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447 Evaluation of efficacy and toxicity of human CD40 agonistic antibodies in C57BL/6-hCD40/hFcγRIIB HuGEMMTM
  1. Demi X Liu1,
  2. Kaixia Lian2,
  3. Xuefei Yan2,
  4. Rong Wang2,
  5. Jie Lin2,
  6. Xinhe Feng2,
  7. Xiaolong Tu2,
  8. Chengcheng Wang2,
  9. Lei Zheng2,
  10. Xiaoxi Xu2,
  11. Annie An2,
  12. Ludovic Bourre2 and
  13. Jessie Wang2
  1. 1Crown Bioscience, Beijing, China
  2. 2Crown Bioscience Inc., San Diego, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background CD40 is a popular target for tumor immunotherapy. However, the clinical development of CD40 agonistic antibodies has been severely hampered by the frequent association of immune-related adverse events (irAE). Engagement of FcγRIIB by the Fc portion of agonistic anti-CD40 was reported as critical for its antitumor efficacy and related irAE in both mouse experiments and clinical trials. To evaluate both the efficacy and the irAE of anti-CD40 agonists in preclinical studies, we established a HuGEMMTM mouse model in which both human CD40 and FcγRIIB were knock-in in C57BL/6 background (C57BL/6-hCD40/hFcγRIIB) mice.

Methods Homozygous C57BL/6-hCD40/hFcγRIIB HuGEMMTM were bred from CRISPR/Cas9-mediated gene editing of human CD40 and FcγRIIB gene single knock-in mice. The cell surface expression of hCD40 and hFcγRIIB on mouse B cells and B cell activation by various agonistic anti-hCD40 antibodies were confirmed by FACS.

To evaluate the antibody efficacy and irAE, homozygous C57BL/6-CD40/FcγRIIB HuGEMMTM were subcutaneously inoculated with MC38 tumor cells. AST and ALT levels were measured by biochemical analysis. Serum TNF-α, IL-12p70, IL-6 and IFN-γ were measured by ELISA. Immune cell infiltration in the liver and tumor was analyzed by immunohistochemistry (IHC) and/or FACS.

Results C57BL/6-CD40/FcγRIIB HuGEMMTM was successfully generated with confirmed expression of human CD40 and FcγRIIB. FcγRIIB engagement enhanced the agonistic activity of the anti-hCD40 antibody as detected by an in vitro B cell activation assay. Human IgG1 isotype anti-hCD40 agonist with hFcγRIIB binding capability (APX005M-S267E) significantly promoted B cell activation compared to its analogue without hFcγRIIB binding capability (APX005M-N297A). Interestingly, a hIgG2a isotype hCD40 antibody, Selicrelumab, which cannot bind hFcγRIIB, also activated the humanized B cells, even at a higher level, suggesting robust hFcγRIIB-independent hCD40 activation. In vivo efficacy and irAE of these agonistic anti-hCD40 antibodies demonstrated that anti-hCD40 antibody with FcγRIIB binding capability significantly inhibited tumor growth and promoted CD8+ T cell infiltration of tumors, compared to the control anti-hCD40 antibody without hFcγRIIB binding capability. In addition, Selicrelumab showed a highly efficient anti-tumor effect with increased T cell infiltration in tumors compared with APX005M-hIgG1(S267E). No significant body weight loss was observed in any of these animals. However, FcγRIIB engagement exacerbated the irAE response with enhanced liver damage, pro-inflammatory cytokine secretion, and immune cell infiltration into livers.

Conclusions This study revealed that human CD40 agonistic antibodies evaluated in C57BL/6-CD40/FcγRIIB HuGEMMTM mice reproduce the efficacy and toxicity phenotypes observed in clinic, demonstrating the improved clinical relevance of this HuGEMMTM model to evaluate future biologic molecules targeting CD40.

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