J Korean Surg Soc.  2010 Jan;78(1):7-16. 10.4174/jkss.2010.78.1.7.

Investigation of the Effective Dose of Agonistic 4-1BB Monoclonal Antibody in a Murine Colon Cancer Metastasis Model

• Kim JM
• Kim SJ
• Joh JW
• Kwon CH
• Park H
• Shin M
• Kim EY
• Moon JI
• Jung GO
• Choi GS
• Lee SK

• KMID: 1775439
• DOI: http://doi.org/10.4174/jkss.2010.78.1.7

Abstract

PURPOSE: The aim of this study was to find the dose of agonistic 4-1BB monoclonal antibody (mAb) that results in optimal T cell activation.
METHODS
Cancer was induced in mice by an intrahepatic parenchymal injection of 1x10(5) cells of CT26 cells. Cancer-carrying mice (n=84) were divided into seven groups and treated with either rat IgG or agonistic 4-1BB monoclonal antibody (mAb) (5microgram, 10microgram, 20microgram, 100microgram, 200microgram, or 300microgram). All treatments were administered intraperitoneally on days 7, 9, and 11. Mice from each group were sacrificed on days 14, 28, and 42. Harvested livers were weighed and the numbers of T cells in the splenocytes were analyzed with a FACS Vantage flow cytometer.
RESULTS
Liver weights increased when 5microgram of agonistic 4-1BB mAb was administered, but showed no additional weight increase for doses greater than 10microgram. The absolute numbers of CD4+ and CD8+ T cells increased in groups treated with low doses of agonistic 4-1BB mAb (5microgram, 10microgram, or 20microgram), but did not increase in the groups treated with high doses of mAb (100microgram, 200microgram, or 300microgram). The levels of CD4/annexin V and CD8/annexin V increased as the dose increased, and the absolute cell numbers of CD4/annexin V were greater than those of CD8/annexin V.
CONCLUSION
Liver weight, including the cancer mass, failed to increase at agonistic 4-1BB mAb doses greater than 10microgram. A high dose (> or =100microgram) of agonistic 4-1BB mAb resulted in lower counts of absolute T cells. This study suggests that a low dose (20microgram) of agonistic 4-1BB mAb can be used for optimal T cell activation in combination with other anti-cancer treatments.

Keyword

4-1BB; 4-1BB monoclonal antibody; Optimal T cell activation; Murine colon cancer metastasis model; Anti-tumor effects; Activation-induced cell death

MeSH Terms

Animals
Cell Count
Colon
Colonic Neoplasms
Immunoglobulin G
Liver
Mice
Neoplasm Metastasis
Rats
T-Lymphocytes
Weights and Measures
Immunoglobulin G

Figure

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  Fig. 1 Change in liver size with dose of agonistic 4-1BB monoclonal antibodies and over time.

• 

  Fig. 2 Liver weights and doses of agonistic 4-1BB monoclonal antibodies. Weight increased for the control and with 5µg of agonistic 4-1BB mAb, but showed no increase at doses ≥10µg. The absolute cell numbers of the control and the 5µg dose group significantly increased over time (*P<0.05).

• 

  Fig. 3 (A) CD4, (B) CD4/CD62L low cell population, and (C) CD4/CD25 cell population. The absolute cell numbers increased in the low dose (≤20µg) groups, but not in the high dose groups (≥100µg). Absolute cell numbers peaked on day 28 in the high dose groups and decreased by day 42. In contrast, the absolute cell numbers on day 42 were significantly higher than those at day 14 for doses of agonistic 4-1BB mAb (≤20µg; *P<0.05).

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  Fig. 4 (A) CD8, (B) CD8/CD62L low cells population, and (C) interferon-gamma (IFN-γ) producing CD8 cell population. The absolute cell numbers of CD8 T cells increased with 5µg of agonistic 4-1BB mAb, but did not increase with 10 µg or 20µg treatments. The absolute cell number of CD8 T cell for 100µg of mAb peaked on day 14, decreased on day 28 and decreased further on day 42 (P<0.05). However, absolute cell numbers of T cells peaked at day 28 for 200µg and 300µg mAb. The absolute cell numbers of IFN-γ producing CD8 T cells gradually decreased over time at high doses of agonistic 4-1BB mAb (≥100µg; P<0.05).

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  Fig. 5 Apoptotic cell proportion. (A) CD4/annexcin V, (B) CD8/annexin V. CD4/annexin V and CD8/annexin V levels in the high dose groups (100µg, 200µg, 300µg of agonistic 4-1BB mAb) peaked on day 28. The absolute cell number for 300µg mAb was higher than that for 100µg (P<0.05). The absolute number of CD4/annexin V cells was greater than that of CD8/annexin V cells (P<0.05).

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  Fig. 6 Innate immunity. (A) CD11b, (B) CD11c, (C) natural killer (NK) cells. The absolute cell numbers increased in the low dose groups (5µg, 10µg, or 20µg of agonistic 4-1BB mAb), but not in the high dose groups (100µg, 200µg, 300µg).

