Tumor Immunotherapy: The mechanism of action of anti-CTLA-4 antibodies requires FcgR-dependant TREG depletion

Ipilimumab is an anti-CTLA-4 antibody used for treatment of metastatic melanoma, one of only two cancer immunotherapeutic drugs approved to date by the FDA.  CTLA-4 is a negative regulatory molecule expressed by activated T cells as well as by negative regulatory T cells (TREGs).  CTLA-4 is related to the T cell co-stimulatory receptor CD28, and acts to suppress T cell function by competing with CD28 for binding to CD80 and CD86 on antigen presenting cells and recruiting inhibitory molecules into the TCR signaling synapse.  Thus, the mechanism of action of Ipilimumab has been presumed to involve releasing anti-tumor effector T cells from CTLA-4-inhibition and/or limiting TREG activity in the tumor and therefore resulting in an increase in the ratio of effector T cells/ TREGs within the tumor.   However, two recent articles demonstrate that Ipilimumab has an additional mechanism of action: FcgR-dependant depletion of intra-tumoral TREGs.

Fcg receptors are a multi-family class of immunoglobulin (IgG)-binding receptors that initiate either activating or inhibitory signals when engaged.  Activating receptors contain cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAM) and activate the FcgR-expressing cell to mediate functions including antibody-dependant cell mediated cytotoxicity (ADCC) and phagocytosis of the antibody-labeled target cell.  FcgRIIB is the single inhibitory Fcg receptor in mice and humans and contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) which instead downregulates cellular responses.  There are four classes of IgG molecules in both humans and mice, and each bind to different Fcg receptors with varying affinity.  Thus differential affinities of IgG subclasses to functionally different Fcg receptors are thought to mediate the variation in clinical effectiveness of different antibodies targeting the same antigen.

Ipilimumab functions to increase the ratio of effector T cells to TREGS in the tumor microenvironment and has been shown to require binding to both types of T cells for maximal anti-tumor effectiveness.  However, how Ipilimumab differentially modulates these cell types remains to be understood.  A recent study published in The Journal of Experimental Medicine by Simpson et. al sought to clarify the mechanism by which Ipilimumab functions to alter the ratio of effector T cells/ TREGs in a murine tumor model.   Interestingly, the effects of Ipilimumab were found to be tissue-dependant.  In tumors which had high levels of infiltrating CD11b+ macrophages expressing the ADCC-activating FcgRIV, TREGS were selectively depleted in an FcgR-dependant manner, while effector T cells were instead expanded.  In lymph nodes lacking significant levels of these macrophages, frequencies of both effector T cells and TREGS were increased.  Tumor-associated TREGS expressedhigher levels of CTLA-4 than their effector T cell counterparts, or than TREGS present in the lymph node, indicating that higher CTLA-4 expression levels mediate ADCC via macrophages in the tumor.  Furthermore, the presence of FcgRs and hence TREG depletion was required for Ipilimumab’s effects.   Thus is appears that the mechanism of action of Ipilimumab on the effector T cell compartment is two-fold: directly targeting effector T cells to release inhibition via blocking CTLA4 activity, as well as by ADCC-mediated depletion of TREGS.

A second article in the same issue of The Journal of Experimental Medicine by Bulliard et al also explored the role of FcgR engagement on the effects of Ipilimumab as well as an agonistic antibody (DTA-1) targeting the T cell activating receptor GITR (TNFR glucocorticoid-induced TNFR-related protein), which is also expressed on both activated T cells and TREGs.  This study concluded that a major mechanism of action for both antibodies involved engagement of activating FcgRs leading to ADCC-mediated TREG depletion from the tumor.  Even though GITR-activation in effector T cells promotes activities including cytokine production and proliferation, the agonistic properties of this antibody alone were not effective in the absence of activating FcgR engagement.  Thus, even for functionally different (antagonistic versus agonistic) immunotherapeutic antibodies targeting these same T cell populations, FcgR-mediated ADCC of TREGs appears to be a critical mechanism for anti-tumor effects.

