Application of a newly-developed cynomolgus macaque BiTE-mediated cytotoxic T-lymphocyte activity assay to various immunomodulatory agents in vitro

Figures & data

Figure 1. Illustration of BiTE^®-mediated CTL assay. (1) EGFR expressing HCT-116 target cells and donor PBMC are co-cultured with α-EGFR × CD3 BiTE^®. (2,3) Upon binding of BiTE^® to both target cell and CD8⁺ T-lymphocyte, immunological synapse forms, and activation ensues. (4,5) Exocytic granules (marked by CD107a) containing perforin and granzymes along with IFNγ are then secreted, resulting in redirected lysis of target cell (5) (Created with BioRender.com).

Figure 2. Immunosuppressive agents targeting T cell-intrinsic signals, proliferation, and activation reduced CTL activity. Percentage CD8⁺ T-cells with CD107a surface staining (left panel) and IFNγ production (right panel) were measured at different drug concentrations. Each data point is mean (±SD) from a single donor run in duplicate. Data are representative individual donors of multiple independent experiments with several donors (n). (A) Dexamethasone (n = 4). (B) Prednisolone (n = 6). (C) Tofacitinib (n = 4). (D) Ruxolitinib (n = 6). (E) Mycophenolic acid (n = 6). (F) Teriflunomide (n = 8). (G) Tacrolimus/FK506 (n = 5). (H) Rapamycin (n = 6). (I) Apremilast (n = 8). Drugs were tested in separate experiments.

Table 1. Immunomodulatory agents and mechanisms of action.

General category of mechanism of action	Agent/active form	Target	Specific mechanism of action	References
Small molecules inhibiting leukocyte signaling, proliferation, and activation	Dexamethasone	Glucocorticoid receptor (GR)	Releases GR to bind glucocorticoid response elements on DNA and modifies gene transcription, resulting in suppression of inflammatory proteins	Barnes 2006; Wust et al. 2008; Shefrin and Goldman 2009
	Prednisolone
	Tofacitinib	Janus kinases (JAKs)	Inhibits JAK1/JAK3 to prevent activation of STATs, resulting in reduction of cell proliferation and activation	Hodge et al. 2016
	Ruxolitinib		Inhibits JAK1/JAK2 to prevent activation of STATs, resulting in reduction of cell proliferation and activation	Elli et al. 2019
	Mycophenolic acid	Inosine-5’-monophosphate dehydrogenase (IMPDH)	Inhibits purine synthesis/DNA replication in T- and B-lymphocytes	Allison and Eugui 2000
	Teriflunomide	Dihydroorotate dehydrogenase (DHODH)	Inhibits pyrimidine synthesis in activated lymphocyte replication	Fox et al. 1999; Bar-Or 2014
	Tacrolimus/FK506	Immunophilin FKBP12	Inhibits calcineurin, resulting in reduced NFAT transduction and IL-2 transcription	Thomson et al. 1995; Jacobson et al. 1998
	Rapamycin	FKBP12	Inhibits mTOR, leading to reduction of IL-2 mediated lymphocyte activation	Dumont and Su 1996
	Apremilast	Phosphodiesterase type 4 (PDE4)	Inhibits PDE4, resulting in a reduction of inflammatory mediators including nitric oxide synthase, TNFα, IL-23, and an increase in IL-10	Schafer 2012
Antibodies inhibiting cytokine signaling and/or costimulatory mechanisms	Adalimumab	TNFα	Disrupts interaction of TNFα with TNF receptor and its inflammatory signals	Moller et al. 1990; Cornillie et al. 2001; Weisman et al. 2003; Buurman et al. 2018
	Infliximab
	Tocilizumab	IL-6R	Binds soluble and membrane bound IL-6 receptors and prevents IL-6/IL-6R mediated pro-inflammatory signaling	Mihara et al. 2005; Ogata et al. 2012
	Ustekinumab	IL-12/IL-23	Binds p40 subunit of IL-12 and IL-23 and inhibits IL-12/23 mediated cell signaling, activation, and cytokine production	Clarke et al. 2010; Benson et al. 2011; Tsakok et al. 2019
	Abatacept	CD80/CD86	Prevents APC from engaging CD28 costimulatory signal required for T-cell activation	Herrero-Beaumont et al. 2012; Douthwaite et al. 2017; Li et al. 2019b
Inhibitors of lymphocyte adhesion, and trafficking	Natalizumab	integrin	Inhibits α4-mediated adhesion on lymphocytes	Leger et al. 1997; Stuve and Bennett 2007; Sehr et al. 2016
	Fingolimod/FTY720	Sphingosine-1-phosphate (S1P) receptor agonist	Internalizes sphingosine-1-phosphate receptors (S1PR) on lymphocytes to prevent egress from lymph nodes	Volpi et al. 2019
Checkpoint inhibitors	Nivolumab	PD-1	Blocks the inhibitory signal from PD-L1/PD-L2 and leads to augmented T-cell activation	Wang et al. 2014
	Atezolizumab	PD-L1	Blocks its interactions with PD-1 and B7.1 and leads to increased T-cell activation	de Sousa Linhares et al. 2019

