In 2011, the FDA approved the first anti-CTLA-4 antibody, Ipilimumab (trademark name YERVOY®), for the treatment of melanoma. Ipilimumab has demonstrated substantial and durable therapeutic effects, and is now undergoing clinical trials in treating many other cancers. According to the checkpoint blockade hypothesis [1], anti-CTLA-4 antibodies cause tumor rejection by promoting priming of naïve T cells through blocking the inhibitory B7-CTLA-4 signaling in peripheral lymphoid organs (Fig. 1). However, this prevailing hypothesis has not been rigorously tested. Ipilimumab was selected according to its ability in blocking the interaction between anchored CTLA-4 and soluble B7 molecules [2]. However, since B7 ligands are co-stimulatory molecules expressed on cell surface, it remains to be tested whether Ipilimumab can effectively block the B7-CTLA-4 interactions under physiological conditions.

Fig. 1
figure 1

The prevailing view: CTLA-4 checkpoint blockade results in tumor immunity. Activation of T cells requires two signals. One is the binding of the T cell receptor (TCR) to the MHC-antigen peptide complex presented by antigen presenting cells (APCs) (signal 1). The other one is the binding of B7 molecules (B7-1 or B7-2) to the co-stimulatory (+) molecule CD28 on the surface of T cells (signal 2). With higher affinity than CD28, inhibitory (−) CTLA-4 binds to B7 ligands on APCs and provides a brake  for T cell activation. Anti-CTLA-4 antibodies were proposed to release brakes of naïve T cells and allow them to be activated in the lymphoid organs and then migrate to tumors to cause tumor rejection

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Recently, we compared multiple anti-CTLA-4 monoclonal antibodies (mAbs) for their abilities to block B7-CTLA-4 interactions under various settings [3]. We found that when B7 molecules were immobilized on solid phases (such as when B7-1 or B7-2 was coated on ELISA plates or expressed on cell surface), Ipilimumab was unable to block the interaction between B7 and CTLA-4 [3]. On the contrary, L3D10, one anti-CTLA-4 antibody generated in our lab [4, 5], can block the in vitro interactions between various forms of CTLA-4 and B7 molecules [3]. Similar trend was observed when CTLA-4-mediated transendocytosis was measured. Consistent with this finding, we found that L3D10, but not Ipilimumab, significantly inhibited CTLA-4-mediated downregulation of B7 on the surface of splenic dendritic cells in CTLA4h/h humanized mice and human CD34+ stem cell reconstituted mice, providing in vivo evidence that Ipilimumab is ineffective in blocking B7-CTLA-4 interaction under physiological conditions [3].

Despite the differences in blocking B7-CTLA-4 interaction, L3D10 and Ipilimumab are comparable in inducing anti-tumor activity [3]. In addition, the fully humanized L3D10 clones, HL12 and HL32, which lose the ability to block B7-CTLA-4 interaction, remain fully active in inducing tumor rejection. Together, the above data demonstrate that blockade of B7-CTLA-4 interaction is unnecessary for immunotherapeutic effect of anti-CTLA-4 antibodies. In CTLA4h/m heterozygous mice, which express mouse and human CTLA-4 molecules in a codominant manner, anti-human CTLA-4 antibodies are unable to engage more than 50% of CTLA-4 as the remaining 50% of the molecules are of mouse origin and thus lack reactivity to antibodies originally made in mice [3]. However, all anti-CTLA-4 antibodies caused robust tumor rejection in CTLA4h/m mice. Therefore, even for blocking antibodies, their ability to completely block B7-CTLA-4 interaction is not required for effective tumor immunotherapy.

Finally, we reasoned that since antibody treatment is initiated after T cell priming has already taken place in the lymphoid environment, it is possible that anti-CTLA-4 antibodies may promote tumor rejection even if their effect in de novo T cell priming is abrogated. We tested if Ipilimumab can cause tumor rejection if de novo T cell priming is shut down by complete blockade of B7 by anti-B7 antibodies. Our results demonstrated that while anti-B7 antibodies effectively blocked de novo T cell priming in lymphoid organs, Ipilimumab remained fully active in causing tumor rejection in the presence of saturating B7 blockade [3]. These data refute the idea that anti-CTLA-4 antibodies cause tumor rejection by promoting T cell priming in the lymphoid organs.

What then is the primary mechanism of anti-CTLA-4 antibody induced tumor rejection? Several groups have established that anti-mouse CTLA-4 antibodies induced tumor rejection through selective depletion of regulatory T (Treg) cells in the tumor microenvironment [6,7,8]. By showing selective Tregs depletion in tumor microenvironment by anti-CTLA-4 antibodies as well as the absolute requirement of Fc receptor (FcR) in Ipilimumab-induced tumor rejection [3], our work demonstrates that the effector mechanism of anti-human CTLA-4 antibodies is similar to that reported by anti-mouse CTLA-4 antibodies (Fig. 2).

Fig. 2
figure 2

New concept: selective depletion of Tregs in the tumor microenvironment results in tumor immunity. Higher levels of CTLA-4 on intratumoral Tregs allow their selective depletion by anti-CTLA-4 antibodies, perhaps through antibody-dependent cellular phagocytosis (ADCP) by macrophages and/or antibody-dependent cellular cytotoxicity (ADCC) by NK cells. Blocking of B7-CTLA-4 interaction is not required for effective depletion of intratumoral Treg cells

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Therefore, rather than focusing on enhancing the blocking of B7-CTLA-4 interaction, other approaches on promoting local intratumoral Tregs clearance should be attempted in order to optimize the therapeutic effect of anti-CTLA-4 antibodies.