These studies highlight several important principles for the field of tumor immunotherapeutics.  Antibody targeting can elicit multiple effects, dependant on expression levels of the target, the isotype of the antibody, and the FcgR-expressing cell types present in the tissue.  Thus knowing the nature of the immune populations present in various types of tumors that are able to mediate ADCC, the FcgRs expressed by these cells, and the expression levels of the target molecule on immune populations that would be ideally targeted for elimination versus activation/inhibition will be critical areas for the forwarding of this field.  Furthermore, as FcgRs are polymorphous in humans, and affect IgG binding affinities, taking these genetic variations into account will be critical in the future of personalized medicine.

Further reading:

Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.  Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA. J Exp Med. 2013 Jul 29.

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies.  Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, Wilson NS, Dranoff G, Brogdon JL. J Exp Med. 2013 Jul 29.

Fcgamma receptors as regulators of immune responses.  Nimmerjahn F, Ravetch JV. Nat Rev Immunol. 2008 Jan;8(1):34-47.


One of the primary roles of the immune system is the specific identification and elimination of tumor cells on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress. This process is referred to as tumor immune surveillance. In this process the immune system recognizes malignant and/or pre-malignant cells and removes them. However, tumor cells do escape from tumor immune surveillance, and therefore, therapies targeted to enhance antitumor immunity is currently in development.

Blockade of immune checkpoints  is the most promising approach to activate therapeutic antitumour immunity. Immune checkpoints refer to a group of inhibitory pathways connected into the immune system that are important for maintaining self-tolerance. In peripheral tissues immune surveillance also modulates the duration and amplitude of physiological immune responses in order to minimize collateral tissue damage. Studies have suggested that tumor cells adopt many immune-checkpoint pathways as a major mechanism of immune resistance. Immune checkpoint receptors cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, also known as CD152) and programmed death 1 (PD-1) receptor appear to play important roles in antitumor immunity and have been most actively studied in the context of clinical cancer immunotherapy.

monoclonal3CTLA-4 is expressed on T cells and down modulates the amplitude of T cell activation. Several preclinical studies demonstrated significant antitumor responses following blockade of CTL4-A with limited immune toxicities. This led to the development of two fully humanized  CTLA-4 antibodies ipilimumab and tremelimumab. In clinical trials, ipilimumab demonstrated survival benefits for patients with metastatic melanoma, and was approved by the US Food and Drug Administration (FDA) for the treatment of advanced melanoma in 2010.

On the other hand, PD-1limits T cell effector functions within tissues. Tumor  cells block antitumor immune responses in the tumor microenvironment by upregulating ligands (PDL1 and PDL2) for PD1. Several studies detected increased PD1 expression by tumor infiltrating lymphocytes and the increased expression of PD1 ligands in melanoma, ovarian, lung, renal-cell cancers and in lymphomas. This provided an important rationale to target PD1 in order to enhance antitumor immunity. The fully human antibody nivolumab was found to produce durable objective responses in patients with melanoma, renal-cell cancer, and non-small-cell lung cancer.

Even though individual blocking of CTLA-4 and PD-1 have shown substantial clinical antitumor activity, studies suggest that blocking a single inhibitory receptor only leads to up-regulation of the unblocked pathway. Therefore, in order  to enhance antitumor immunity within the tumor microenvironment it appears to require simultaneous blockade of multiple negative co-stimulatory receptors. In preclinical studies, concurrent inhibition of CTLA-4 and PD-1 resulted in more pronounced antitumor activity than blockade of either pathway alone. On the basis of these observations, a phase I study was conducted to investigate the safety and efficacy of combined inhibition of CTLA-4 and PD-1in advanced melanoma patients and published recently in The New England Journal of Medicine (July 11, 2013). In their study, Wolchok and collagues (2013) treated 53 patients concurrently, and 33 patients sequentially with nivolumab and ipilimumab. Rapid responses were observed in concurrent-regimen cohorts as compared with sequential-regimen cohorts. The objective response rate in the concurrent-regimen cohorts was 40% along with 53% patients exhibited tumor regression of 80% or more. The objective response rate in the sequenced-regimen cohorts was 20% and 13% patients had tumor regression of 80% or more. In both groups, treatment related adverse events were managed with the use of immunosuppressants.

Collectively this study suggested that combined blockade of CTLA-4 and PD-1 would be more effective to enhance antitumor immunity compared to single inhibition of either CTLA-4 or PD-1.


