-dependent manner. To determine whether OVA-HBc NCs can induce the maturation of DCs, we incubated OVA257-264 peptide or OVAHBc NCs with immature BMDCs respectively and examined typical DCs maturation markers (e.g. CD80, CD83 and CD86) by flow cytometry. The results showed that OVA-HBc NCstreated DCs indeed increased the expression of those maturation markers compared to OVA257-264 peptide-treated BMDCs (Figure S6). To trace the transportation of OVA-HBc NCs in vivo, we subcutaneously injected C57BL/6 mice with Cy5.5-labelled OVA257-264 peptide or OVA-HBc NCs respectively, and imaged the mice by in vivo fluorescent imaging system. As shown in Figure 1F and 1G, compared with OVA257-264 peptide, OVA-HBc NCs were more efficiently accumulated in the peripheral lymph nodes 24 hours after injection. This ﬁnding implied that the enhanced accumulation of


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OVA-HBc NCs in the lymph nodes could consequently facilitate antigen presentation and T cell activation. Cytotoxic CD8+ T cells are known to mediate anti-tumor immunity by directly lysing tumor cells and releasing cytokines.25 To examine whether OVA-HBc NCs influence the function of antigen-specific CD8+ T cells in vivo, we immunized C57BL/6 mice with OVA-HBc NCs and then determined the antigen specific killing activity of OVA-specific CD8+ T cell. For this purpose we did in vivo cytotoxic T lymphocyte (CTL) killing assay as outlined (Figure S7). The results showed that OVA-HBc NCs could induce the strongest specific target cell killing (62.6 ± 6.4%), compared to that induced by OVA257-264 peptide (18.9 ± 2.8%) or PBS (5.3 ± 4.7%) (Figure 2A). Namely, OVA-HBc NCs administration can elicit approximately 3.3 times more killing of target cells than OVA257-264 peptide administration. Next, we sought to analyze the number of antigen specific CD8+ T cells in the peripheral blood by OVA-tetramer staining. The results showed that the number of OVA-tetramer positive CD8+ T cells in the periferal blood was increased by 2.8-fold in the OVA-HBc NCs group compared with OVA257-264 peptide group (Figure 2B), indicating that OVA-HBc NCs can much stronger stimulate T cells to clonal expansion than OVA257-264 peptide. To further characterize the dynamics of OVA-HBc NCs induced T cell proliferation in depth, we conducted an in vivo T cell proliferation assay by using CFSE label. C57BL/6 mice were injected intravenously with CFSE-labeled OT-I T cells and then immunized with OVAHBc NCs or OVA257-264 peptide subcutaneously 1 day later. The proliferation dynamics of OT-I T cells was measured by CFSE dilution by flow cytometry 3.5 days post immunization. We observed a more robust proliferation of OT-I CD8+ T cell in response to OVA-HBc NCs. Numerically, OT-I CD8+ T cells showed a 5.9-fold higher proliferation in OVA-HBc NCs group than that in OVA257-264 peptide group (Figure 2C). Cytokine production is a major mechanism for CD8+ T cells to exert their effector function. Therefore we measured cytokine 7 ACS Paragon Plus Environment

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production of OT-I CD8+ T cells isolated from the peripheral lymph nodes. The result showed that the proportion of IFN-γ-secreted OT-I CD8+ T cells is 13-fold higher in OVA-HBc NCs group than that in OVA-peptide group (Figure 2D). Thus the increased proliferation and IFNγ production of CD8+ T cell altogether demonstrated that OVA-HBc NCs is a very potent stimulator of cytotoxic T cells. To assess the potential of using OVA-HBc NCs as cancer vaccine carriers, we adopted a prophylactic vaccination model. We chose the well-studied B16 melanoma model and examined whether the OVA-HBc NCs can induce anti-tumor immunity in this setting. As the protocol outlined in Figure 3A, we first immunized naïve C57BL/6 mice with OVA-HBc NCs, OVA257-264 peptide, or PBS respectively, then boosted with the same antigen on day 7 and day 14. The immunzied mice were subsequently s.c. inoculated with 1 × 106 B16-OVA-Luc tumor cells 3 weeks post the initial immunization. The result showed that immunization with OVAHBc NCs can significantly delay and suppress tumor growth (Figure 3B, C). Accordingly, the size of the tumor was much smaller in OVA-HBc NCs group (132.4 ± 62.7 mm3) than that in OVA257-264 peptide group (1705.6 ± 1066.3 mm3) (p < 0.05). To further explore the potential correlation between the improved survival and OVA-HBc NCs induced CD8+ T cell mediated anti-tumor immunity, we quantified OVA-specific CD8+ T cells in vivo by OVA tetramer staining. The results showed that there were much more OVA-specific CD8+ T cell cells (22.8 ± 3.3%) in OVA-HBc NCs group compared to OVA257-264 peptide group (5.5 ± 0.4%) (Figure 3D, E). More importantly, the survival rate of mice treated with the OVA-HBc NCs was higher than those of other groups. Whereas all mice in the PBS group (7/7) and OVA257-264 peptide group (6/6) died by day 33 and 37 respectively, only 3 mice died in the OVA-HBc NCs group (3/6) in this 40 day period of observation (Figure 3F, G). Although in our vaccination model OVA-HBc NCs immunization could effectively prevent the occurrence or progression of primary melanoma, in clinical settings many patients are 8 ACS Paragon Plus Environment