Table 2. IC₅₀ values of immunosuppressant agents.

Agent	CD107a (nM)	IFNγ (nM)	Number of donors
Dexamethasone	3.6 ± 1.6	8.0 ± 4.0	n = 4
Prednisolone	32.2 ± 18.0	31.8 ± 10.3	n = 6
Tofacitinib	12.9 ± 3.4	11.7 ± 4.0	n = 4
Ruxolitinib	6.9 ± 2.6	5.0 ± 1.2	n = 6
Mycophenolic acid	83.9 ± 25.0	128.8 ± 29.5	n = 6
Teriflunomide	290.6 ± 99.4	284.5 ± 156.2	n = 8 (3*)
Tacrolimus (FK506)	0.6 ± 0.2	0.3 ± 0.1	n = 5
Rapamycin	0.4 ± 1.4	0.5 ± 0.2	n = 6
Apremilast	16.8 ± 28.3	67.4 ± 62.3	n = 8 (2*)

*Number of donors whose cells did not show CTL suppression in the assay.

Figure 3. Biologics and compounds targeting cytokine or cytokine receptors, co-stimulation, or lymphocyte migration did not impact CTL activity in vitro. Percentage CD8⁺ T-cells with CD107a surface staining (left panel) and IFNγ production (right panel) were measured at different drug concentrations. Each data point is mean (±SD) from a single donor run in duplicate. Data are representative of individual donors of multiple independent experiments with several donors (n). (A) Adalumumab (n = 3). (B) Infliximab (n = 3). (C) Tociluzumab (n = 3). (D) Ustekinumab (n = 3). (E) Abatacept (n = 6). (F) Nataluziumab (n = 3). (G) Fingolimod/FTY720 (n = 6). Drugs were tested in separate experiments.

Figure 4. Anti-PD-1/PD-L1 immune checkpoint blockades enhanced CTL activity in vitro. Percentage CD8⁺ T-cells with CD107a surface staining (left panel) and IFNγ production (right panel) were measured at different drug concentrations. Each data point is mean (±SD) from a single donor run in duplicate. Data are representative of individual donors of multiple independent experiments with several donors (n). (A) Anti-PD-1 (n = 3). (B) Anti-PD-L1 (n = 3). Drugs were tested in separate experiments.

Barnes P. 2006. How corticosteroids control inflammation: Quintiles Prize Lecture 2005. Br J Pharmacol. 148(3):245–254.

 PubMed Web of Science ®Google Scholar

Wust S, van den Brandt J, Tischner D, Kleiman A, Tuckermann J, Gold R, Luhder F, Reichardt H. 2008. Peripheral T-cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis. J Immunol. 180(12):8434–8443.

 PubMed Web of Science ®Google Scholar

Shefrin A, Goldman R. 2009. Use of dexamethasone and prednisone in acute asthma exacerbations in pediatric patients. Can Fam Physician. 55(7):704–706.

