Melittin-lipid nanoparticles target to lymph nodes and elicit a systemic anti-tumor immune response

Targeted delivery of a nanovaccine loaded with a tumor antigen and adjuvant to the lymph nodes (LNs) is an attractive approach for improving cancer immunotherapy outcomes. However, the application of this technique is restricted by the paucity of suitable tumor-associated antigens (TAAs) and the sophisticated technology required to identify tumor neoantigens. Here, we demonstrate that a self-assembling melittin-lipid nanoparticle (α-melittin-NP) that is not loaded with extra tumor antigens promotes whole tumor antigen release in situ and results in the activation of antigen-presenting cells (APCs) in LNs. Compared with free melittin, α-melittin-NPs markedly enhance LN accumulation and activation of APCs, leading to a 3.6-fold increase in antigen-specific CD8+ T cell responses. Furthermore, in a bilateral flank B16F10 tumor model, primary and distant tumor growth are significantly inhibited by α-melittin-NPs, with an inhibition rate of 95% and 92%, respectively. Thus, α-melittin-NPs induce a systemic anti-tumor response serving as an effective LN-targeted whole-cell nanovaccine.

antigens are inconvenient. It can be useful to mention the ease/cost of melittin synthesis or purification in the discussion section as a comparison.
-Since melittin is a major component of bee venom, and some individuals are allergic to bee venom, will this be a consideration when stratifying patients if this strategy is to be translated to the clinic? The authors should address the issue of melittin as a general allergen. - Fig. S5a is difficult to interpret, as the fluorescence intensity of the "fluorescent model antigens" look similar in the PBS control image compared with that of the α-melittin-NP. A clarification is needed.
-The statement in line 192-193 regarding the idea that the melittin "induces release of whole tumor cell antigens" has not yet been fully demonstrated at that point in the paper. This can be moved to later in the manuscript, after the appropriate results have been shown. Minor comments -The figure numbers are not sequential in a few cases. For instance, Fig. 1d and 1e are discussed prior to 1b and 1c.
-The cancer type used for in vivo studies can be mentioned in the abstract and earlier upon introduction of the cell line name, clarifying that it is melanoma.
-The middle panels of Fig. 1a were not mentioned in the main text. -In Fig. 1b, there is no indication that these agents are FITC-conjugated.
-The MTS assay results in Fig. 2 c, d can be described clearly by the IC50 values.
-A few of the word choices can be improved (i.e. "fantastic" in line 55, "just like killing two birds with one stone" in line 116).

Reviewer #3 (Remarks to the Author): Expert in melittin and nanoparticles
These authors are investigating the ability of peptide-lipid nanoparticles to enhance the presentation of tumor antigens. This particular type of nanoparticle has been available for some time and is known to be of small size (10-100 nm) which is desirable to provide good tissue access. This manuscript presents the preparation only briefly and does not address the particle composition and structure over the size range that was used. In the first experiments presented fluorescein labeled melittin and NPs were used to demonstrate that formulation into nanoparticles was critical to permit transit to lymph nodes. However what is not presented is the comparative activity of the FITC substituted nanoparticles. This could have been accomplished using the effect on contra lateral tumors but that was not tested with the FITC nanoparticles. The authors show that cultured tumor cells are more sensitive to αmelittin NPs than isolated cultured antibody presenting cells. It is not clear to this reviewer that this experiment contributes to the authors hypothesis. There is no development of the basis for this difference. This cultured cell protocol could be used to determine if the FITC labeled NPs are as active as the underivatized probes that were used. The significance of the tfRFP expressing cells is not clear in that if apoptosis is the mechanism of cultured cell demise the expressed antigen would be lost in the small vesicles formed during apoptosis. Furthermore the authors use TUNEL assays to indicate that apoptosis is prominent in cultured cells exposed to the melittin protocols. The TUNEL assay can be applied to tissue, cultured cells and in flow cytometry. Data for the initial tumor, draining lymph nodes, distant tumor and induced lymphocytes would speak directly to the mechanism of the "one stone -two birds" hypothesis. The major data in this manuscript is a series of bilateral flank tumor experiments that are intended to verify "the whole cell vaccine" importance to melittins action on cancer cells. The results in this manuscript are striking

Point-by-point responses to the reviewers' comments
We would like to express our sincere thanks to the editor and all reviewers for their critical and constructive comments. We have performed substantial additional experiments to respond point-by-point to their concerns. We feel that their comments have helped us to significantly improve and strengthen the manuscript, as well as clarify some of the important issues of our work. We hope that the revision has addressed their major concerns. The revised sections are marked in red, and the data that were not added to the manuscript and are presented only below in the response to the reviewers' concerns are labelled with Figs. R1-4. This work is of high importance and should be published, but requires significant clarifications and discussion of the issues in the major comments before publication.