1.  Swann, J.B. and M.J. Smyth, Immune surveillance of tumors. J Clin Invest, 2007. 117(5): p. 1137-46.

2.   Pardoll, D.M., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012. 12(4): p. 252-64.

3.   Topalian, S.L., et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med, 2012. 366(26): p. 2443-54.

4.   Wolchok, J.D., et al., Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med, 2013. 369(2): p. 122-33.

PD-1 re-expression is differentially regulated by IL-12 vs IFNα in CD8 T cells

TUMOR_immunotherapyDownregulation of immune functions following responses to pathogen infections is critical for limiting damage to the host by the immune system.  T cell activity is known to be downregulated by a variety of negative regulatory mechanisms including negative checkpoint regulatory proteins, a family of CD28-related molecules.  PD-1 is one such molecule that is transiently expressed on activated T cells.   The ligands for PD-1 are PD-L1 and PD-L2, members of the B7 family of molecules which are upregulated on antigen presenting cells and tumor cells.  Interaction of PD-1 with its ligand leads to inhibition of TCR-mediated signaling via recruitment of SHP1 and SHP2 phosphatases to the TCR synapse.  In the August 2013 edition of The Journal of Immunology, Gerner et al., demonstrate that CD8 T cells initially activated in the presence of IL-12 and IFNα differentially re-express PD-1 upon antigen restimulation.

Cytokines play roles in regulation of nearly every aspect of immune responses.  The cytokine milieu present during T cell activation directs differentiation into the different functional classes of CD4 T helper or CD8 T cells.  This study sought to determine the differences in anti-tumor CD8 T cell effector functions mediated when T cells are activated in the presence of various cytokines.  IL-12 and IFNα activate both overlapping and distinct gene programs and promote cytotoxic CD8 T cell responses.  Thus, these cytokines were chosen for comparison in this study.

In this system, CD8+ OT-1 cells were activated ex-vivo in the presence of either IL-12 or IFNα, and transferred into B16-OVA tumor-bearing mice.  T cells activated in the presence of IL-12 were found to mediate tumor-growth inhibition significantly better than if they had been activated in the presence of IFNα.  Over time in tumor-bearing mice, transferred IFNα-matured OT-1 cells were observed to decline in number and lost the ability to produce IFNγ ex vivo upon restimulation, indicating these cells may be exhausted.

Because PD-1 is known to be a marker and mediator of T cell exhaustion, PD-1 expression was examined.  Initial induction levels of PD-1 were comparable on OT-1 cells following ex vivo activation with IFNα or IL-12.  Following transfer into tumor-bearing mice, PD-1 levels declined over time on both types of cells isolated from the spleen and on IL-12 matured cells isolated from the tumor.  However, PD-1 expression was high on transferred IFNα-matured cells when isolated from the tumor.  Similar results were seen when cells were transferred into mice that subsequently received an injection of the OVA peptide.  Thus, CD8+ T cells matured in the presence of IFNα appear to re-express significantly higher levels of PD-1 upon antigen restimulation than IL-12 matured T cells.

PD-1 and PD-L1 targeting with inhibitory antibodies have emerged as promising avenues in tumor immunotherapy.  In this study, anti-PD-1 antibody administration had no additional anti-tumor effect in mice that received IL-12-matured T cells, while in mice that received IFNα-matured T cells, anti-PD-1 antibodies led to inhibition of tumor-growth to a level similar to that in mice that had received IL-12-matured T cells.  Thus, the relatively poor ability of IFNα-matured T cells to efficiently inhibit tumor growth appears to be largely due to PD-1 upregulation.  Finally, when T cells were matured with both IL-12 and IFNα, the effect of IL-12 was dominant.

Many questions remain regarding the mechanisms mediating PD-1 re-expression in IFNα vs. IL-12 matured T cells.  However, since IL-12 activity was dominant over IFNα on regulating PD-1 expression, IL-12 administration during immunotherapy regimens may enhance anti-tumor T cell responses by blocking the mechanisms by which IFNα enhances PD-1 re-expression.

Further Reading:

Cutting Edge: IL-12 and Type I IFN Differentially Program CD8 T Cells for Programmed Death 1 Re-expression Levels and Tumor Control.  Gerner MY, Heltemes-Harris LM, Fife BT, Mescher MF. J Immunol. 2013 Aug 1;191(3):1011-5. doi: 10.4049/jimmunol.1300652. Epub 2013 Jun 26.