The Antitumor Activity of TCR-Mimic Antibody-Drug Conjugates (TCRm-ADCs) Targeting the Intracellular Wilms Tumor 1 (WT1) Oncoprotein

Round 1

Reviewer 1 Report

The authors describe the development of novel TCR-Mimic antibody-drug conjugates targeting tumor-specific peptide/MHC complexes. This is an interesting approach for targeting intracellular tumor-specific proteins, but the low number of targets per cell is a major challenge.

The article requires extensive English editing.

Some important pieces of data are missing in the results section:

- doses of ADCs administered in the in vivo studies

- data on the generation and evaluation of the ESK-Q2L bispecific

In figures, some axis should be properly re-labelled:

- Figures 4a and 6c - concentrations should be expressed as real numbers, rather than powers of 10

Authors perform prediction of WT1 protein epitope peptides, but do not validate their presentation in vitro.

The advantage of using bispecific antibody should be confirmed by more experiments (ideally in vivo and with the bispecific targeting two WT1-derived peptides).

Overall, the article describes an interesting and challenging approach, but the manuscript has to be thoroughly revised and more experiments are needed to demonstrate the superiority of bispecific ADCs.

Author Response

Responses to Reviewer 1

1. The article requires extensive English editing.

Response:

We thank the reviewer for the suggestion. In the revised version, we have modified the statement in the manuscript. English native speakers have helped us to improve the phrasing and diction.

2. Some important pieces of data are missing in the results section:

- doses of ADCs administered in the in vivo studies

Response:

  We thank the reviewer for the suggestion. We provided the doses of ADCs administered in the method section but didn’t in the results section in the previous manuscript, and we added the doses of ADCs administered in the results section in the revised manuscript.

- data on the generation and evaluation of the ESK-Q2L bispecific

Response:

We thank the reviewer for the suggestion. But, we didn’t generate ESK-Q2L bispecific because of the competition-binding of ESK and Q2L (Figure S3) which can’t meet our requirements to increase the number of targets.

3. In figures, some axis should be properly re-labelled:

- Figures 4a and 6c - concentrations should be expressed as real numbers, rather than powers of 10

Response:

We thank the reviewer for the suggestion. In the revised version, the concentrations have been expressed as real numbers.

4. Authors perform prediction of WT1 protein epitope peptides, but do not validate their presentation in vitro.

Response:

We are very grateful to the reviewer for this meaningful comment. NetMHC and NetCTL are mainstream software for predicting peptide presented by MHC I. NetMHC was trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles[1]. For predictions of HLA class I affinity, NetMHC method has been judged to be one of the two best methods in a comparative study of the performance of 30 methods for HLA class I affinity prediction [2]. NetMHC has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75–80% confirmed MHC binders[3]. NetCTL is a web-based tool designed for predicting human cytotoxic T lymphocyte (CTL) epitopes in any given proteins[4]. It does so by integrating predictions of proteasomal cleavage and TAP transport efficiency. The reliability of NetCTL has previously been shown to be as high as or higher than other publicly available methods for CD8⁺ T cell epitope predictions[4-5].

We used NetMHC and NetCTL to predict potential presenting peptides of WT1 protein but didn’t validate their presentation in vitro. Because we just want to convey the concept that several peptides from WT1 can be presented by HLA I molecules in this manuscript. In addition, these results improve the practicability of Bi-TCRm-ADCs. For the further study, we will select several peptides to validate their presentation in vitro and screen TCRm antibody targeting these epitopes. Then, we will generate Bi-TCRm-ADCs targeting two WT1-derived peptides which enhance the application of Bi-TCRm-ADCs.

5. The advantage of using bispecific antibody should be confirmed by more experiments (ideally in vivo and with the bispecific targeting two WT1-derived peptides).

