To the Reviewers: POINT-BY-POINT REPLY

Transcription

1 To the Reviewers: We changed our manuscript according to the Reviewer s suggestions. We used a red character for the novel information added or for the sentences deleted from the original version of the manuscript. We are grateful to the Reviewers for their extremely useful suggestions, which improved, in our opinion, the overall quality of the manuscript. We think that all the major and minor points raised by the Reviewers were discussed within the text. POINT-BY-POINT REPLY Reviewer 1: OK SISTEMARE I 2 PUNTI IN GIALLO We are grateful to the Reviewer for the interest on our work and for the kind comments. We are also grateful for his minor comments that were very helpful for improving the manuscript. Major issue: No major issue from the reviewer. Minor issues, point-by point reply: Paragraph 2.1.2: Differentiation of acute leukemia into DC and induction of CTLs against leukemia is not referenced (I would suggest PMID: , and PMID: ). Suggested references have been added. Paragraph 2.4.2: it should be emphasized that the only clinical setting where CIK cells have shown some clinical activity is their infusion after allogeneic transplantation (ref 87 + Laport et al, PMID: ), where they have shown very low GVH potential and some efficacy. We have emphasized this point in the paragraph. Paragraph : in addition to chimeric antigen receptor approach with chimeric antibodies targeting tumor surface antigens, I would mention the possibility of targeting cytoplasmic or nuclear antigens by TCR transfer (I suggest PMID: and PMID: ) Accordingly to Reviewer s suggestions, a short paragraph about these preclinical data has been included. In agreement, Figure 1 has been implemented. Paragraph 2.4.4: this paragraph contains an introductory part (line ) which is redundant, considering what already outlined in paragraphs 1.1 and 1.2. We significantly reduced the introductory part to this paragraph. Line 269: Change CD-33 into CD33. Line 278: eliminate space after CD (change CD 33 into CD33). Line 293 eliminate space after CD (change CD 66 into CD66). Line 314: Please balance the comparatives: highest expressed.lower in AML. Line : Please correct the sentence the strong T-cell activation lead by BiTE trigger a pleiotropic secretion (led instead of lead, triggers instead of trigger). Line 408: I would suggest to change upregulatable into upregulable. Changed.

2 Line : please rephrase the sentence ( RESULTING the antileukemic activity of anti-cd33 targeting therapies RESULTS.. ). We rephrase the sentence that was not clear as suggested by the Reviewer. Reviewer 2: We are grateful to the reviewer for his suggestions. Regarding the general comments, we rewrote the manuscript according the Reviewer s suggestion. We thus added as much details as we can, considering also the comments from the other reviewers who suggested shortening the paper, focusing only in important things. Point-by point reply to Reviewer 2 Line 63 Provide what percentage of patients relapse after SCT. We added the percentage (35 to 45%). Line 67 List one or two examples of poor rick cytogenetics. We added few examples. Line 68 It is not clear what molecular-based targeted therapies is meant by the authors. We clarified this point Line 71 List examples of molecular aberrations. We listed examples of aberrations and specific therapies. The sections, 2.2 Monoclonal antibodies, 2.3 Biospecific T-cell engagers, and CAR T cells are written more specifically and provide sufficient information to the readers. Nothing to reply to this point. Other places that need descriptions to be more specific include, but not limited to; Abstract is too general and needs more specific descriptions. Novel is used 5 times in the abstract, and it is annoying. We rewrote the abstract according to the Reviewer s suggestions. Novel is used numerous times in the body of the manuscript as well. Partly removed and corrected Table 1, 3a, 3b Novel should be deleted from the titles. Line 189 Specify what cell adhesion molecules expressions and cytokines are altered. Accordingly to the Reviewer s suggestion, we better clarified this point and expanded the paragraph Line 193 List examples of genes that were expressed abnormally. Examples of altered genes were included and a more detailed description of the correlation between gene expression profiling of MSCs and AML was added. Line Immunological responses were observed, but clinical results are unsatisfactory? Need more clarifications. We rephrased the sentence and better clarified both the type of immune response and the significance of unsatisfactory clinical response. Line 230 Explain some immunologial results We added a sentence to better explain the type of tests used for evaluating leukemia-specific immune response

