Adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer

Abstract

Administration of anti-4-1BB mAb has been found to be a potent adjuvant when combined with other therapeutic approaches, e.g. chemotherapy, cytokine therapies, anti-OX40 therapy, and peptide or DC vaccines. However, the adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer has not been fully evaluated. In this report, effector T cells were generated in vitro by anti-CD3/anti-CD28 activation of tumor-draining lymph node (TDLN) cells and used in an adoptive immunotherapy model. While T cells or anti-4-1BB alone showed no therapeutic efficacy in mice bearing macroscopic 10-day pulmonary metastases, T cells plus anti-4-1BB mediated significant tumor regression in an anti-4-1BB dose dependent manner. Mice bearing microscopic 3-day lung metastases treated with T cells alone demonstrated tumor regression which was significantly enhanced by anti-4-1BB administration. NK cell depletion abrogated the augmented therapeutic efficacy rendered by anti-4-1BB. Cell transfer between congenic hosts demonstrated that anti-4-1BB administration increased the survival of adoptively transferred TDLN cells. Using STAT4(-/-) mice, we found that modulated IFN gamma secretion in wt TDLN cells after anti-CD3/CD28/4-1BB activation in vitro was lost in similarly stimulated STAT4(-/-) TDLN cells. Additionally, anti-4-1BB administration failed to augment the therapeutic efficacy of T cell therapy in STAT4(-/-) mice. Together, these results indicate that administered anti-4-1BB mAb can serve as an effective adjuvant to augment the antitumor reactivity of adoptively transferred T cells by recruiting the host NK cells; increasing the persistence of infused effector T cells, and modulating the STAT4 molecular signaling pathway.

Keywords: Adoptive immunotherapy; Anti-4-1BB; Cancer; NK cells; STAT4; T cells.

Figure 1

Anti-4-1BB mAb administration augments the efficacy of adoptive T cell therapy in the treatment of 10-day established MCA 205 pulmonary metastases. B6 mice were inoculated i.v. with 2 x 10⁵ MCA 205 tumor cells to induce pulmonary metastases. Mice with 10-day established pulmonary metastases were treated either by the adoptive transfer of anti-CD3/anti-CD28-activated MCA 205 TDLN cells alone, or with anti-4-1BB alone at 25 μg or 100 μg i.p. on days 0 and 3 following T cell adoptive transfer on day 0, or with T cells plus anti-4-1BB administration. Commencing on the day of the cell transfer (day 0), intraperitoneal (i.p.) injections of IL-2 (40,000IU) were administered in 0.5 ml of PBS and continued twice daily for eight doses. *p<0.05 compared with any other groups. Data are representative of two independent experiments.

Figure 2

Anti-4-1BB mAb-enhanced therapeutic efficacy is partially mediated by host NK cells. Treatment of 3-day established MCA 205 pulmonary metastases was performed as in Figure 1, but neutralizing anti-NK1.1 mAb was used to deplete NK cells. MCA 205 tumor cells were injection on day -3 to establish pulmonary metastasis. Anti-NK1.1 (400 μg/mouse) was injected i.p. to deplete host NK cell on day -1 and day 5. Activated and expanded TDLN cells (2x10^6) were transferred on day 0 with IL-2 administration. Anti-4-1BB mAb (100 μg) was administrated i.p. on day 0 and day 3. Lungs were harvested two weeks later for enumeration of pulmonary metastatic nodules. *p < 0.05 versus all other groups. Data are representative of two independently performed experiments.

Figure 3

Anti-4-1BB administration enhances the survival of adoptively transferred TDLN cells. CD45.2 mice received intravenous adoptive transfer of activated and expanded CD45.1 TDLN cells on day 0. Anti-4-1BB (100μg/mouse) was administrated i.p on day 0 and day 3. Spleens were harvested and blood samples were collected on days 1, 4, and 9 after cell infusion. The absolute number of donor CD45.1⁺CD3⁺ cells was determined using fluorochrome-conjugated mAb staining and flow cytometry analysis as described in Materials and Methods. A, Data are reported as the mean number of CD45.1⁺CD3⁺ cells of 5 mice/group in the spleen. Two independently performed experiments are shown. B, Mean number of CD45.1⁺CD3⁺ cells detected per 70 microliter of blood per mouse. There were 5 mice each group.

Figure 3

Anti-4-1BB administration enhances the survival of adoptively transferred TDLN cells. CD45.2 mice received intravenous adoptive transfer of activated and expanded CD45.1 TDLN cells on day 0. Anti-4-1BB (100μg/mouse) was administrated i.p on day 0 and day 3. Spleens were harvested and blood samples were collected on days 1, 4, and 9 after cell infusion. The absolute number of donor CD45.1⁺CD3⁺ cells was determined using fluorochrome-conjugated mAb staining and flow cytometry analysis as described in Materials and Methods. A, Data are reported as the mean number of CD45.1⁺CD3⁺ cells of 5 mice/group in the spleen. Two independently performed experiments are shown. B, Mean number of CD45.1⁺CD3⁺ cells detected per 70 microliter of blood per mouse. There were 5 mice each group.

Figure 4

Lack of modulated IFNγ secretion in STAT4^-/- TDLN cells post CD3/CD28/4-1BB activation in vitro. TDLN cells from wt or STAT4^-/- mice were activated with anti-CD3/anti-CD28 with or without anti-4-1BB followed by culture in IL-2. Supernatants at the end of cell culture were collected to examine the IFNγ secretion using ELISA. Two independently performed experiments are shown.

Figure 5

Anti-4-1BB administration fails to augment the therapeutic efficacy of T cell therapy in STAT4^-/- mice. MCA 205 TDLN cells were induced in STAT4^-/- mice and were activated with anti-CD3/anti-CD28 followed by IL-2 expansion to generate effector T cells for adoptive therapy. Three-day MCA205 pulmonary metastases were established in the STAT4^-/- mice as in Figure 3. Activated and expanded STAT4^-/- TDLN cells (2x10^6) were adoptively transferred into tumor bearing STAT4^-/- mice followed by IL-2 injection (4x10^4 IU twice daily for 4 days) with or without anti-4-1BB administration (100μg on day 0 and day 3 relevant to T cell transfer on day 0). Fifteen to 17 days after tumor injection, all mice were euthanized for enumeration of pulmonary metastasis nodules. Data are representative of two independently performed experiments.