Journal Article
. 2021 Nov; 22(21):.
doi: 10.3390/ijms222111302.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells

Madhu S Pandey 1 Chunlei Wang 2 Scott Umlauf 1 Shihua Lin 1 
Affiliations
  • PMID: 34768776
  •     31 References

Abstract

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Keywords: bioassay; bispecific antibody; mechanism of action; potency; qualification.

PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer.
Zhiqiang Guo, Xin Wang, +3 authors, Shulan Zhang.
PLoS One, 2014 Mar 04; 9(2). PMID: 24586709    Free PMC article.
Highly Cited.
CD40/CD154 interactions at the interface of tolerance and immunity.
Sergio A Quezada, Lamis Z Jarvinen, Evan F Lind, Randolph J Noelle.
Annu Rev Immunol, 2004 Mar 23; 22. PMID: 15032580
Highly Cited. Review.
Sufficiency of CD40 activation and immune checkpoint blockade for T cell priming and tumor immunity.
Alexander H Morrison, Mark S Diamond, +2 authors, Robert H Vonderheide.
Proc Natl Acad Sci U S A, 2020 Mar 28; 117(14). PMID: 32213589    Free PMC article.
A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity.
Shiming Ye, Diane Cohen, +16 authors, Debra T Chao.
Cancer Immunol Res, 2019 Aug 30; 7(11). PMID: 31462409
Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.
G J Freeman, A J Long, +16 authors, T Honjo.
J Exp Med, 2000 Oct 04; 192(7). PMID: 11015443    Free PMC article.
Highly Cited.
The use of agonistic anti-CD40 therapy in treatments for cancer.
Andrea Khong, Delia J Nelson, +2 authors, Bruce W S Robinson.
Int Rev Immunol, 2012 Jul 19; 31(4). PMID: 22804570
Review.
Costimulatory and Coinhibitory Receptor Pathways in Infectious Disease.
John Attanasio, E John Wherry.
Immunity, 2016 May 19; 44(5). PMID: 27192569    Free PMC article.
Highly Cited. Review.
Advances in bispecific biotherapeutics for the treatment of cancer.
Chad May, Puja Sapra, Hans-Peter Gerber.
Biochem Pharmacol, 2012 Aug 04; 84(9). PMID: 22858161
Review.
A CD40 Agonist and PD-1 Antagonist Antibody Reprogram the Microenvironment of Nonimmunogenic Tumors to Allow T-cell-Mediated Anticancer Activity.
Hayley S Ma, Bibhav Poudel, +9 authors, Elizabeth M Jaffee.
Cancer Immunol Res, 2019 Jan 16; 7(3). PMID: 30642833    Free PMC article.
4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function.
Seung-Joo Lee, Lara Myers, +5 authors, Anthony T Vella.
J Immunol, 2004 Aug 24; 173(5). PMID: 15322159
Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.
E M Sotomayor, I Borrello, +6 authors, H I Levitsky.
Nat Med, 1999 Jul 08; 5(7). PMID: 10395323
Highly Cited.
Innate and adaptive immune cells in the tumor microenvironment.
Thomas F Gajewski, Hans Schreiber, Yang-Xin Fu.
Nat Immunol, 2013 Sep 21; 14(10). PMID: 24048123    Free PMC article.
Highly Cited. Review.
CD40 ligand-mediated activation of the de novo RelB NF-kappaB synthesis pathway in transformed B cells promotes rescue from apoptosis.
Nora D Mineva, Thomas L Rothstein, +2 authors, Gail E Sonenshein.
J Biol Chem, 2007 Apr 21; 282(24). PMID: 17446175
Clinical and biological effects of an agonist anti-CD40 antibody: a Cancer Research UK phase I study.
Peter Johnson, Ruth Challis, +14 authors, Anthony Williams.
Clin Cancer Res, 2015 Jan 16; 21(6). PMID: 25589626
PD-1/PD-L1 blockage in cancer treatment-from basic research to clinical application.
Masaki Mandai.
Int J Clin Oncol, 2016 Mar 13; 21(3). PMID: 26968586
