M860, a Monoclonal Antibody against Human Lactoferrin, Enhances Tumoricidal Activity of Low Dosage Lactoferrin via Granzyme B Induction
Round 1
Reviewer 1 Report
Please find the attached file.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1 Comments
Previously, the authors analyzed an anti-LF monoclonal antibody (M860), which induced an amount of proinflammatory cytokines in human monocytes by LF-M860 immune complex formation (LF-IC). In this paper, authors investigated the effect of LF-IC on lysis of Jurkat and Raji cells via Granzyme B production.
Major comments
Point 1:What is the isotype (subclass) of M860? I think the M860 is a mouse monoclonal antibody. However, experiments were carried out using human monocytes. Authors should be confirmed that M860 interacts with Ig receptors in human monocytes.
Response 1:The isotype of M860 is mouse IgG1. As stated in ref.36, mIgG1 could interact with hFcgRIIa, which has been added to the P12, line 3-7.
Point 2:In Figure 1A and 1B, the specific lysis effect by LF-IC is less than 10%. Is it significant as an antitumoricidal activity?
Response 2:We have confirmed the anti-tumoricidal activity of LF-IC primed monocyte for more than ten times, although the specific lysis effect is around 10%. In my opinion, the limited lysis effect in vitromay be the result of the assay system. The lysis effect was determined by PI insertation which indicated the late phase apoptosis or necrosis, while the cells undergoing early apoptosis and total lysed cells were not included.
Point 3:In Figure 1A and 1B, the effects of mixing OVA-IC and LF should be observed.
Response 3:The mixing OVA-IC with LF control has been shown in Fig.1 in the revised MS. OVA-IC+LF did not show any anti-tumor effect. The synergisitic effect of FcgRIIa and CD14 is indispensible for empowerment for tumoricidal activity of monocytes by LF-IC. Although OVA-IC plus LF could activate signaling pathway of both FcgRIIa and CD14, they could not show synergisitic effect as well as LF-IC (P13, Lines 3-4).
Point 4: In Figure 4A, authors used mIgG1 and mIgG2b as the normal mouse IgG. I think that human normal IgG (IgG1, IgG2, IgG3) should be used for the experiment.
Response 4: mIgG1 and mIgG2b were used as the isotype control of anti-CD16,anti-CD32 and anti-CD64 in the blocking assay. The further application of LF-IC in human need optimization of the mouse IgG1 monoclonal antibody M860, such as humanization.
Point 5:Authors should show Western blotting data as the change (phosphorylation) of signal molecules (PI3K, AKT, mTOR) in the condition of Granzyme B induction.
Response 5: As suggested, the activation of syk, PI3K and AKT were determined by Western Blotting Assay. The data has been added in Fig.6A.
Point 6 :Authors described concerning the effect of LF-IC on the growth of tumor cell in hFcgammaRIIa transgenic mice (data not public) in the Discussion section. The results should be shown in results.
Response 6:The tumor inhibition activity of LF-IC in vivo mentioned in the MS was conducted using melanoma cell lines B16 and the result was included in another MS which is under reviewing. In our work, we have performed the experiment using a mouse leukemia cell line EL4 in mouse system in the revised MS. However, LF-IC primed mouse monocyte isolated from the hFcgRIIa mice could not show any tumoricidal activity in vitro. In the in vivoassay, LF-IC injection i.p. could not inhibit tumor growth of EL4 inoculated intravenously (Fig.5). For the contradiction effect of LF-IC in the two models, the treatment route of LF-IC would be the main reason. Since LF-IC could reverse M2 macrophage into M1 phenotype, LF-IC was injected intra-tumor in the B16 model, where it can targeting tumor asscociate macrophage (TAM) directly.
FcgRs expressed differently in mouse monocytes and human monocytes. FcgRIIa was specific expressed in human monocytes. FcgRIIb, an inhibitory FcgR, expressed high level in mouse monocytes while low level in human monocytes. (Ref 37-38). The interference of FcgRIIb may be the main reason for the failure that LF-IC prime mouse monocytes could not show tumoricidical activity, which needed to be further explored in future P9, Lines 20-22; P10, Lines 1-17; P12, Lines 10-15).
