The Classical B71 B72 and CD28 CTLA4 Pathways

Constitutively expressed on naïve peripheral T-cell, CD28 plays an essential role for effective T-cell activation. It delivers a primary costimulatory signal upon the engagement of its ligands B7-1 (CD80) or B7-2 (CD86) expressed on APCs (37). CD28 signaling promotes antigen-specific T-cell proliferation, produces of an array of cytokines, and leads to the differentiation of effector T-cells (37-40). To the contrary, ligation of the homologous CTLA-4 receptor on activated T-cells with the same B7-1 or B7-2 ligands will attenuate T-cell response (41-43). Unlike CD28, surface expression of CTLA-4 is only induced on activated T-cells. CTLA-4 also has a much higher binding affinity with B7 ligands than CD28 (44) and, thus, could effectively compete for ligand binding. The critical negative regulatory role of CTLA-4 for T-cell activation was best demonstrated in CTLA-4-deficient mice, which had massive enlargement of lymphoid organs resulting from uncontrolled T-cell proliferation, and fatal multiorgan destruction caused by lymphocyte infiltration (45,46).

More than a decade ago, introduction of the costimulatory molecule B7-1 into tumor cells was shown to enhance antitumor immunity (47). An immunogenic melanoma (K1735) expressing E7 viral gene product could grow progressively in immunocompetent murine hosts. Transfection of E7+ K1735 with B7-1 resulted in tumor regression by a B7-1-depen-dent response mediated by CD8+ T-cells. The induced host antitumor response also led to the regression of a parental B7-1-negative tumor at distal site, indicating a therapeutic potential for even metastatic tumors. Mechanistic studies demonstrated that enhanced priming of tumor-specific CTLs, through both direct priming (antigen presented by tumor) (47) and cross-priming (antigen presented by tumor site APCs) (48), played major roles in B7+ tumor-induced immunity. Subsequent studies, however, revealed that the effectiveness of T-cell immunity enhanced by B7-1 transfection correlates directly to tumor immu-nogenicity. Poorly immunogenic tumors, such as sarcomas MCA101 and melanoma B16, remained progressive despite expression of the B7-1 (49).

To treat less immunogenic tumors and achieve a better response against large established tumors, several independent groups have reported synergistic antitumor effects with combined strategies of B7 overexpression with either systemic treatment or overexpression of immune response stimulatory cytokines, including interferon (IFN)-y (50), interleukin (IL)-2 (51,52), IL-7 (53), IL-10 (54), IL-12 (55), IL-15 (56), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (57). In all of these models, the combined strategies led to effective tumor rejection and establishment of immunity against the parental unmodified tumor. An even greater antitumor effect was observed when both MHC class II and B7-1 were cotransfected into mouse sarcoma I tumor, indicating that the induction of both CD4+ and CD8+ tumor-specific T-cell response will boost the antitumor effects (58).

In addition to enhancement of positive signals, suppression of coinhibitory pathways has also been explored and successfully implemented to induce strong T-cell-mediated antitumor response in animal models. The ability of CTLA-4 to prevent continuous T-cell activation makes it an ideal target. First shown by Leach and colleagues in 1995, then further investigated by other groups, systemic administration of CTLA-4 blocking antibody could enhance the potency of tumor vaccine and generate a stronger antitumor immunity (59-62). Another potential mechanism behind the CTLA-4 mAb antitumor effect is that CD4+ CD25+T-reg cells constitutively express a high level of cell surface CTLA-4, which is essential for T-reg cell function in some models (63,64). Application of CTLA-4 mAb will directly inhibit T-reg cell or cause T-reg cell depletion in vivo. Similar to the combined strategy with B7 transfection, systematic administration of GM-CSF also has a syn-ergistic effect when applied with CTLA-4 blockade (60,65). This reveals a promising approach that is undergoing clinical trials: enhancement of host APC function together with modulation of costimulatory pathway to maximize antitumor immunity. Soluble antibodies hold practical advantages clinically because they bypass the requirement of complicated in vitro tumor manipulation. In theory, agonistic CD28 antibody should also have antitumor effects because it could significantly costimulate T-cell proliferation and cytokine production in vitro. Ironically, there is no in vivo antitumor activity reported for CD28

mAb to date. One possible explanation is that CD28 antibody could not fully mimic the binding of B7 ligands, which might depend on cell-cell contact and coordination of other costimulatory molecules.

Two recent reports showed that significant autoimmune responses could be induced by infusion of anti-CTLA-4 mAb in cancer patients with metastatic melanoma (66,67), although autoreactivity was relatively mild in mouse tumor models (60). Six out of fourteen patients manifested severe autoimmune responses, which were mediated by massive infiltration of lymphoid cells into various organs with the symptoms of dermatitis, colitis, and hepatitis. The antitumor effect of the anti-CTLA-4 mAb was moderate: Out of fourteen total patients, two experienced complete tumor regression and one had partial response (67). These studies thus highlighted the critical role of CTLA-4 in the control of autoimmune responses and demand further studies on mechanistic aspects of CTLA-4-mediated suppression before further extensive clinical trials.

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