Review.
Molecular basis and therapeutic implications of CD40/CD40L immune checkpoint.
TingTing Tang, Xiang Cheng, +3 authors, Hong Wang.
Pharmacol Ther, 2020 Oct 23; 219. PMID: 33091428    Free PMC article.
Review.
The Diverse Function of PD-1/PD-L Pathway Beyond Cancer.
Weiting Qin, Lipeng Hu, +4 authors, Xu Wang.
Front Immunol, 2019 Oct 23; 10. PMID: 31636634    Free PMC article.
Highly Cited. Review.
Bispecific antibodies in cancer immunotherapy.
Eva Dahlén, Niina Veitonmäki, Per Norlén.
Ther Adv Vaccines Immunother, 2018 Jul 13; 6(1). PMID: 29998217    Free PMC article.
Highly Cited. Review.
Antibiotics are associated with attenuated efficacy of anti-PD-1/PD-L1 therapies in Chinese patients with advanced non-small cell lung cancer.
Sha Zhao, Guanghui Gao, +10 authors, Caicun Zhou.
Lung Cancer, 2019 Mar 20; 130. PMID: 30885328
Highly Cited.
Mechanistic basis of co-stimulatory CD40-CD40L ligation mediated regulation of immune responses in cancer and autoimmune disorders.
Tikam Chand Dakal, Bhanupriya Dhabhai, +9 authors, Amit Sharma.
Immunobiology, 2020 Jan 04; 225(2). PMID: 31899051
Review.
Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain.
Manisha Singh, Christina Vianden, +15 authors, Willem W Overwijk.
Nat Commun, 2017 Nov 14; 8(1). PMID: 29129918    Free PMC article.
Bispecific antibodies for cancer immunotherapy: Current perspectives.
Dafne Müller, Roland E Kontermann.
BioDrugs, 2010 Mar 05; 24(2). PMID: 20199124
Review.
Molecular mechanism and function of CD40/CD40L engagement in the immune system.
Raul Elgueta, Micah J Benson, +3 authors, Randolph J Noelle.
Immunol Rev, 2009 May 12; 229(1). PMID: 19426221    Free PMC article.
Highly Cited. Review.
Transformation of the tumour microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumour model.
Nadia M Luheshi, Jane Coates-Ulrichsen, +8 authors, Robert W Wilkinson.
Oncotarget, 2016 Feb 27; 7(14). PMID: 26918344    Free PMC article.
CD40/CD40 ligand interactions in immune responses and pulmonary immunity.
Tsutomu Kawabe, Miyoko Matsushima, +2 authors, Yoshinori Hasegawa.
Nagoya J Med Sci, 2011 Sep 21; 73(3-4). PMID: 21928689    Free PMC article.
Review.
The blockade of immune checkpoints in cancer immunotherapy.
Drew M Pardoll.
Nat Rev Cancer, 2012 Mar 23; 12(4). PMID: 22437870    Free PMC article.
Highly Cited. Review.
Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis.
Cornelia Hömig-Hölzel, Caroline Hojer, +8 authors, Ursula Zimber-Strobl.
J Exp Med, 2008 May 21; 205(6). PMID: 18490492    Free PMC article.
PD-1/PD-L1 Blockade Therapy in Advanced Non-Small-Cell Lung Cancer: Current Status and Future Directions.
Liliang Xia, Yuanyong Liu, Ying Wang.
Oncologist, 2019 Mar 02; 24(Suppl 1). PMID: 30819829    Free PMC article.
Review.
CD40 signaling to the rescue: A CD8 exhaustion perspective in chronic infectious diseases.
Rajarshi Bhadra, Dustin A Cobb, Imtiaz A Khan.
Crit Rev Immunol, 2013 Aug 27; 33(4). PMID: 23971530    Free PMC article.
Bispecific antibodies: a mechanistic review of the pipeline.
Aran F Labrijn, Maarten L Janmaat, Janice M Reichert, Paul W H I Parren.
Nat Rev Drug Discov, 2019 Jun 09; 18(8). PMID: 31175342
Highly Cited. Review.
Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.
Weimin Chen, Madhu Pandey, +7 authors, Shihua Lin.
MAbs, 2021 Apr 20; 13(1). PMID: 33870864    Free PMC article.