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indeed dignosed when the tumor had already metastasized to other tissues and organs. For such advanced progressive tumors, combining immunotherapy with other agents, such as chemotherapy, may hold greater promise for the patients. We thus investigated the possibility of OVA-HBc NCs in conjuction with low dose paclitaxel (PTX) (equivalent to one-fifth of the regular dose PTX) to treat advanced metastatic cancer.26 To test this, we adopted an experimental metastasis model by intravenously injecting B16-OVA-Luc melanoma cells into the mice (Figure 4A). After lung metastases were established, we treated mice with OVAHBc NCs and PTX on day 4, 10, and 16 respectively. The development of lung metastatic nodule was monitored by bioluminescence imaging (Figure 4B). Whereas either OVA-HBc NCs or PTX single treatment can reduce the lung metastasis to some extent, combining OVAHBc NCs and PTX can further significantly reduce the tumor burden of mice (Figure 4B, C). We also analyzed CD31 expression on those lung tissue sections as a surrogate measurement for lung metastasis. CD31, a specific and sensitive marker of endothelial cells, is widely used to identify tumor angiogenesis in histological tissue sections. Our results showed that in the metastasis sites CD31 expression level was signifcantly reduced in OVA-HBc NCs and PTX combination group (Figure 4D). To understand the mechanisms of how OVA-HBc NCs and PTX act together to enhance the anti-tumor immune response, we analyzed T cell compartment in this scenario in detail. The results showed that the proportion of OVA-specific CD8+ T cells are further increased in the lungs of mice under combination therapy (OVA-HBc NCs + PTX) compared to OVAHBc NCs or PTX singly treated mice (Figure 5A, D). Next we examined the status of regulatory T cells (Tregs) in these mice. Tregs are considered to play an inhibitory role in anti-tumor immunity by suppressing CTL response via multiple cellular and molecular mechanisms. We found that the proportion of Foxp3+ Tregs decreased in PTX alone or OVAHBc NCs/PTX treated groups (Figure 5B, E). As a result, the CD8+/Tregs ratio was significantly higher in OVA-HBc NCs/PTX group compared with PBS group (Figure 5F). 9 ACS Paragon Plus Environment

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These results suggested that low dose PTX can beneficially modulate tumor microenvironment through reducing regulatory T cells and expanding CD8+/Treg ratio. We also examined the memory CD8+ T cell subsets in our combination therapy model. Memory T cells is critical for long term T cell mediated protection against the same pathogen upon future infections.27, 28 Memory CD8+ T cells can be phenotypically grossly subdivided, based on their CD62L and CD44 expression pattern, into central memory (TCM, CD62L+CD44+) and effector memory (TEM, CD62L-CD44+) subsets.29,

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ahthough both TCM and TEM can contribute to antitumor immunity, TCM has been shown to be more superior than TEM in mediating protective immunity.28 We found that whereas the fraction of TCM cells (CD62L+CD44+) was increased to a similar level in the PTX group and OVA-HBc NCs group, the fraction of TCM cells in the OVA-HBc NCs/PTX combination group was further increased and much larger than that in singly treated group (Figure 5C, G). To evaluate whether OVA-HBc NCs single or combined usage have any side effect, we also examined multiple organs by hematoxylin and eosin (H&E) staining, and found no sign of obvious lesions in the organs examined (Figure S8). Overall our findings indicated that treatment with OVA-HBc NCs and PTX combination could effectively elicite antitumor immunity by modulating the T cell compartent. We finally carried out the therapeutic vaccination model and found that combining OVAHBc NCs with PTX could further inhibit the progression of tumor cells compared to either OVA-HBc NCs or PTX alone (Figure S9). Together, these results demonstrated the potent synergic effect of OVA-HBc NCs and PTX when used together for tumor prevention and treatment.