Response:
We thank the reviewer for their constructive comments. In the original manuscript, the percent values in the text represent the average of several samples, and the values in the gates in Fig. 1f indicate the percentage of the representative sample. According to the reviewer's suggestion, the values in the gates in Fig. 1f

Response:
We thank the reviewer for their detailed comments. The bilateral flank tumor model is often used to characterize local and abscopal effects of intratumoral therapy, and the time interval for inoculating two tumors is not standard. According to the corresponding references, tumor cells could be injected in the right flank (contralateral tumor) on days 0 (simultaneously) 1 , 2 2,3 , 3 4 , 4 5-7 , and 6 8

Response:
We thank the reviewer for their constructive suggestion. Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has been extensively used to label siRNA, peptides and proteins for analyzing distribution 9-12 . In our original manuscript (Supplementary Fig. 7), FITC were extracted from organs by ultrasonic extraction with methanol and analyzed by a microplate reader. Essentially, the FITC content was quantified with fluorescence (excitation: 495 nm; emission: 525 nm). Our data showed that organs seemed to retain an undetectable level of FITC, especially in the liver, kidney and heart. Organs emitted autofluorescence signals because of the presence of endogenous fluorochromes, such as nicotinamide adenine dinucleotide (NADH), riboflavin, aromatic amino acids, etc. 13 . Riboflavin is the most likely fluorophore inducing an emission signal in the range of 520-540 nm and is rich in the liver, kidney and heart 14,15 . We speculate that the organ autofluorescence interferes with the detection of FITC. However, by subtracting the organ autofluorescence signal, we still detected tiny amounts of FITC in the organs ( Supplementary Fig. 11). Therefore, we concluded that a very small proportion of FITC-α-melittin-NPs entered the bloodstream. As discussed in the legend in  In addition, the ratio of FITC to peptide was determined by spectrophotometric analysis and calculated using the following formula according to the manufacturer's instructions: where MW is the molecular weight of the peptide, 389 is the molecular weight of

Response:
We thank the reviewer for their constructive suggestion. In the revised manuscript, we have added a discussion of the incomplete response.
We modified the manuscript as follows: Page 15, line 411 of the manuscript (Discussion) now states the following: "However, the incomplete response occurred in both α-melittin-NP and melittin groups. Tumor progression involves the co-evolution of neoplastic cells together with tumor microenvironment, and heterologous cell types within tumors can actively influence the therapeutic response and shape resistance, even in cases in which immune cell actively drive the initial response to targeted therapies 16

Response:
We thank the reviewer for their very constructive suggestion. In the revised manuscript, we have added background information on tumor-associated antigens in melanoma and a discussion about the translatability potential of this strategy.
We modified the manuscript as follows:

Response:
We thank the reviewer for their constructive suggestion. In the revised manuscript, we have added a discussion about the ease/cost of melittin-lipid nanoparticle synthesis and purification.
We modified the manuscript as follows:

Response:
We thank the reviewer for their detailed suggestion. Although melittin is a major component of bee venom, it is a relatively weak allergen 30 . It was reported that the best characterized allergen of bee venom is phospholipase A2 [31][32][33] . In addition, the prevalence of systemic allergic reactions to Hymenoptera stings ranges from 0.3 to 7.5% in adults and up to 3.4% in children 34 . If allergic reactions occur, the effective treatment to prevent further systemic sting reaction is venom immunotherapy (VIT).
The European Academy of Allergy and Clinical Immunology's (EAACI) Taskforce prepared a detailed guideline on VIT. According to the formal systematic review and meta-analysis of a large sample, VIT proved to be highly effective and safe, and no fatalities were recorded 34 .
8. Fig. S5a is difficult to interpret, as the fluorescence intensity of the "fluorescent model antigens" look similar in the PBS control image compared with that of the α-melittin-NP. A clarification is needed.

Response:
We thank the reviewer for their constructive comments. In the original Supplementary

Response：
We thank the reviewer for their constructive suggestions. As shown above ( Supplementary Fig. 9), melittin itself has the ability to kill B16F10 tumor cells and induce the release of whole tumor cell antigens. When melittin was loaded onto the scaffold of α-peptide-NP, it could form an ultrasmall melittin-lipid nanoparticle, named as α-melittin-NP. This α-melittin-NP maintained the ability of melittin to directly induce tumor cell necrosis/apoptosis. More importantly, the α-melittin-NP has the required size for an optimal LN-targeted nanovaccine that can efficiently drain into lymphatic capillaries and lymph nodes.
Minor comments: 1. The figure numbers are not sequential in a few cases. For instance, Fig. 1d and 1e are discussed prior to 1b and 1c.