Reviewer 2 Report
In this study, the authors demonstrate that LF-IC primed monocytes mediate apoptosis of JurkatandRaji cells via Granzyme B. The study is very interesting but unfortunately some major revisions are required.
Major comments
Although the authors demonstrate the in vitro effect of LF-IC primed monocytes on Jurkat and Raji cells, it is essential that the authors perform in vivo experiments to validate their findings. This is an important step as it could have important therapeutic implications. The authors suggest that LF-IC induces granzyme B expression in human monocytes via syk-PI3K-AKT pathway. However, a detailed analysis of the pathway is required. The manuscript contains several grammatical errors and requires further editing.Author Response
Response to Reviewer 2 Comments
In this study, the authors demonstrate that LF-IC primed monocytes mediate apoptosis of Jurkat and Raji cells via Granzyme B. The study is very interesting but unfortunately some major revisions are required.
Major comments
Point 1: Although the authors demonstrate the in vitro effect of LF-IC primed monocytes on Jurkat and Raji cells, it is essential that the authors perform in vivo experiments to validate their findings. This is an important step as it could have important therapeutic implications.
Response 1: As suggested, the empowerment for tumoricidal activity of monocytes by LF-IC has been determined on mouse model. Since we have proved that the tumoricidalof LF-IC primed monocyte depended on hFcgRIIa, which however does not have homology genes in mouse, the hFcgRIIa transgenic mice (hFcgRIIa TG) were employed. However, we proved that LF-IC primed hFcgRIIa TG mouse monocyte could not induce cell death of mouse leukemia cells EL4 in vitroand that the survival curve of tumor bearing mice inoculated with EL4 intravenously was not improved when treated with LF-IC intraperitoneally (Fig. 5). FcgRs were expressed differently in mouse monocytes and human monocytes. FcgRIIa was specific expressed in human monocytes. FcgRIIb, an inhibitory FcgR, was expressed at a high level in mouse monocytes while at a low level in human monocytes (Ref 36-37). The interference of FcgRIIb may be the main reason for the failure for LF-IC empowering tumoricidal activity of mouse monocytes, which needed to be further explored in future. The data has been added as Fig.5, with according description in Materials and Methods ( P14, Lines 18-24; P16, Lines 1-6), Results (P9, Lines 20-22; P10, Lines 1-17) and Discussion (P12, Lines 10-15).
Point 2: The authors suggest that LF-IC induces granzyme B expression in human monocytes via syk-PI3K-AKT pathway. However, a detailed analysis of the pathway is required.
Response 2: As suggested, the activation of syk, PI3K and AKT were determined by Western Blotting Assay. The data has been added in Fig.6A.
Point 3: The manuscript contains several grammatical errors and requires further editing.
Response 3:The manuscript has been double checked and grammatical errors have been corrected.
Round 2
Reviewer 1 Report
The manuscript seems to be revised appropriately.
Author Response
Although the reviewer commented that "the MS seems to be revised appropriately", "Can be improved" was checked across the sections including Introduction, Experimental Design, Methodology, Results and Discussion. For this reason we thoroughly revised the whole MS again for further improvement. The MS has also been double checked for mistakes in English grammar, spelling errors and typos. We sincerely hope that the latest version is acceptable for publication in Molecules.
Reviewer 2 Report
The authors must include the comments provided in response to point 1 into the discussion. This will allow the the readers to evaluate theprogress made by this study.
Author Response
Following the reviewer's suggestion that our response to Point 1 must be included in Discussion so that the readers can better evaluate the progress made by this study, we have expanded the Discussion section to incorporate the related comments (Page 12, Lines 12-21).
In addition, the whole manuscript, particularly the Discussion section, has been carefully revised for further improvement. We sincerely that the latest version is satisfactory for publication in Molecules.
