Enhancement of Antitumor Immunity by CTLA-4 Blockade
Our immune system protects us daily against thousands of pathogens that try to invade. But when our own cells go rogue, the immune system is not always capable of protecting us. Have you ever wondered how cancer can go undetected by the immune system’s T cells? Nobel laureate James Allison and the co-authors of this paper take a deeper look at CTLA-4, a molecule found on the surface of T cells. CTLA-4 is one of the brakes of the immune system. Is it the key to developing an immune response against cancer?
One reason for the poor immunogenicity of many tumors may be that they cannot provide signals for CD28-mediated costimulation necessary to fully activate T cells. It has recently become apparent that CTLA-4, a second counterreceptor for the B7 family of costimulatory molecules, is a negative regulator of T cell activation. Here, in vivo administration of antibodies to CTLA-4 resulted in the rejection of tumors, including preestablished tumors. Furthermore, this rejection resulted in immunity to a secondary exposure to tumor cells. These results suggest that blockade of the inhibitory effects of CTLA-4 can allow for, and potentiate, effective immune responses against tumor cells.
Despite expressing antigens recognizable by a host's immune system, tumors are very poor in initiating effective immune responses. One reason for this poor immunogenicity may be that the presentation of antigen alone is insufficient to activate T cells. In addition to T cell receptor engagement of an antigenic peptide bound to major histocompatibility complex (MHC) molecules, additional costimulatory signals are necessary for T cell activation (1). The most important of these costimulatory signals appears to be provided by the interaction of CD28 on T cells with its primary ligands B7-1 (CD80) and B7-2 (CD86) on the surface of specialized antigen-presenting cells (APCs) (2-4). Expression of B7 costimulatory molecules is limited to specialized APCs. Therefore, even though most tissue-derived tumors may present antigen in the context of MHC molecules, they may fail to elicit effective immunity because of a lack of costimulatory ability. Several studies support this notion. In a variety of model systems, transfected tumor cells expressing costimulatory B7 molecules induced potent responses against both modified and unmodified tumor cells (5-8). It appears that tumor cells transfected with B7 are able to behave as APCs, presumably allowing direct activation of tumor-specific T cells.
Recent evidence suggests that costimulation is more complex than originally thought and involves competing stimulatory and inhibitory signaling events (3, 9-12). CTLA-4, a homolog of CD28, binds both B7-1 and B7-2 with affinities much greater than does CD28 (13-16). In vitro, antibody cross-linking of CTLA-4 has been shown to inhibit T cell proliferation and interleukin-2 production induced by antibody to CD3 (anti-CD3), whereas blockade of CTLA-4 with soluble intact or Fab fragments of antibody enhances proliferative responses (17, 18). Similarly, soluble intact or Fab fragments of anti-CTLA-4 greatly augment T cell responses to nominal peptide antigen or the superantigen Staphylococcus enterotoxin B in vivo (19, 20). It has also been suggested that CTLA-4 engagement can induce apoptosis in activated T cells (21). Finally, mice deficient in CTLA-4 exhibit severe T cell proliferative disorders (22). These results demonstrate that CTLA-4 is a negative regulator of T cell responses and raise the possibility that blockade of inhibitory signals delivered by CTLA-4-B7 interactions might augment T cell responses to tumor cells and enhance antitumor immunity.
We first sought to determine whether CTLA-4 blockade with nonstimulatory, bivalent antibody (18, 20) would accelerate rejection of B7-positive tumor cells. Previously, we showed that B7-1 expression was partially successful at inducing rejection of the transplantable murine colon carcinoma 51BLim10 (23). We reasoned that CTLA-4 blockade would remove inhibitory signals in the costimulatory pathway, resulting in enhanced rejection of the tumor cells. We injected groups of BALB/c mice with B7-1-transfected 51BLim10 tumor cells (B7-51BLim10) (23). Two groups were treated with a series of intraperitoneal injections of either anti-CTLA-4 or anti-CD28 (18, 24). Treatment with anti-CTLA-4 inhibited B7-51BLim10 tumor growth as compared with the antiCD28-treated mice or the untreated controls (Fig. 1). All mice in the untreated and antiCD28-treated groups developed small tumors that grew progressively for 5 to 10 days and then ultimately regressed in 8 of the 10 mice by about day 23 after injection. The two small tumors that did not regress remained static for more than 90 days. In contrast, three of five mice treated with anti-CTLA-4 developed very small tumors, all of which regressed completely by day 17. Although these results were encouraging and were consistent with our hypothesis, they were not very dramatic because B7-1 expression resulted in fairly rapid rejection of transfected 51BLim10 cells even in the absence of CTLA-4 blockade; however, these results confirmed that anti-CTLA-4 did not inhibit tumor rejection.