Response:
We thank the reviewer for their constructive suggestions. According to the reviewer's suggestions, we have described Fig. 1b and 1c earlier in the revised manuscript.

Response:
We thank the reviewer for their constructive suggestions. According to the reviewer's suggestions, we mentioned the cancer type earlier in the revised manuscript.
We modified the manuscript as follows: Page 2, line 30 of the manuscript (Abstract) now states the following: "Time-lapse imaging showed that α-melittin-NPs displayed low toxicity to APCs but maintained the toxicity of melittin to B16F10 melanoma cells".
3. The middle panels of Fig. 1a were not mentioned in the main text.

Response:
We thank the reviewer for their critical comments. In our revised manuscript, we have mentioned the middle panels of Fig. 1a.
We modified the manuscript as follows: Page 5, line 130 of the manuscript (Results: α-melittin-NPs enhance the LN uptake of melittin and activate APCs) now states the following: "Wide-field fluorescence imaging data showed that α-melittin-NPs as well as the α-peptide-NPs scaffold led to their substantial accumulation in inguinal LNs (ILNs) and axillary LNs (ALNs) (Fig.   1a, lower and middle panels)". Fig. 2 c, d can be described clearly by the IC50 values.

Response:
We thank the reviewer for their constructive suggestions. According to the reviewer's suggestions, we have described IC50 values in the revised manuscript.
We modified the manuscript as follows:

A few of the word choices can be improved (i.e. "fantastic" in line 55, "just like
killing two birds with one stone" in line 116).

Response:
We thank the reviewer for their constructive suggestions. According to the reviewer's suggestions, the word "fantastic" has been changed to "great" in the revised manuscript. In addition, "killing two birds with one stone" is an old proverb meaning that we can achieve two things in a single action, and has been used in research papers [35][36][37][38] .

Comments to authors:
This work is carefully planned and strongly suggestive but incomplete in at least three ways. This limits the potential for mechanistic interpretations that can be supported.
This is reflected in their discussion which re-presents the phenomena reported in the Results.

Response：
We thank the reviewer for their detailed comments. According to the reviewer's suggestions, we provided additional detailed information about the preparation of nanoparticles in the method sections. In addition, we compared the basic characteristics (such as surface charge, particle size, and cytotoxic activity) of fluorescently labelled or unlabelled α-melittin-NPs in Fig. R2. Apolipoprotein A1 (ApoA1) is a main protein component in high-density lipoprotein (HDL) particles. In spherical HDL particles, the amphipathic α-helix in ApoA1 can interact with phospholipids and cover ~80% of the total surface, underlining the major role of this apolipoprotein in stabilizing HDL particle structure and shape 39,40 . Therefore, the ApoA1-packing density increase indicates a reduction in the spherical particle size 41 .

The melittin nanoparticles (α-melittin-NPs) consisted of phospholipid [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)], cholesterol oleate (CO), and
α-melittin, among which α-melittin was designed by hybridizing melittin and an ApoA1-mimetic peptide (D4F, noted as α-peptide). More importantly, α-melittin displayed a strong α-helical configuration. As shown in our previous study, the interaction between the α-melittin network and the lipid monolayer resulted in nanoparticles that appeared spherical in shape and possessed a small particle size (10~20 nm) 42 . Therefore, α-melittin is a crucial factor in precisely controlling the structure and size of the α-melittin-NPs. In the methods section, we introduced the preparation of the nanoparticles as well as the function of the α-melittin peptide in detail.
Dynamic light scattering (DLS) data indicated that FITC-α-melittin-NPs (13.6 ± 0.72 nm) had a slightly smaller size than unlabelled α-melittin-NPs (15.41 ± 0.6 nm) but both were smaller than 100 nm (Fig. R2b). This result means that both have the required sizes to efficiently drain into lymphatic capillaries and lymph nodes. In addition, the zeta potential also decreased after FITC labelling (Fig. R2c). However, despite that, both formulations had similar killing effects on B16F10 cells (Fig. R2d).
In our previous study, we found that melittin was buried in the phospholipid monolayer, which avoids the impact of FITC on melittin. It is worth noting that seven C-terminal residues (DWFKAFY) of α-peptide (ApoA1-mimetic peptide in NPs) are not involved in lipid-binding status and are exposed outside the particle 43 , and the lysine (primary amine) in the seven residues is most likely the binding site of FITC.
Not surprisingly, therefore, the conjugation of FITC did not affect the cytotoxic activity of melittin. Above all, FITC labelling has no remarkable influence on the size and cytotoxicity of α-melittin-NPs except the zeta potential of intact NPs.
We modified the manuscript as follows:  indicate that apoptosis is prominent in cultured cells exposed to the melittin protocols.
The TUNEL assay can be applied to tissue, cultured cells and in flow cytometry. Data for the initial tumor, draining lymph nodes, distant tumor and induced lymphocytes would speak directly to the mechanism of the "one stone -two birds" hypothesis.

