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106 Development of RO assay for anti-PD-1 mAb and anti-PD-1×CD47 BsAb utilizing hPD-1/hCD47 dual humanized mice, at preclinical setting to facilitate clinical validation
  1. Xiaoyu An1,
  2. Mingfa Zang1,
  3. Ke Hang2,
  4. Xianfei He3,
  5. Ruilin Sun3,
  6. Jian Fei3,
  7. Henry Li2 and
  8. Haijuan Yu1
  1. 1Crown Bioscience Inc, san diego, CA, USA
  2. 2Hanx Biopharmaceuticals, Wuhan, China
  3. 3Shanghai Model Organisms Center, Inc, Shanghai, China


Background One of the most relevant pharmacokinetic (PK) parameters for antibody-based therapy in the clinics is receptor occupancy (RO). Each specific therapeutics usually requires a tailored RO assay, an PD-1 RO assay for anti-PD-1 antibody treatment. For bispecific antibody (BsAb) treatment, the needed RO assays can include each receptor RO and total RO combining the two different receptors. Due to the complexity and challenges in developing such assays using clinical samples, it would be prudent to develop a model system to test RO assay methods in vivo at the preclinical stage prior to clinical validation, since it may simulate dosing process in human and also provide opportunity to correlate RO to tumor model efficacy if tumor pharmacology is also conducted simultaneously. Surface expressing CD47 and PD-1 receptors are recognized two most important checkpoints for both innate and adaptive immunity suppressing tumor immunity, and have been explored for immunotherapies, in the cases of investigational drugs, HX008 (PD-1 mAb) and HX009 (CD47×PD1 BsAb).1–4 In order to facilitate development of RO assay for the clinics, we explored preclinical modelling RO assays using hPD-1/hCD47 dual humanized mouse model for testing these two species-restricted investigational drugs.

Methods The brief procedures are as followed: 1) Non-tumor bearing hPD1×hCD47 dual humanized mice were single administrated via i.p. injection with test antibodies at different dose levels; 2) PBMCs were collected and prepared from the animals at different timepoints (pre-dosing, 2 hours post dosing and 7 days post dosing; 3) one half of the PBMC samples were added into the vials containing over-saturating amount of test antibody, and the other half were added into the vials containing PBS, followed by mixing; 4) add defined amount of fluorescence-labeled test antibody, followed by washing and flow cytometry analysis, and the%RO being determined. The test antibodies could be anti-human PD-1 antibody, anti-human CD47 antibody or anti-human PD-1×CD47 BsAb for measuring the respective RO values.

Results Preliminary results demonstrated that HX008 RO assays showed anticipated dose dependent RO values at both timepoints, suggesting this assay development is highly doable and predictive using the preclinical humanized models. Further detailed to be analyzed, including HX009 RO values for PD-1 receptor only, CD47 receptor only and total for combining both receptors.

Conclusions Supposing further data from HX-009 RO modelling proven valid, RO assay using humanized mice could become a standard method of choice for RO assay development at the preclinical stage, enabling simpler/efficient clinical development.


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  2. Liu, B., Guo, H., Xu, J., Qin, T., Guo, Q., Gu, N., Zhang, D., Qian, W., Dai, J., Hou, S., et al. (2018). Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses. MAbs 10, 315–324.

  3. Liu, X., Pu, Y., Cron, K., Deng, L., Kline, J., Frazier, W. A., Xu, H., Peng, H., Fu, Y. X., and Xu, M. M. (2015). CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat Med 21, 1209–1215.

  4. Roohullah, A., et al., (2021). First-in-human phase I dose escalation study of HX009, a novel recombinant humanized anti-CD47/PD-1 bispecific antibody, in patients with advanced malignancies. ASCO 2021 Annual Meeting.

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