Response:

We thank the reviewer for the suggestion. The reason why we generated the ESK-1G4-MMAE was to convey that the target amount is one of the factors which influence the efficiency of TCRm-ADCs. The results of affinity and cytotoxicity evaluation experiments had confirmed this idea. Otherwise, in vivo activity in solid tumors is affected by many factors, such as molecular weight, metabolism, etc., and in vitro experiments can better control the variables to verify the idea we conveyed. The reviewer's suggestion is very meaningful. But the ESK-1G4-MMAE isn’t a good Bi-TCRm-ADC due to the limit of targets which derived from two proteins. ESK-1G4-MMAE only paves the way for Bi-TCRm-ADCs targeting two WT1-derived peptides. For the further study, we will screen TCRm antibodies targeting other epitopes and generate Bi-TCRm-ADCs targeting two WT1-derived peptides. At that time, we will consider these factors comprehensively and evaluate the Bi-TCRm-ADCs both in vitro and in vivo.

References

[1] Lundegaard C, Lamberth K, Harndahl M, et al. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11[J]. Nucleic Acids Research, 2008, 36:509-512.

[2] Lin H H, Ray S, Tongchusak S, et al. Evaluation of MHC class I peptide binding prediction servers: Applications for vaccine research[J]. BMC Immunology, 2008, 9(1):8.

[3] Sylvester-Hvid C, Nielsen M, Lamberth K, et al. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation[J]. Tissue Antigens, 2004, 63(5):395-400.

[4] Larsen M V, Lundegaard C, Lamberth K, et al. An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions[J]. European journal of immunology, 2005, 35(8):2295-2303.

[5] Larsen M V, Lundegaard C, Lamberth K, et al. Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction[J]. Bmc Bioinformatics, 2007, 8(1):424.

Reviewer 2 Report

Ying et al constructed a well-worked Bi-TCRm-ADCs, the experimental design is reasonable and the results are reliable. Here are some questions should be improved .

1.      Fig 2C and L119, how do you know the DAR was 3.0, do you have a standard eluted curve? It's better to tell more information here.

2.      Fig 2D, the affinity of Q2L-MMAE was decreased more than ESK-MMAE, not slightly attenuated (L122), can you give a explanation.

3.      The IC50 of in vitro antitumor activity of TCRm-ADCs were high,  which were probably caused by the low peptide presentation. Is there other higher-expressed cells  that you can verify this?

4.      In vitro cytotoxicity of ESK-1G4-MMAE was better than others, have you finished  in vivo experiments, which will be more convincing.

5.      Just an advice for the curve colors, it will be more clear if they are consistent in whole manuscript. Such as Fig 5B/5D, the colors of ESK and ESK-IG4, also Fig 4A/B/C and else.

6.      Small mistake, L101, that should be those. Others in the manuscript should be revised further.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

In this manuscript, Shen .etc described the design of TCR-mimic ADCs. In general, the paper is well-written, it represents a relatively new field of ADC development. Please see below my questions to address before publication:

(1) Why the authors chose MMAE as the payload? The authors need to measure the MHC complex number and report it in this work to justify the choice of the payload. (I am worried the MHC complex number may be lower than 1000, it can not reach the level of HER2 in breast cancer, then a more potent payload e.g. DNA-damaging agents should be considered)

(2) The internalization figure (confocal images) is really blurred, 300 dpi has to be used in this case to make the conclusion of trafficking, it is really misleading to show only one lysosome per cell.

(3) I suggest the authors to try CART if ADC can not provide good efficacy.

Author Response

Responses to Reviewer 3

(1) Why the authors chose MMAE as the payload? The authors need to measure the MHC complex number and report it in this work to justify the choice of the payload. (I am worried the MHC complex number may be lower than 1000, it can not reach the level of HER2 in breast cancer, then a more potent payload e.g. DNA-damaging agents should be considered)

Response:

We thank the reviewer for the suggestion. Currently, five ADCs have been approved by the FDA or EMA, the payload of Adcetris and Polivy is MMAE, and largest group of ADCs in clinical trials are based on MMAE[1]. MMAE has been selected for its high potency, water solubility, stability under physiological conditions and suitability for the attachment of stable linkers[2]. Besides, the target in this manuscript is WT1, which is highly expressed in many tumor cells but also expressed in some normal cells. More potent payload leads to stronger side effects. Tumor specific antigens (TSAs) are expressed only on the surface of a certain tumor cell and not on normal cells, which are the ideal targets for ADCs. In the further study, we will choose tumor TSAs as the targets of TCRm-ADCs and consider selecting DNA-damaging agents, such as duocarmycins and calicheamicin, to improve the efficacy of TCRm-ADCs.

