PONE-D-21-10696

Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

PLOS ONE

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Hassan Issafras, Chi-Ling Tseng, Yunchih Cheng, Peihua Lin, Lisa Xiao, Yun-Ju Huang, Chih-Hsiang Tu, Ya-Chin Hsiao,  Yen-Hsiao Chen, Chien-Hsin Ho, Ou Li, Yanling Wang, Sandra Chen, Zhenyu Ji, Eric Zhang, Yi-Ting Mao, Eugen Liu, Shumin Yang and Weidong Jiang were employees of either Hengenix Inc or Shanghai Henlius Biotech, Inc., P. R. China.

Shilong Fan and Min Li received funding from Shanghai Henlius Biotech, Inc."

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors present an extensive study describing the generation of an anti-PD-1 IgG4 monoclonal antibody with a remarkable activity in vitro (binding to PD-1, to PD-1 expressing cells, inhibition of PD-1/PD-L1 binding, activity on humanT cells) and in vivo in several tumor models. Also a pharmacokinetic study in cynomolgus monkeys has been performed is here reported. The antibody named HLX10 appears as a promising new candidate for immunotherapy.

The work is well designed, well executed and well presented and can be accepted with minor revisions.

A more detailed description of the phage display library constructiuon and screening should be provided in terms of complexity, panning rounds, number of new sequences etc.

Reviewer #2: In this paper, author reported that HLX10, a novel fully humanized anti PD-1 IgG4 mAb, blocked PD1/PD-L1 interaction and activated T-cell proliferation and cytokine secretion in vitro. HLX10 demonstrated significant antitumor efficacy in several in 5 mouse models and synergized with Avastin biosimilar to promote robust tumor activity. Finally, author showed a co-crystal structure of the antigen-binding fragment (Fab) of HLX10 in complex with PD-1 at a 1.78-Å resolution. Besides, HLX10 was well tolerated by Monkey single dose pharmacokinetics assay. Altogether, comprehensive study proved that HLX10 had better bioactivity to FDA approved nivolumab and pembrolizumab.

Minor questions:

1. As author described “The EMT-6 is considered an anti-PD-1 resistant tumor, due to an immune excluded phenotype, macrophage-derived suppressive infiltration, and epithelial-to-mesenchymal transition (EMT) [22, 23]. Upon treatment of established EMT-6/ tumors in BALB/c mice, HLX10 was able to significantly inhibit tumor growth with some responding and nonresponding mice. Please add a short discussion. You may refer recent study on anti-PD-L1/TGF-b bispecific antibody (Yi M, et al. The construction, expression, and enhanced anti-tumor activity of YM101: a bispecific antibody simultaneously targeting TGF-β and PD-L1. J Hematol Oncol, 2021;14:27).

2. HLX10 was shown to synergize with Avastin biosimilar to promote robust tumor activity. For background information as to the combination of ICI with anti-angiogenesis, please refer recent review and make short discussion (Yi M, et al. Synergistic effect of immune checkpoint blockade and anti-angiogenesis in cancer treatment. Mol Cancer, 2019; 19:60).

3. Could you please show CD8+ T-cells and blood vessel for tumor tissues from in vivo study of Fig 8A, B?

Reviewer #3: The research article by Hassan Issafras, Shilong Fan et al. deals with the structural, biological and pharmacological characterizations of HLX10, a humanized IgG4 monoclonal antibody against PD-1 receptor.

A comparative analysis of binding activity of HLX10 and several approved mAb to PD-1 was carried out, showing a similar efficiency of HLX10 to the reference antibody Nivolumab in enhancing T-cell responses and cytokine production in vitro. The anticancer activity of HLX10 was then demonstrated using several syngeneic and xenograft models. Moreover, an increase of antitumor efficacy of HLX10 was observed when used in combination with HLX10 enhanced an anti-angiogenic antibody (HLX04).

Crystallographic studies of HLX10/PD-1 complex disclose a similar binding mode to the approved Pembrolizumab mAb and identify Arg86 of PD-1 as key residue for HLX10 binding. PK studies in cynomolgus monkey showed that HLX10 is endowed with a small volume of distribution and long elimination half-life, which is common to recombinant monoclonal antibodies. Toxicity studies show also that HLX10 is well tolerated up to 100 mg/kg for 4 weeks. Overall, the work is well conceived, the English language is very good, and results are of interest to researchers working in the field of cancer immunotherapies. The methodological part and experiments are well executed and compliant to a high technical standard. Conclusions are appropriately written and supported by the results.

However, before accepting the paper for publication in PLOS One, authors should address the following minor issues.

- Line 303-305: Authors should mention the technique used to evaluate HLX10 binding to PD-1 in CHO transfected cells (in the same way as it is reported for the BLI assay to check the monomeric binding affinity).

- Line 412: The text ‘Error! Reference source not found’ appears, the author should insert the citation properly.

- Line 428: Authors should mention that crystallographic methods are reported in supplementary Material

- The legend and clarity of figure 6 should be improved. It is indeed unclear the meaning of the nine

growth curves (solid black lines) representing HLX10 15 mg/kg. Do they show the growth of EMT breast cancer syngeneic tumors following the injections (6 or 9 injections) ?

- In the legend of figure 9 it is stated that “Arg86 of hPD-1 is involved in several hydrogen bonds, salt bridge and hydrophobic interaction with HLX10.” Being a polar positively charged residue, Arg86 cannot form hydrophobic interactions with HLX10.

- In the conclusion part, authors should specify that the combination therapy HLX10/HLX04 (NCT03757936) is a phase I clinical study.

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Reviewer #1: Yes: Menotti Ruvo

Reviewer #2: No

Reviewer #3: Yes: Antonio Macchiarulo

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Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

PONE-D-21-10696R1

Dear Dr. Issafras,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Irina V. Balalaeva, PhD

Academic Editor

PLOS ONE

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