3 Line 363 Should describe what are the specific component of AML cell surface that are being targeted. Changed. Also because CAR is described here for the first time in this review and more details are provided in the following section, authors should indicate something like details of chimeric antigen receptor will be provided in the next section. Ok, done. In the first part of this review, Pathways and targets, the authors discuss 5 pathways that are more relevant. However, this 5 pathways includes various levels of potential targets, such as cell surface molecules, intracellular molecules and subpopulation of cells all together. It would be more reasonable to have a sub-heading such as surface molecule, etc. Revision of Table 1 is also recommended. Accordingly to the Reviewer s suggestions, we subdivided the first part of the review in three major subheadings, each containing the different items. The same modifications have been done in Table 1. Some overlapped descriptions are found such as, Line Descriptions are overlapping and can be cut short. In agreement, we cut the paragraph, avoiding overlaps. Line Explanations of naturally occurring Tregs and induced Tregs this part has overlapping descriptions and should be organized more. Better organized,avoiding overlaps There are numerous typographic errors and grammatical errors that need to be corrected. Line 104 in experimental murine WHAT? Experimental murine models. Line 225 Wilms tumor gen should be Wilms tumor 1 gene.. Line 228 WT-1 should be WT1.. Line This part, especially does not make sense as English and difficult understand what the authors are trying to describe. In addition, this does not cite references. To better clarify this point, we added some pertinent references. Line 268 Company should start with lowercase. Line 269 CD-33 should be CD33. Line 278 CD 33 should be CD33. Line 293 CD 66 should be CD66. Line 374 Check the font here. Line 388 phase 1 should be phase I. Line 395 Mardiros et al. Line 414 predominant should be predominantly. Lacks consistency of using PD-L2 or PDL2 AMG330 or AMG 330. Line Separating the long sentence to make a clear statements would be recommended.. Figure 1 CD80/86 to CTLA4 signaling is indicated to induce proliferation. It is a consensus that CTLA4 signaling is inhibitory. /substituted with inhibition

4 Reviewer 3: We are grateful to the Reviewer for the interest on our work and for the kind comments. We are also grateful for his minor comments that were very helpful for improving the manuscript. Point-by-point reply Generally the authors have done a great job, but I think this review is also too long, even though many areas are covered, and it will be possible to shorten it without excluding the important things: there is too much introduction to each class of the immune therapies, especially the sections about CAR-T cells and checkpoint inhibitors the former has been used extensively in also other hematological malignancies and the latter in many solid cancers, so both will be familiar to most readers from the field. We reduced these subchapters. Further I think all the sub-sections in the chapter 2-part will benefit from focusing more directly on the use in hematological malignancies and AML, thus shorten the description of a particular treatments in other cancers and mouse models in accordance with the title. We agree with the Reviewer. Most of the sub-sections are now focusing only on hematological malignancies and AML. Where discussed, the data on other tumors or mice models are relevant for understanding the basis for the development of clinical strategies in hematological cancers. On the other hand, I was surprised that there is nothing about the interesting indirect immunological effects of treatment with the hypomethylating agents as 5-azacytidine, decitabine and the newer Sapacitabine all used as standard in AML treatment, please refer to i.e. Goodyear O et al., Blood Apr 5;119(14):3361-9; Goodyear O et al., Blood Sep 16;116(11): ; Gang & Frøsig et al Blood Cancer J Mar 28;4:e197 and Stübig Mediators Inflamm. 2014;2014: We added a short subchapter (2.4.5) in which we briefly discussed the indirect immunological effect of hypomethylating agents in AML. It would increase the readability of the paper if the authors include their criteria for choosing the pathways and therapy classes that are presented. Obviously, and fully understandable, they haven t included every possible immune therapy used in the treatment of AML as is also stated shortly in lines We indicated that we described the most important targets for an up-to date therapeutical intervention that can help to improve the clinical outcome for patients with AML Lines The content of this article is overall well-written, although it would benefit from further language editing correcting typos here and there: including, but not limited to: Line 64: worst performance, should be worse performance.

5 Line 130: activity on innate immune by in vitro reducing NK cell cytolytic function should read in vitro immune activity by reducing NK cell cytolytic function. Line 149: I agree part of the MDSC suppressive effect is related to arginase I expression, but this is among other mechanisms. We inserted the sentence among other mechanisms. Line 157: Phisiologically, should read physiologically. Line 161: high-levesl, should read high-levels. Line 374: layout mistakes, the greek letters are not shown in the version I ve got. Figure 1 provides a good overview on the different immune cell populations playing against or together with the AML cells, but it could be better explained in the figure text. A minor note is the parallel use of MHC and HLA in this figure I guess HLA would be most appropriate. As MDSCs are mentioned in the text (line 149) they should also be included in this figure. Accordingly, MHC was substituted with HLA. As for the suggestion to include MDSC, we agree with the Reviewer it would be consistent with the text. However, we would ask the Reviewer to take into consideration that the addition of a further item in the figure is likely to make it very difficult to access. Moreover, as explained in the text, the reference to MDSC as a relevant player in AML BM microenvironment is an indirect finding from a preclinical study. Similar considerations may be drawn for figure legend. I would like references included in table 1 as well as they already are in tables 3a-b. References have been included Table 2 is only referred to once (lines ) and I would prefer this table is excluded and the content is just displayed directly in the text. I suggest tables 3a and 3b are merged to one table just marking the drugs in pre-clinical and clinical development differently. We deleted table 2. We merged tables 3a and 3b (now table 2)