 PubMed Web of Science ®Google Scholar

Hodge J, Kawabata T, Krishnaswami S, Clark J, Telliez J, Dowty M, Menon S, Lamba M, Zwillich S. 2016. The mechanism of action of tofacitinib – an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis. Clin Exp Rheumatol. 34(2):318–328.

 PubMed Web of Science ®Google Scholar

Elli E, Barate C, Mendicino F, Palandri F, Palumbo G. 2019. Mechanisms underlying the anti-inflammatory and immunosuppressive activity of ruxolitinib. Front Oncol. 9:1186.

 PubMed Web of Science ®Google Scholar

Allison A, Eugui E. 2000. Mycophenolate mofetil and its mechanisms of action. Immunopharmacology. 47(2–3):85–118.

 PubMed Web of Science ®Google Scholar

Fox R, Herrmann M, Frangou C, Wahl G, Morris R, Strand V, Kirschbaum B. 1999. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 93(3):198–208.

 PubMed Web of Science ®Google Scholar

Bar-Or A. 2014. Teriflunomide (Aubagio(R)) for the treatment of multiple sclerosis. Exp Neurol. 262:57–65.

 PubMed Web of Science ®Google Scholar

Thomson A, Bonham C, Zeevi A. 1995. Mode of action of tacrolimus (FK506): Molecular and cellular mechanisms. Ther Drug Monit. 17(6):584–591.

 PubMed Web of Science ®Google Scholar

Jacobson P, Uberti J, Davis W, Ratanatharathorn V. 1998. Tacrolimus: A new agent for the prevention of graft-versus-host disease in hematopoietic stem cell transplantation. Bone Marrow Transplant. 22(3):217–225.

 PubMed Web of Science ®Google Scholar

Dumont F, Su Q. 1996. Mechanism of action of the immunosuppressant rapamycin. Life Sci. 58(5):373–395.

 PubMed Web of Science ®Google Scholar

Schafer P. 2012. Apremilast mechanism of action and application to psoriasis and psoriatic arthritis. Biochem Pharmacol. 83(12):1583–1590.

 PubMed Web of Science ®Google Scholar

Moller A, Emling F, Blohm D, Schlick E, Schollmeier K. 1990. Monoclonal antibodies to human TNFα: In vitro and in vivo application. Cytokine. 2(3):162–169.

 PubMedGoogle Scholar

Cornillie F, Shealy D, D'Haens G, Geboes K, van Assche G, Ceuppens J, Wagner C, Schaible T, Plevy S, Targan S, et al. 2001. Infliximab induces potent anti-inflammatory and local immunomodulatory activity but no systemic immune suppression in patients with Crohn's disease. Aliment Pharmacol Ther. 15(4):463–473.

 PubMed Web of Science ®Google Scholar

Weisman M, Moreland L, Furst D, Weinblatt M, Keystone E, Paulus H, Teoh L, Velagapudi R, Noertersheuser P, Granneman G, et al. 2003. Efficacy, pharmacokinetic, and safety assessment of adalimumab, a fully human anti-TNFα monoclonal antibody, in adults with rheumatoid arthritis receiving concomitant methotrexate: Pilot study. Clin Ther. 25(6):1700–1721.

 PubMed Web of Science ®Google Scholar

Buurman D, Blokzijl T, Festen E, Pham B, Faber K, Brouwer E, Dijkstra G. 2018. Quantitative comparison of the neutralizing capacity, immunogenicity and cross-reactivity of anti-TNFα biologicals and an Infliximab-biosimilar. PLOS One. 13(12):e0208922.

 PubMed Web of Science ®Google Scholar

Mihara M, Kasutani K, Okazaki M, Nakamura A, Kawai S, Sugimoto M, Matsumoto Y, Ohsugi Y. 2005. Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family. Int Immunopharmacol. 5(12):1731–1740.

 PubMed Web of Science ®Google Scholar

Ogata A, Hirano T, Hishitani Y, Tanaka T. 2012. Safety and efficacy of tocilizumab for the treatment of rheumatoid arthritis. Clin Med Insights Arthritis Musculoskelet Disord. 5:27–42.

