For antitumor x anti-CD3 bifunctional antibody (BFA) therapy to be clinically relevant in solid tumors, activated lymphocytes must be present within tumors. Toward that end, three uniquely different in vivo activation approaches were investigated in a p97 human antigen expressing syngeneic murine melanoma model. beta-Glucan (200 micrograms), staphylococcal enterotoxin B (SEB) (50 micrograms), and F(ab')2 BFA (10 micrograms) were tested for their ability to activate lymphocytes, neutralize pulmonary metastases, and treat established tumors. Systemic activation, measured as the ability of splenocytes to lyse tumor cells in vitro in the presence of BFA, was enhanced by the in vivo administration of SEB but not by beta-glucan or F(ab')2 BFA. Despite lacking a systemic effect, F(ab')2 BFA increased both direct and BFA-mediated cytotoxicity in fresh tumor infiltrating lymphocytes. beta-Glucan did not increase systemic or intratumor T cell activation. However, it significantly enhanced the ability of splenocytes to lyse NK-sensitive YAC-1 cells. When tested in a pulmonary metastases model, all three forms of immune modulation combined with F(ab')2 BFA significantly reduced the number of metastases. BFA were more effective at tumor neutralization when combined with SEB compared with adoptively transferred, in vitro-activated splenocytes. These studies demonstrate that immune modulators when combined with F(ab')2 BFA can provide effective antitumor therapy. Several clinical obstacles may be overcome by the application of these reagents.

The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor x anti-CD3 F(ab')2 bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 gamma g intraperitoneal) with or without bsAb (5 micrograms i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P = 0.02 and P < 0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97-parental cell line, K1735(P = 0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and non-targeted tumor antigens.

In vitro-activated T lymphocytes can be retargeted with anti-CD3 X anti-tumor bispecific antibodies (BsAb) t o kill tumor cells in vitro and in vivo. The purpose of the present study was to examine the systemic and intra-tumor effects of in vivo T-cell activation with BsAb, staphylococcal enterotoxin B (SEB), and P-glucan in combination with BsAb as a retargeting agent. CL-62 melanoma cells were injected subcutaneously into C3H/ HeN mice. Mice were subsequently treated with BsAb alone or with SEB or P-glucan plus BsAb. Fresh splenocytes, lymph-node cells and tumor-infiltrating lymphocytes (TlL) were tested for their proliferative response using incorporation of 3H-thymidine, and for their ability to lyse CL-62 cells in the presence or absence of BsAb in 4-hr S'Chromium release assays. Toxicity of treatments was assessed in a D-galactosamine model. BsAb, alone or combined with P-glucan, had essentially no effect on systemic T-cell cytotoxicity, and essentially no effect on systemic proliferation, unless exogenous IL-2 was provided. A t the tumor site, BsAb alone, BsAb plus P-glucan, and SEB plus BsAb all significantly increased BsAb-mediated TIL cytotoxicity. In contrast to the TIL-limited effects of BsAb and of BsAb plus p-glucan, SEB plus BsAb markedly increased both systemic and intra-tumor T-lymphocyte activation. Toxicity correlated with measures of systemic activation, with limited effects from BsAb alone and from P-glucan plus BsAb, and with marked lethality from SEB plus BsAb. Overall, these results suggest moderate intra-tumor activation of TIL, but no measurable systemic activation after in vivo treatment with BsAb or p-glucan plus BsAb. SEB plus BsAb results in complete T-cell activation in both systemic and intra-tumor compartments, but at the expense of increased systemic toxicity. In conclusion, TIL can be activated in situ with different combinations of in vivo activants. In vivo-activated TIL can be retargeted with bispecific antibodies to lyse tumor cells, and may be an alternative to ex vivo T-cell activation and adoptive transfer therapy.o 1996 Wiley-Liss, Inc.

In vitro-activated T lymphocytes can be retargeted with anti-CD3 X anti-tumor bispecific antibodies (BsAb) t o kill tumor cells in vitro and in vivo. The purpose of the present study was to examine the systemic and intra-tumor effects of in vivo T-cell activation with BsAb, staphylococcal enterotoxin B (SEB), and P-glucan in combination with BsAb as a retargeting agent. CL-62 melanoma cells were injected subcutaneously into C3H/ HeN mice. Mice were subsequently treated with BsAb alone or with SEB or P-glucan plus BsAb. Fresh splenocytes, lymph-node cells and tumor-infiltrating lymphocytes (TlL) were tested for their proliferative response using incorporation of 3H-thymidine, and for their ability to lyse CL-62 cells in the presence or absence of BsAb in 4-hr S'Chromium release assays. Toxicity of treatments was assessed in a D-galactosamine model. BsAb, alone or combined with P-glucan, had essentially no effect on systemic T-cell cytotoxicity, and essentially no effect on systemic proliferation, unless exogenous IL-2 was provided. A t the tumor site, BsAb alone, BsAb plus P-glucan, and SEB plus BsAb all significantly increased BsAb-mediated TIL cytotoxicity. In contrast to the TIL-limited effects of BsAb and of BsAb plus p-glucan, SEB plus BsAb markedly increased both systemic and intra-tumor T-lymphocyte activation. Toxicity correlated with measures of systemic activation, with limited effects from BsAb alone and from P-glucan plus BsAb, and with marked lethality from SEB plus BsAb. Overall, these results suggest moderate intra-tumor activation of TIL, but no measurable systemic activation after in vivo treatment with BsAb or p-glucan plus BsAb. SEB plus BsAb results in complete T-cell activation in both systemic and intra-tumor compartments, but at the expense of increased systemic toxicity. In conclusion, TIL can be activated in situ with different combinations of in vivo activants. In vivo-activated TIL can be retargeted with bispecific antibodies to lyse tumor cells, and may be an alternative to ex vivo T-cell activation and adoptive transfer therapy.o 1996 Wiley-Liss, Inc.

Bifunctional Abs (BFA) with specificity for the TCR/CD3 complex of T cells and tumor Ag can bridge T lymphocytes and tumor cells and, thereby, trigger activation events. The ability of intact and F(ab')2 anti-CD3 (500A2) x anti-p97 (96.5) BFA to induce activation of T lymphocytes in the presence of murine melanoma tumor cells (CL-62) expressing human melanoma-associated Ag (p97) was investigated in vitro and in vivo. Intact and F(ab')2 BFA induced significant proliferation of T lymphocytes in the presence of p97+ tumor cells. Incubation of splenocytes with intact or F(ab')2 BFA and p97+ tumor cells increased BFA-mediated cytotoxicity against relevant tumor cells. Intact BFA, in contrast to F(ab')2 BFA, induced some activation of T cells in vitro even in the absence of p97+ target cells. In nontumor-bearing mice, administration of F(ab')2 BFA, in contrast to intact BFA, did not increase cytotoxic activity of lymph node (LN) cells and splenocytes. However, when F(ab')2 BFA was administrated into CL-62-bearing mice, an increase of BFA-mediated cytotoxicity of tumor-infiltrating lymphocytes, but not splenocytes nor LN cells, was observed. Moreover, in D-galactosamine-sensitized mice, injection of intact BFA (1 microgram/mice) induced 100% lethality, whereas the same dose of F(ab')2 BFA was not toxic. These results demonstrate that F(ab')2 BFA can induce activation of cytotoxic lymphocytes only in the presence of relevant tumor cells, both in vitro and in vivo. That these activated lymphocytes can be redirected to lyse relevant tumor cells by the same BFA has important implications for the clinical application of BFA anti-tumor therapies.