Vaccines for tumor immunotherapy have shown great promise, but clinical applications are hampered by the complexity of large-scale manufacturing, quality control and safety.31 Exampled by OVA-HBc NCs in this study, we showed that HBc NCs is a very promising 10 ACS Paragon Plus Environment

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tumor vaccine carrier for several reasons. First, OVA-HBc NCs can be readily manufactured in large quantities meanwhile maintaining their original properties. Second, OVA-HBc NCs with approximate 40-nm spherical size can be more efficiently recognized and endocytosed by DCs, facilitating tumor-antigen presentation to T cells.32, 33 In our study, model antigen (OVA257-264) inserted into the major immunogenic loop region of HBc protein could be clustered and displayed on the surface of formed nanocages via HBc self-assembly. Functional studies showed that, compared to soluble OVA-peptide, OVA-HBc NCs can induce stronger CD8+ T cell responses. This result suggested that nanocarrier can effectively enhance the immunogenicity of candidate antigen by adapting a native virion like morphology, suited for displaying high density repetitive array of epitopes in a limited space. Emerging data indicated that neoantigen derived from tumor play a central role in the initiation of anti-tumor immunity. Recent technological advances have made the rapid identification of neoantigens derived from tumor-specific mutations possible. As demonstrated by OVA-HBc NCs in this study, HBc NCs based carrier system would be an ideal cargo delievery platform to accomodate antigenic peptide drevied from those neoantigen and used as personalized vaccines to patients in time. How to rationally combine different treatments to achieve the best therapeutic effect for patients with different tumor burden, type and different immune status remains as a challenge need to be addressed urgently. We found that coupling chemotherapeutic drug PTX at low dose with OVA-HBc NCs treatment could further enhance the antitumor immune response induced by the latter. This benefit was evidenced in vivo through reducing regulatory T cells and expanding CD8+ T cells with a functional phenotype of central memory cells. Although standard-dose chemotherapy can destroy cancer cells, their side effects can be intolerable and difficult to control in some cases. Our results showed that combining non-toxic OVA-HBc NCs with low dose PTX can not only alleivate the side effect of conventional chemotherapy,


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lower the risk of developing drug resistance, but also induce a better immunotherapy outcomes. In conclusion, we presented here the design of the HBc NCs-based nanocages used as a tumor vaccine carrier for cancer immunotherapy. OVA257-264 peptide was introduced to the major immunogenic loop region of HBc protein to yield a monodispersed well-defined OVAHBc NCs. Consequently, when administered in vivo OVA-HBc NCs could accumulate in the draining lymph nodes, effectively induce DC maturation and subsequent antigen presentation to naive CD8+ T cells, resulting in robust OVA-specific CD8+ T cells proliferation and IFN-γ production. Moreover OVA-HBc NCs immunization can effectively induce antigen specific CTL responses, resulting in targeted killing of cancer cells. In B16-OVA melanoma mouse models, OVA-HBc NCs immunization enhanced antitumor immunity and resulted in a significant delay in tumor growth and prolongs mouse survival. Finally, when given in combination with chemotherapy, such as low dose PTX, the tumor progression and metastasis was further inhibited compared to the single usage of either PTX or OVA-HBc NCs. It is worth noting that the combination of OVA-HBc NCs and low dose PTX was well-tolerated with no significant cytotoxic side effects. Thus exampled by OVA-HBc NCs in this study, HBc NC is a promising nanovaccine vector for cancer immunotherapy.


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Scheme 1. Schematic of OVA-HBc nanocage-mediated antitumor immunity in combination cancer immunotherapy.


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Figure 1. (A) Schematic representation of OVA-HBc genetic fusion construct. (B) 3Dreconstruction of OVA-HBc NCs where the major immunogenic loop region (MIR) is simulated. The HBc core protein region is shown in green or yellow (PDB ID: 1QGT); OVA257-264 and linkers are shown in red and pink, respectively. Dimer of OVA-HBc (Left); Self-assembled OVA-HBc nanocage (right). (C) TEM image of OVA-HBc NCs. (D) DLS measurement of OVA-HBc NCs. (E) Western blot analysis of HBc-183 and OVA-HBc proteins with antibody against HBc, respectively. (F) Near infrared imaging of Cy5.5-labeled OVA257-264 peptide and OVA-HBc NCs accumulation in lymphoid organs and (G) the ex vivo images of major lymph nodes of mice sacrificed at 24 h after subcutaneous injection at the tail base of C57BL/6 mice, respectively.


