BLOCKADE OF CTLA-4 AND PD-1 ENHANCED TUMOR REGRESSION IN MELANOMA

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.

References:

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.

Computers meet T cells: in silico identification of mutated tumor antigens targeted by T cells

It is well accepted that T cells can recognize and kill tumors that arise in individuals but that tumor cells escape immune surveillance due to the immunosuppressive tumor microenvironment that renders these T cells dysfunctional is less understood.  Only a relatively small number of antigens that T cells recognize for tumor-killing have been identified, and the methods used to identify these antigens are quite cumbersome.  In a recent article in Nature Medicine, Robbins et al. utilize informatics methods to identify mutated tumor antigens in melanoma patients that allowed effective targeting by anti-tumor T cells.

Genome sequencing T cells

In an effort to identify clinically relevant mutated tumor cell epitopes recognized by T cells, Robbins et al. first performed whole-exome sequencing of tumor cells and matched normal cells from melanoma patients who demonstrated tumor regression following adoptive transfer of autologous tumor infiltrating lymphocytes (TILs).  Mutations in tumor cells that resulted in amino acid changes were identified and then screened using an MHC binding algorithm that predicts high affinity binding of peptide sequences to specific HLA alleles.  Candidate peptides of 9-10 amino acids in length were synthesized and pulsed with specific HLA-expressing target cell lines to load the peptides into the MHC complex.  Peptide-pulsed target cells or autologous tumor cell lines were then cultured with autologous TILs from the same donor and IFN-gamma production was assessed as a read out of T cell activation.

Three metastatic melanoma patients were assessed using this methodology.  The first patient was homozygous for HLA-A*0201, and thus mutated melanoma cell line peptides predicted to bind to the HLA-A*0201 allele were identified by the MHC-binding algorithm.  From this donor, 4 out of 55 candidate peptides elicited IFN-gamma responses from autologous T cells cultured with peptide-pulsed target cells.  Two of these mutated peptides were found to correspond to the casein kinase1α1 protein (CSNK1A1), one peptide was mapped to the growth arrest specific 7 gene (GAS7) gene, and the fourth was a fragment of the HAUS augmin-like complex, subunit 3 (HAUS3) protein.  The wild-type versions of each of these peptides bound very poorly (100-10,000 fold less) or not at all to the HLA and were not recognized by T cells.  Two other donors were assessed for predicted binding of mutated peptides to HLA-A*0101 and HLA-A*1101.  Autologous T cell responses were found to be activated in response to mutated peptides from pleckstrin homology domain containing, family M member 2 (PLEKHM2), protein phosphatase 1 regulatory subunit 3B (PPP1R3B), matrilin 2 (MATN2), and cyclin-dependent kinase 12 (CDK12) genes, but not their wild-type counterparts.  Furthermore, tumor lines were validated to express these mutated proteins.

Finally, the authors compared the reactivity of peripheral blood mononuclear cells (PBMCs) drawn before and after adoptive TIL transfer into two of these patients to determine if anti-tumor reactive T cell clones persisted in vivo.  T cells that recognized the same tumor antigens as the TILs were identified post-adoptive transfer at greater levels than prior to adoptive transfer.  Thus, T cells that recognize mutated tumor epitopes may play a clinically relevant role in mediating tumor regression.  Many questions remain, including a direct demonstration that such tumor-reactive TILs are responsible for mediating the observed tumor regression in these patients, and whether further mutation of these residues might facilitate immune escape later it the course of disease. 

Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.  Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA. Nat Med. 2013 May 5. doi: 10.1038/nm.3161.

NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence.  Nielsen M, Lundegaard C, Blicher T, Lamberth K, Harndahl M, Justesen S, Røder G, Peters B, Sette A, Lund O, Buus S. PLoS One. 2007 Aug 29;2(8):e796.