Response:
We thank the reviewer for their detailed comments. According to the reviewer's suggestions, we added new data in Fig. 2d, e and Supplementary Fig. 6-8.
We modified the manuscript as follows: Page 7, line 177 of manuscript (Results: α-melittin-NPs have more cytotoxic effects on tumor cells than on APCs) now states the following: "Because negatively charged phosphatidylserine and O-glycosylated mucins are overexpressed in the plasma membrane of many cancer cells, thereby causing these membranes to carry a slightly higher net negative charge than those of normal eukaryotic cells 44 , we speculated that the reason for the differential killing effects of α-melittin-NPs is probably related to membrane potential-mediated cellular binding. To further confirm this hypothesis, the cell membrane potential was measured using a Malvern Zetasizer Nano ZS90 instrument. The results showed that the zeta potential of the B16F10 cell membrane (-27.65 ± 0.93 mV) was more negative than that of BMDCs (-14.64 ± 1.87 mV) and BMDMs (-10.78 ± 1.57 mV) (Supplementary Fig. 6). Next, we compared the cellular-binding ability of α-melittin-NPs to these three different cells using flow cytometry. The results revealed that the B16F10 cells captured a dramatically greater amount of FITC-α-melittin-NP than BMDMs at various concentrations during 1 h and 3 h incubation ( Fig. 2d and Supplementary Fig. 7). We also noted that there was no difference in the mean fluorescent intensity (MFI) of FITC between B16F10 cells and BMDCs during 1 h incubation (except 1.25 μM) (Supplementary Fig. 7), but as the incubation time was prolonged to 3 h and the concentration increased, B16F10 cells also displayed a significantly higher MFI value than BMDCs (Fig. 2d).
To observe the cellular distribution of FITC-α-melittin-NPs in these three different cells in vitro, BMDCs and BMDMs were isolated from mT/mG mice that express a strong red fluorescence protein (tdTomato) in the membrane systems (plasma membrane, lysosome, etc) of all cell types. Confocal imaging data showed that FITC-α-melittin-NPs displayed remarkably stronger fluorescent intensity in B16F10 cells than in BMDCs and BMDMs after incubation for 3 h in a 10 μM concentration (Fig. 2e).
More interestingly, the FITC-α-melittin-NPs were mainly distributed in the cell membrane of B16F10 cells, but they were distributed in the intracellular membranes of BMDCs and BMDMs. This finding means that FITC-α-melittin-NPs quickly move through endocytosis to the intracellular membranes after binding, making APCs resistant to the plasma membrane permeabilization-dependent necrosis." Page 13, line 368 of manuscript (Discussion) now states the following: "Although the lytic activity of melittin is mainly associated with its ability to disturb cell membrane integrity by incorporating into phospholipid bilayers 45,46 , nanoparticle-delivered melittin might elicit apoptosis after trafficking to intracellular membranes via activation of the intrinsic pathway 11 . Therefore, the different spatial distribution of α-melittin-NPs in B16F10 cells and APCs (BMDCs and BMDMs) seem to not explain simply the differential killing effects. A plausible possibility is the existence of a mechanism of anti-apoptosis. After all, necrosis is mainly an irreversible event, but apoptosis can be regulated. We also previously found that Birc5 (also known as survivin), a member of the inhibitor of apoptosis (IAP) gene family encoding negative regulatory proteins that prevent apoptotic cell death, was upregulated in liver sinusoidal endothelial cells (LSECs, another APC type in the liver) after the administration of α-melittin-NPs 47 . However, it is worth noting that α-melittin-NPs also exhibited cytotoxicity in APCs with the increase of melittin concentration." Page 8, line 208 of manuscript (Results: α-melittin-NPs have more cytotoxic effects on tumor cells than on APCs) now states the following: "In addition to zeta potential, cholesterol has been reported to have an inhibiting effect on the lytic activity of melittin to erythrocytes 48 . Next, we estimated the cholesterol content in the three different cells using an Amplex® Red reagent-based assay. However, the data showed that the cholesterol level in B16F10 cells was significantly higher than that in BMDCs and BMDMs (Supplementary Fig. 8). These results suggest that membrane potential-mediated cellular binding and subsequent spatial distribution are, at least in part, the main reasons for differential killing effects in a certain concentration range."

