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The potential of T cells to attack and destroy tumors has long been recognized. Recent experimental evidence has also demonstrated that T cell surveillance throughout the life of an organism functions to prevent tumor development. Nonetheless, T cells face an uphill battle in their attempts to recognize and eliminate tumors, as many of the same mechanisms which prevent autoimmunity also impair T cell responses to the “altered self” of tumors. In addition, it has become apparent that tumors are capable of selectively co-opting multiple of the inhibitory mechanisms that prevent sustained T cell responses to self-tissue. Selective blockade of these natural inhibitory checkpoints might provide a means of releasing the brakes on T cell activation and promoting potent anti-tumor responses.
Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in many cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumor response. Inhibitors of CTLA4, PD1 or PDL1 administered as single agents have resulted in durable tumor regression in some patients, and combinations of CTLA4 and PD1 inhibitors may enhance antitumor benefit. Numerous additional immunomodulatory pathways, as well as inhibitory factors expressed or secreted by myeloid and stromal cells, are potential targets for synergizing with immune checkpoint blockade.
Expression pattern of CTLA-4 and PD-1
Expression of CTLA-4 is primarily restricted to T cells, although the expression on B cells and other cell types has been described. Compared with CD28 which is expressed on the surface of resting and activated T cells, CTLA-4 exhibits minimal expression in resting T cells (Figure 1). CTLA-4 is induced at the mRNA level and protein level in response to TCR activation. Expression of CTLA-4 is enhanced by co-stimulation through IL-2 and/or CD28. PD-1 exhibits minimal expression on resting cells of the immune system. But upon activation, PD-1 expression is broadly induced in B cells, T cells, NK cells, NKT cells, DCs, and macrophages (Figure 1). Induction of PD-1 in peripheral T cells occurs downstream of TCR signaling. PD-1 is highly expressed by dysfunctional T cells in the setting of chronic infections but is not expressed on resting memory T cells which arise after an acute infection.
Figure 1. Unique spatiotemporal regulation of PD-1 and CTLA-4.
Intracellular signaling of CTLA-4 and PD-1
The cytoplasmic tail of CTLA-4 interacts with many signaling molecules that inhibit proximal signaling via the CD28 and TCR (Figure 2). TCR/CD28 signaling results in activation of numerous kinases, including Lyn, Lck, Fyn, Rlk, and Jak2, which are capable of phosphorylating Y165 and Y182 of the cytoplasmic domain. Initial studies demonstrated that phosphorylation at Y165 creates a docking site for the protein tyrosine phosphatase SHP-2, which subsequently inhibits proximal TCR signaling, linker for activation of T cells, and the Ras regulator p52SHC. Moreover, CTLA-4 stabilizes expression of the ubiquitin ligase Cbl-b, an important negative regulator of signaling via the CD28 and TCR.
PD-1-mediated signals can inhibit T lymphocyte glucose consumption, cytokine production, proliferation, and survival (Figure 2). Upon TCR stimulation, PD-1 undergoes phosphorylation of the tyrosine residues in the ITSM and ITIM motifs of the cytoplasmic tail, allowing recruitment of the phosphatases SHP-1 and SHP-2, which in turn, dephosphorylate proximal signaling molecules downstream of the CD28 and TCR. Positional mutagenesis studies demonstrated that the ITSM motif is necessary for the inhibitory function of PD-1. In addition, PD-1 ligation and recruitment to the immune synapse appear to be necessary to mediate the inhibitory effects on proximal TCR signaling.
Figure 2. Distinct mechanisms of intracellular signaling by PD-1 and CTLA-4.