Reference

1. Vinay DS, Cha K, Kwon BS. Dual immunoregulatory pathways of 4-1BB signaling. J Mol Med. 2006. 84:726–736.

2. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol. 2005. 23:515–548.

3. Watts TH. TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol. 2005. 23:23–68.

4. Cheuk AT, Mufti GJ, Guinn BA. Role of 4-1BB:4-1BB ligand in cancer immunotherapy. Cancer Gene Ther. 2004. 11:215–226.

5. Kwon BS, Kozak CA, Kim KK, Pickard RT. Genomic organization and chromosomal localization of the T-cell antigen 4-1BB. J Immunol. 1994. 152:2256–2262.

6. Pollok KE, Kim YJ, Zhou Z, Hurtado J, Kim KK, Pickard RT, et al. Inducible T cell antigen 4-1BB. Analysis of expression and function. J Immunol. 1993. 150:771–781.

7. Melero I, Johnston JV, Shufford WW, Mittler RS, Chen L. NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell Immunol. 1998. 190:167–172.

8. Futagawa T, Akiba H, Kodama T, Takeda K, Hosoda Y, Yagita H, et al. Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int Immunol. 2002. 14:275–286.

9. Laderach D, Movassagh M, Johnson A, Mittler RS, Galy A. 4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8 (+) T cells. Int Immunol. 2002. 14:1155–1167.

10. Croft M. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat Rev Immunol. 2003. 3:609–620.

11. Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med. 1997. 186:47–55.

12. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellstrom KE, et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997. 3:682–685.

13. Halstead ES, Mueller YM, Altman JD, Katsikis PD. In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses. Nat Immunol. 2002. 3:536–541.

14. Choi BK, Kim YH, Kang WJ, Lee SK, Kim KH, Shin SM, et al. Mechanisms involved in synergistic anticancer immunity of anti-4-1BB and anti-CD4 therapy. Cancer Res. 2007. 67:8891–8899.

15. Sun Y, Chen JH, Fu Y. Immunotherapy with agonistic anti-CD137: two sides of a coin. Cell Mol Immunol. 2004. 1:31–36.

16. Kim J, Choi WS, La S, Suh JH, Kim BS, Cho HR, et al. Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells. Blood. 2005. 105:2206–2213.

17. Miller RE, Jones J, Le T, Whitmore J, Boiani N, Gliniak B, et al. 4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner. J Immunol. 2002. 169:1792–1800.

18. Murillo O, Arina A, Hervas-Stubbs S, Gupta A, McCluskey B, Dubrot J, et al. Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma. Clin Cancer Res. 2008. 14:6895–6906.

19. Seo SK, Choi JH, Kim YH, Kang WJ, Park HY, Suh JH, et al. 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat Med. 2004. 10:1088–1094.

20. Sun Y, Chen HM, Subudhi SK, Chen J, Koka R, Chen L, et al. Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease. Nat Med. 2002. 8:1405–1413.

21. Taraban VY, Rowley TF, O'Brien L, Chan HT, Haswell LE, Green MH, et al. Expression and costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses. Eur J Immunol. 2002. 32:3617–3627.

22. Corbett TH, Griswold DP Jr, Roberts BJ, Peckham JC, Schabel FM Jr. Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure. Cancer Res. 1975. 35:2434–2439.

23. Takahashi C, Mittler RS, Vella AT. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J Immunol. 1999. 162:5037–5040.

24. Wilcox RA, Tamada K, Flies DB, Zhu G, Chapoval AI, Blazar BR, et al. Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo. Blood. 2004. 103:177–184.

25. Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol. 2002. 169:4882–4888.

26. Lee HW, Nam KO, Park SJ, Kwon BS. 4-1BB enhances CD8+ T cell expansion by regulating cell cycle progression through changes in expression of cyclins D and E and cyclin-dependent kinase inhibitor p27kip1. Eur J Immunol. 2003. 33:2133–2141.

27. Cannons JL, Lau P, Ghumman B, DeBenedette MA, Yagita H, Okumura K, et al. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol. 2001. 167:1313–1324.

28. Kwon B, Lee HW, Kwon BS. New insights into the role of 4-1BB in immune responses: beyond CD8+ T cells. Trends Immunol. 2002. 23:378–380.

29. Kim YH, Choi BK, Kim KH, Kang SW, Kwon BS. Combination therapy with cisplatin and anti-4-1BB: synergistic anticancer effects and amelioration of cisplatin-induced nephrotoxicity. Cancer Res. 2008. 68:7264–7269.

30. Li Q, Pan PY, Gu P, Xu D, Chen SH. Role of immature myeloid Gr-1+ cells in the development of antitumor immunity. Cancer Res. 2004. 64:1130–1139.

31. Gray JC, French RR, James S, Al-Shamkhani A, Johnson PW, Glennie MJ. Optimising anti-tumour CD8 T-cell responses using combinations of immunomodulatory antibodies. Eur J Immunol. 2008. 38:2499–2511.