We next examined the effects of CTLA-4 blockade on the growth of V51BLim10, a vector control tumor cell line that does not express B7 (23). All mice either injected with 4 x 106 V51BLim10 tumor cells and left untreated, or treated with anti-CD28, developed progressively growing tumors and required euthanasia by 35 days after inoculation (Fig. 2A). In contrast, all mice treated with anti-CTLA-4 completely rejected their tumors after a short period of limited growth. Similarly, control mice injected with 2 x 106 tumor cells developed rapidly growing tumors and required euthanasia by day 35 (Fig. 2B). Anti-CTLA-4 treatment had a dramatic effect on tumor growth, but one mouse did develop a tumor quickly (accounting for a majority of the growth indicated in Fig. 2B) and another developed a tumor much later (Fig. 2C). Anti-CTLA-4 appeared to be less effective at a tumor dose of 1 x 106 cells, where treatment resulted in significantly reduced tumor growth rates, but four of five mice developed progressively growing tumors (25). Thus, although curative responses were not obtained in all cases, it is clear that CTLA-4 blockade significantly enhanced rejection of B7-negative tumor cells.
We next sought to determine whether tumor rejection as a consequence of CTLA-4 blockade was associated with enhanced immunity to a secondary challenge. Mice that had rejected V51BLim10 tumor cells as a result of treatment with anti-CTLA-4 were challenged with 4 x 106 wild-type 51BLim10 cells 70 days after their initial tumor injections. These mice showed significant protection against a secondary challenge as compared with naive controls (Fig. 2D). All control animals had progressively growing tumors by 14 days after injection, developed massive tumor burdens, and required euthanasia by day 35. Only one of the previously immunized mice had a detectable tumor by day 14, and growth of this tumor was very slow. Ultimately, two more tumors developed in the immunized mice 42 days after challenge. Two mice remained tumor-free throughout the course of the experiment. These results demonstrate that tumor rejection mediated by CTLA-4 blockade results in immunologic memory.
To determine whether anti-CTLA-4 treatment could have an effect on the growth of established tumors, we injected groups of mice with 4 x 106 wild-type 51BLim10 tumor cells and treated them with anti-CTLA-4 beginning on day 0 as before, or beginning 7 days later at which time most mice had palpable tumors. Mice treated with anti-CTLA-4 at either time period had significantly reduced tumor growth compared with untreated controls (Fig. 3). In fact, delaying treatment appeared to be more effective, with two of five mice remaining tumor-free beyond 30 days after inoculation.
The effects of anti-CTLA-4 treatment were not limited to variants of the murine colon carcinoma 51BLim10. Similar results were obtained with a rapidly growing fibrocarcinoma of A/JCr mice, SA1N (26) (Fig. 4). All control mice injected subcutaneously with 1 x 106 Sa1N cells developed measurable, rapidly growing tumors within 7 days, whereas only two mice treated with anti-CTLA-4 had tumors by day 30, and one additional mouse developed a tumor around day 40 after injection. The remaining mice were still tumor-free 70 days after injection. In another experiment, control mice injected with 4 x 105 Sa1N tumor cells also developed rapidly growing tumors, whereas 7 of 10 mice treated with anti-CTLA-4 were tumor-free by day 25 after injection (25).
Our results indicate that removing inhibitory signals in the costimulatory pathway can enhance antitumor immunity. Although it has been shown that anti-CTLA-4 interferes with signals that normally down-regulate T cell responses in vivo (17, 18), the exact mechanisms of antitumor immunity elicited by CTLA-4 blockade are not clear. In the case of B7-negative tumors, antigens are most likely transferred to and presented by host APCs (27), where CTLA-4 blockade might effect T cell responses in two nonexclusive ways. First, removal of inhibitory signals may lower the overall threshold of T cell activation and allow normally unreactive T cells to become activated. Alternatively, CTLA-4 blockade might sustain proliferation of activated T cells by removing inhibitory signals that would normally terminate the response, thus allowing for greater expansion of tumor-specific T cells.
Regardless of the mechanism, it is clear that CTLA-4 blockade enhances antitumor responses. Most importantly, we have observed these effects against unmanipulated, wild-type tumors. Current methods of enhancing antitumor immunity generally require the engineering of tumor cells (8). Some of these methods, such as the induction of B7 expression, rely on enhancing the costimulatory activity of the tumor cells themselves. Others, such as engineering tumor cells to express MHC class II molecules (26, 28, 29) or to produce granulocyte-macrophage colony-simulating factor (27, 30, 31) or pulsing dendritic cells with antigen ex vivo (32, 33), seek to enhance antigen presentation, antigen transfer, or both. Thus, CTLA-4 blockade, by removing potentially competing inhibitory signals, may be a particularly useful adjunct to other therapeutic approaches involving the costimulatory pathway.
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34. We thank S. Ostrand-Rosenberg and R. Warren for providing tumor lines. Supported by NIH grants CA57986, CA09179, and CA40041.