CTLA-4 blockade in cancer immunotherapy
Shortly after the inhibitory functions of CTLA-4 were discovered, antibody-mediated blockade of CTLA-4 function was found to enhance the rejection of transplanted mouse colon carcinoma and fibrosarcoma tumors and additionally, delay growth of established tumors. Moreover, anti-CTLA-4 treatment resulted in immune memory, such that previously challenged mice could reject subsequently implanted tumors without additional CTLA-4 blockade. The anti-tumor effects of CTLA-4 blockade have been extended to numerous other murine tumor models, including lymphoma, prostate carcinoma and renal cell carcinoma. Importantly, the antibody used in these studies was a nonstimulatory, bivalent antibody that did not induce optimal cross-linking of CTLA-4 but rather, blocked the interaction of CTLA-4 with B7 ligands without affecting the activity of CD28.
These preclinical findings encouraged the production and testing of two fully humanized CTLA4 antibodies, tremelimumab and ipilimumab, which began clinical testing in 2000. Both antibodies produced objective clinical responses in ~10% of patients with melanoma, but immune-related toxicities involving various tissue sites were also observed in 25–30% of patients, with colitis being a particularly common event. The first randomized Phase III clinical trial to be completed was for tremelimumab in patients with advanced melanoma. In this trial, 15 mg per kg tremelimumab was given every three months as a single agent and compared with dacarbazine, a standard melanoma chemotherapy treatment. The trial showed no survival benefit with this dose and schedule relative to dacarbazine.
PD-1 blockade for cancer immunotherapy
Another immune-checkpoint receptor, PD1, is emerging as a promising target, thus emphasizing the diversity of potential molecularly defined immune manipulations that are capable of inducing antitumor immune responses by the patient’s own immune system. Compared with CTLA4, the major role of PD1 is to limit the activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity (Figure 3). This translates into a major immune resistance mechanism within the tumor microenvironment.
Figure 3. Immune checkpoints regulate different components in the evolution of an immune response.
PD-L1 pathway confirms its importance in immune evasion by tumors. Overexpression of PD-L1 on a mouse mastocytoma tumor inhibits tumor-directed T cell cytotoxicity in vitro and promotes immune evasion leading to enhanced growth of the tumor in vivo. These effects are abrogated by antibody-mediated blockade of PD-L1, which promotes immune-mediated tumor rejection. Likewise, blockade of PD-L1 promotes immune-mediated destruction of tumors that naturally express PD-L1, including melanoma, myeloma, and mammary carcinoma. In some settings, PD-L1 expression by the tumor promotes apoptosis of tumor-specific T cells, whereas in other systems, tumor expression of PD-L1 inhibits tumor cell lysis by cytotoxic T cells in the absence of apoptosis or anergy.
Note that the effects of PD-1 blockade as an anti-cancer therapeutic may be mediated partially by immune cells other than T cells, including B cells or NK cells. Tumor-mediated production of IL-18 enhances PD-1 expression and inhibits the function of NK cells, another type of immune cell important for anti-tumor immunity. In fact, the lupus-like disease and dilated cardiomyopathy observed in PD-1-deficient mice may be, at least in part, a result of aberrant B cell function, given the predominant, antibody-mediated pathology. Blockade of PD-1 in chronic SIV infection enhanced proliferation of memory B cells and boosted anti-SIV antibody titers. Thus, restoration of B cell and NK cell function might also play a role in the anti-tumor activity of PD-1 blockade.
There is nothing like a clinical success to open up a new area of therapeutics. The FDA approval of anti‑CTLA4 therapy, quickly followed by reports of encouraging preliminary clinical data for anti‑PD1 therapy, has engendered a new-found awareness among oncologists of the potential antitumor activity of a patient’s endogenous immune system once the ‘brakes’ elicited by the immune system have been released. Besides CTLA-4 and PD-1, numerous other immune inhibitory receptors and ligands are being investigated as potential targets for cancer immunotherapy, including T and B lymphocyte attenuator, lymphocyte activation gene 3, and T cell membrane protein 3. The potential for combined blockade of immune checkpoints offers new hope for cancer patients. A sound understanding of the cellular and molecular mechanisms whereby these immune modulators function will be essential to guide their optimal use in the clinic.