(2) The internalization figure (confocal images) is really blurred, 300 dpi has to be used in this case to make the conclusion of trafficking, it is really misleading to show only one lysosome per cell.

Response:

We thank the reviewer for the suggestion. In the revised manuscript, we provide clearer fluorescence confocal images (Figure 2B and 6B), the white arrows showed the TCRm antibody/ADCs gathered at aggregate of lysosomes rather than one lysosome.

(3) I suggest the authors to try CAR-T if ADC can not provide good efficacy.

Response:

We thank the reviewer for the suggestion. CAR-T is a good way to enhance the efficacy of TCRm antibodies. Rafiq[3] had reported a TCRm CAR against WT1 RMF/HLA-A*02:01 utilizing the single-chain variable fragment (scFv) of ESK, termed WT1-28z. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01⁺ WT1⁺ tumors and enhanced survival of mice engrafted with HLA-A*02:01⁺, WT1⁺ leukemia or ovarian tumors. However, the severe side effects, such as cytokine‐release syndrome (CRS) and neurotoxicity, limited the application of CAR-T[4]. In addition, CAR-T therapy requires T cells isolated from the patient which is too personalized. We develop ADCs to provide an alternative to antitumor therapy and are not a substitute for CAR-T. Moreover, the operating conditions required by CAR-T are very complicated and our laboratory is currently unable to achieve it.

References

[1] de Goeij, B.E.; Lambert, J.M. New developments for antibody-drug conjugate-based therapeutic approaches. Curr. Opin. Immunol. 2016, 40, 14–23.

[2] Senter, P. D.; Sievers, E. L. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol.2012, 30, 631–637. 

[3] Rafiq, S.; Purdon, T.J.; Daniyan, A.F.; Koneru, M.; et al. Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen. Leukemia 2017, 31, 1788–1797.

[4] Hartmann J,; Schüßler-Lenz M,; Bondanza A,; et al. Clinical development of CAR T cells—challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med., 2017, 9(9): 1183–1197.

Reviewer 4 Report

The authors have reported on the development of the　TCR-mimic antibody-drug conjugate with MMAE (TCRm-ADC) against WT1-positive tumor. Two types of TCRm-ADCs; ESK17-MMAE and Q2L-MMAE, which have different affinity property to bind WT1 RMF/HLA-A*02:01, were evaluated in vitro and in vivo. In addition, both WT-1 and NY-ESO-1 targeting bispecific TCRm-ADC were newly-developed in order to enhance the anti-tumor effect. This work is so unique that it provides important information and insight into the research field of antibody therapeutics. However, there are some issues which should be clarified.

1.    Current article structure is incomplete. In particular, in vivo efficacy of the bispecific TCRm-ADC was not shown, despite it being produced in order to improve the anti-tumor effect in the animal model over ESK17-MMAE or Q2L-MMAE. Otherwise, the content should be focused on TCRm-ADCs with the exception of the bispecific TCRm-ADC.

2.    If possible, the anti-tumor effects of ESK17-MMAE or Q2L-MMAE should be evaluated in a smaller sized tumor to compare them precisely. The antibody with a higher dissociation rate can show efficient penetration into the large size tumor as compared with the antibody with a lower one. By contrast, in a smaller sized tumor, the result might be different.

3.    The number of targeted molecules on the tumor cell is a critical factor in ADCs achieving good efficacy. It is important to examine it especially in the clinical samples. Moreover, in vitro study using several tumor cells with a different number of the targeted molecule, e.g. from about 10^3 to 10^5 molecules per single cell, would be helpful to validate the potential activity.

4.    In Figure 4 A, IC50 of free MMAE should be provided. In addition to the antibody delivery, targeted molecule, internalization and drug release, the payload-sensitivity of the tumor cells is a key factor in determining the ADC efficacy.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have made all the necessary corrections and the publications is acceptable in its current form.

Reviewer 4 Report

I have understood the responses. I'm also looking forward to the further study.