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Figure 2. Antigen specific T cell response induced by OVA-HBc NCs. (A)-(B) C57BL/6 mice (n = 3) were injected s.c. with OVA-HBc, OVA257-264 peptide, and PBS respetively on day 0, 7, and 14, followed by analysis on day 21. (A) Representative flow cytometry histograms of OVA-specific CD8+ T cell mediated target cell killing in vivo (Left). Quantitative comparison of OVA-specific CTL responses induced by OVA-HBc, OVA257-264 peptide, and PBS (Right). (B) Representative flow cytometry dot plots of H-2kb/SIINFEKL tetratmer staining of CD8+ T cells in the blood (Left) and percentage of OVA (SIINFEKL)specific CD8+ T cells summarized (Right). (C)-(D) C57BL/6 mice (n = 3) were adoptively transferred with CFSE-labeled OT-I cells, followed by s.c. injection of OVA-HBc NCs, OVA257-264 peptide, and PBS the next day. (C) Representative flow cytometry histograms of 15 ACS Paragon Plus Environment

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OVA-specific CD8+ T cells proliferations (Left) and quantitative comparison of OVAspecific CD8+ T cells proliferation (Right). (D) Representative flow cytometry dot plots of intracellular IFN-γ staining of OVA-specific CD8+ T cells in lymph nodes (Left) and percentage of OVA (SIINFEKL)-specific CD8+ IFN-γ+ T cells in comparison (Right). (*p < 0.05, **p < 0.01, ***p < 0.001).


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Figure 3. Induction of antitumor immunity by OVA-HBc NCs. (A) Schematic timeline of immunisation protocol. (B) Tumor growth curve of mice bearing B16-OVA-Luc tumor (n = 5). (C) Bioluminescence image of B16-OVA-Luc tumor-bearing mice on day 28 (n = 5). (D) Representative flow cytometry dot plots of OVA-tetramer staining of CD8+ T cells in spleens. (E) Percentage of OVA (SIINFEKL)-specific CD8+ IFN-γ+ T cells in comparison. (F) Schematic timeline of vaccination immunisation protocol. (G) Survival curve of mice after treatment with OVA-HBc NCs (n = 6), OVA257-264 peptide (n = 6), and PBS (n = 7), respectively. 17 ACS Paragon Plus Environment

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Figure 4. OVA-HBc NCs and low-dose PTX combined treatment in metastatic melanoma model. (A) Schematic outline of experimental protocol. (B) Bioluminescence images of metastatic B16-OVA-Luc tumor-bearing mice on day 15, 18, and 21 (n = 4). (C) Quantification of lung matastasis nodules (n = 5, p < 0.05). (D) The images of isolated metastatic lung, and anti-CD31 antibody stained tissue sections by immunohistochemistry.


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Figure 5. PTX enhance CTL response induced by OVA-HBc NCs. Induction of OVAspecific CD8+ T cell responses in TILs derived from the lungs of melanoma metastatic mice. (A) Representative dot plots for OVA-tetramer staining and (D) cumulative data for OVAspecific CD8+ T cells are shown. (n = 3, *p < 0.05, **p < 0.01). (B) Representative dot plots for Foxp3 staining in the spleen and (E) cumulative data for Foxp3+ CD4+ T cells are shown. (C) Representative flow cytometry results and (G) percentage of effector memory T cells 19 ACS Paragon Plus Environment

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(Tem, CD62L− CD44+), and central memory T cells (Tcm, CD62L+ CD44+) in spleen. (n = 3). (F) The CD8+/Treg ratio are shown. (n = 3, *p < 0.05).

AUTHOR INFORMATION Corresponding authors *(L.R.) E-mail: [email protected]. Tel: +86-592-2188530. *(G.F.) E-mail: [email protected]. Tel: +86-592-2880353. Author Contributions W.S. and H.Z. contributed equally. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by National Natural Science Foundation of China (grants U1505228 and 31870994 to L.R., 81571764 to L.S., 31770952, 31570911 and 2017ZX10202203-003001 to G.F.). We thank Wenzhu Fan, Guangxi Wu and Yazhen Hong for technical help.. Supporting Information The Supportig Information is available free of charge on ACS Publications website at DOI: 10.1021/acs.nanolett.xxxxxxx. Experimental Section, Figure S1-S9 (PDF)

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Bioengineered Nanocage from HBc Protein for Combination Cancer

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