Supplementary Figure 6. Zeta potential measurement of B16F10 cells, BMDCs and BMDMs.
Data are shown as the mean ± SEM. ***P < 0.001 and ****P < 0.0001, as analyzed by one-way ANOVA with Bonferroni's post hoc test.  [redacted] [redacted] We modified the manuscript as follows:  Fig. 9). The immunofluorescence imaging of TUNEL staining indicated that TUNEL positive cells were also induced by melittin and α-melittin-NPs.
It is noticeable, however, that TUNEL-positive cells also displayed the disappearance of fluorescent model antigens (plasma membrane permeabilization

Response:
We thank the reviewer for their constructive suggestion. According to the reviewer's suggestion, we performed these experiments at different time points (14 days and 21 days), and we added new data in Fig. 6b-d and Supplementary Fig. 13 in the revised manuscript.

Cellular and humoral immune response:
As shown in Supplementary Fig. 13, α-melittin-NPs had no effect on the frequencies of cytokine + (TNF-α + and IFN-γ + ) T cells 14 days after left tumor implantation. In addition, there was also no difference in the percentage of IgG + cells between all the groups. The data at 21 days are shown in the original manuscript (Fig. 5). These results indicate that specific cellular immunity and humoral immunity did not occur in the early stages. Many studies also suggest that the time points to evaluate antigen-specific T cell responses and antibody responses should be selected at 21 days (or longer) after immunization or tumor implantation [56][57][58] .
We modified the manuscript as follows: Page 10, line 289 of manuscript (Results: α-melittin-NPs elicit tumor antigen-specific cellular and humoral immune response) now states the following: "We used flow cytometry to analyze the cytokine expression in T cells. The data showed that compared with PBS group, α-melittin-NPs induced increases in the frequencies of IFN-γ + CD8 + (12.2-fold) and IFN-γ + CD4 + (7.2-fold) T cells at day 21 after tumor implantations but no differences emerged at 14 days. (Fig. 5a-c and Supplementary   Fig. 13a, b)."

Lymphocyte infiltration:
Furthermore, we also analyzed lymphocyte infiltration in distant tumors at different time points (14 days and 21 days).
We modified the manuscript as follows: Page 11, line 311 of manuscript (Results: α-melittin-NPs induce lymphocyte infiltration and dramatic changes in the cytokine/chemokine milieu in the distant tumor) now states the following: "The flow cytometry data showed that, compared to the PBS, α-melittin-NPs induced an increase in the numbers of innate immune components, including natural killer (NK) cells, monocytes and neutrophils, but not the adaptive components, including CD4 + and CD8 + T cells at 14 days after left tumor implantation (Fig. 6b, c). However, at 21 days, the α-melittin-NP group exhibited a significant increase in the number of CD4 + (4.4-fold, p = 0.0074) and CD8 + T cells (3.7-fold, p = 0.00243) in addition to NK cells and monocytes. Immunofluorescence analysis of the distant tumors also revealed that CD4 + and CD8 + T cells were present at high density after α-melittin-NP treatment (Fig. 6d)." These results suggest that inflammatory infiltration induced by α-melittin-NPs was shifting from mainly innate immune cells in the early stage to mainly adaptive immune cells in the late stage. C57BL/6 mice (n = 4 per group) were treated as described above (Fig. 3a). On day 14, the lymphocytes isolated from the tumor-draining LNs were restimulated with DCs pulsed with B16F10 tumor lysates and were analyzed by flow cytometry with intracellular cytokine staining.
Cytokine + cell frequencies from each group are shown. (c) B16F10 tumor cells were incubated with 5% serum that was collected from treated mice and age-matched naïve mice. Subsequently, these cells were stained with a DyLight649-conjugated mouse IgG-specific secondary antibody and analyzed by flow cytometry. IgG + cell frequencies from each group are shown. Error bars indicate the SEM. Statistical analysis was performed with one-way ANOVA by Bonferroni's post hoc test. 3). n.s. not significant, *P < 0.05, **P < 0.01 and ***P < 0.001, as analyzed by one-way ANOVA with Bonferroni's post hoc test.

Minor comments：
1. In the Discussion the authors refer to work by Dezfuli but I did not find a reference to this work in the manuscript.

Response:
We thank the reviewer for their constructive suggestion. In the revised manuscript, we provided the exact author's name (Page 13, line 358).