 PubMedGoogle Scholar

Clarke A, Poulton L, Wai H, Walker S, Victor S, Domagala T, Mraovic D, Butt D, Shewmaker N, Jennings P, et al. 2010. A novel class of anti-IL-12p40 antibodies: Potent neutralization via inhibition of IL-12-IL-12Rβ2 and IL-23-IL-23R. MAbs. 2(5):539–549.

 PubMed Web of Science ®Google Scholar

Benson J, Sachs C, Treacy G, Zhou H, Pendley C, Brodmerkel C, Shankar G, Mascelli M. 2011. Therapeutic targeting of the IL-12/23 pathways: Generation and characterization of ustekinumab. Nat Biotechnol. 29(7):615–624.

 PubMed Web of Science ®Google Scholar

Tsakok T, Wilson N, Dand N, Loeff F, Bloem K, Baudry D, Duckworth M, Pan S, Pushpa-Rajah A, Standing J, et al. 2019. Association of serum ustekinumab levels with clinical response in psoriasis. JAMA Dermatol. 155(11):1235–1243.

 PubMed Web of Science ®Google Scholar

Herrero-Beaumont G, Martinez Calatrava M, Castaneda S. 2012. Abatacept mechanism of action: Concordance with its clinical profile. Reumatol Clin. 8(2):78–83.

 PubMedGoogle Scholar

Douthwaite J, Moisan J, Privezentzev C, Soskic B, Sabbah S, Cohen S, Collinson A, England E, Huntington C, Kemp B, et al. 2017. A CD80-biased CTLA4-Ig fusion protein with superior in vivo efficacy by simultaneous engineering of affinity, selectivity, stability, and FcRn Binding. J Immunol. 198(1):528–537.

 PubMed Web of Science ®Google Scholar

Li X, Roy A, Murthy B. 2019b. Population pharmacokinetics and exposure-response relationship of intravenous and subcutaneous abatacept in patients with rheumatoid arthritis. J Clin Pharmacol. 59(2):245–257.

 PubMed Web of Science ®Google Scholar

Leger O, Yednock T, Tanner L, Horner H, Hines D, Keen S, Saldanha J, Jones S, Fritz L, Bendig M. 1997. Humanization of mouse antibody against human α4 integrin: Potential therapeutic for the treatment of multiple sclerosis. Hum Antibodies. 8(1):3–16.

 PubMedGoogle Scholar

Stuve O, Bennett J. 2007. Pharmacological properties, toxicology and scientific rationale for the use of natalizumab (Tysabri) in inflammatory diseases. CNS Drug Rev. 13(1):79–95.

 PubMedGoogle Scholar

Sehr T, Proschmann U, Thomas K, Marggraf M, Straube E, Reichmann H, Chan A, Ziemssen T. 2016. New insights into pharmacokinetics and pharmacodynamics of natalizumab treatment for patients with multiple sclerosis, obtained from clinical and in vitro studies. J Neuroinflammation. 13:164.

 PubMedGoogle Scholar

Volpi C, Orabona C, Macchiarulo A, Bianchi R, Puccetti P, Grohmann U. 2019. Preclinical discovery and development of fingolimod for the treatment of multiple sclerosis. Expert Opin Drug Discov. 14(11):1199–1212.

 PubMed Web of Science ®Google Scholar

Wang C, Thudium K, Han M, Wang X, Huang H, Feingersh D, Garcia C, Wu Y, Kuhne M, Srinivasan M, et al. 2014. In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer Immunol Res. 2(9):846–856.

 PubMed Web of Science ®Google Scholar

de Sousa Linhares A, Battin C, Jutz S, Leitner J, Hafner C, Tobias J, Wiedermann U, Kundi M, Zlabinger G, Grabmeier-Pfistershammer K, et al. 2019. Therapeutic PD-L1 antibodies are more effective than PD-1 antibodies in blocking PD-1/PD-L1 signaling. Sci Rep. 9(1):11472.

 PubMedGoogle Scholar