Modulation of CTLA4 and GITR for Cancer Immunotherapy

Francesca Avogadri, Jianda Yuan, Arvin Yang, David Schaer, and Jedd D. Wolchok


1 Introduction 212

2 CTLA-4 Preclinical Data 213

2.1 CTLA-4: A "Brake" on T Cell Activation 214

2.2 Preclinical Studies Using CTLA-4 Blocking Antibodies 217

3 GITR Preclinical 219

3.1 GITR: "Accelerator" of Effector Function 220

3.2 Preclinical Studies Using GITR Agonist Antibody 220

4 Clinical Experiences with CTLA-4 Blockade 222

4.1 CTLA-4 Monotherapy 223

4.2 Postsurgical Adjuvant Therapy 225

4.3 Combinations 226

4.4 Tremelimumab 226

4.5 Immune Related Adverse Effects 227

4.6 Novel Criteria for Antitumor Response to Ipilimumab and Increased Duration of Response 227

4.7 Other Malignancies 228

5 From Clinical Trials, Back to the Bench 229

5.1 Monitoring Antigen-Specific CD4+ and CD8+ T Cell

Activity and Polyfunctionality 229

F. Avogadri and D. Schaer

Immunology Program, Sloan-Kettering Institute, New York, NY 10065, USA J. Yuan

Immunology Program, Sloan-Kettering Institute, New York, NY 10065, USA

Ludwig Center for Cancer Immunotherapy, Immunology Program, Sloan-Kettering Institute, New

York, NY 10065, USA

Immunology Program, Sloan-Kettering Institute, New York, NY 10065, USA Melanoma/Sarcoma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA

Ludwig Center for Cancer Immunotherapy, Immunology Program, Sloan-Kettering Institute, New York, NY 10065, USA e-mail: [email protected]

G. Dranoff (ed.), Cancer Immunology and Immunotherapy, 211

Current Topics in Microbiology and Immunology 344, DOI 10.1007/82_2010_49 © Springer-Verlag Berlin Heidelberg 2011, published online: 19 June 2010

5.2 Cellular Phenotype Analysis: ICOS, Foxp3, HLA-DR and IDO

231 231

233 233 236

5.3 ICOS

5.4 Foxp3

5.5 HLA-DR

5.7 Antibody Responses

5.8 The "Immunogram": A Tool to Synthesize Immune Monitoring

6 Future Directions


Abstract The rational manipulation of antigen-specific T cells to reignite a tumor-specific immune response in cancer patients is a challenge for cancer immunotherapy. Targeting coinhibitory and costimulatory T cell receptors with specific antibodies in cancer patients is an emerging approach to T cell manipulation, namely "immune modulation." Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor family receptor (GITR) are potential targets for immune modulation through anti-CTLA-4 blocking antibodies and anti-GITR agonistic antibodies, respectively. In this review, we first discuss preclinical findings key to the understanding of the mechanisms of action of these immunomodu-latory antibodies and the preclinical evidence of antitumor activity which preceded translation into the clinic. We next describe the outcomes and immune related adverse effects associated with anti-CTLA-4 based clinical trials with particular emphasis on specific biomarkers used to elucidate the mechanisms of tumor immunity in patients. The experience with anti-CTLA-4 therapy and the durable clinical benefit observed provide proof of principle to effective antitumor immune modulation and the promise of future clinical immune modulatory antibodies.

1 Introduction

In the late nineteenth century, the antitumor effects of Coley's toxin provided the first suggestive evidence that the immune system could be harnessed to combat cancer. Over 100 years later, we now possess a better understanding of mechanisms of T cell activation and the technology to manipulate these findings in the clinical setting. Current clinical, immunotherapeutic treatments, although not exclusively effective in one disease, have been most successful in melanoma. FDA approved treatments for melanoma include the adjuvant use of high-dose interferon alpha and high-dose IL-2 in the metastastic setting. The complete list of approved therapies for melanoma only requires the addition of dacarbazine (DTIC), the only FDA approved chemotherapeutic agent for melanoma. The modest response rates for both IL-2 and DTIC coupled with recent epidemiological data demonstrating an increasing incidence of melanoma provide incentive for alternative strategies. Recent advances in immunology have led to a more profound insight regarding the function of costimulatory and coinhibitory receptors expressed by different T cell subsets, providing a novel approach to optimize immunotherapies through immune modulation.

During the primary activation of naive T lymphocytes, the immune system utilizes various checks and balances to maintain tolerance to self while assuring appropriate activation against foreign and self antigens. Although primary antigen recognition occurs through the interactions of the T cell receptor (TCR) and peptide-MHC complexes, without costimulation through CD28 binding to either B7-1 (CD80) or B7-2 (CD86), cognate antigen recognition will result only in T cell anergy induction (Linsley and Ledbetter 1993). This first check is followed by additional signals mediated by coinhibitory/costimulatory receptors, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor family receptor (GITR), which further shape the resulting effector function and dictate its efficacy and duration. Since their discovery, much effort has been put into understanding the immunomodulatory properties of CTLA-4 and GITR in mice. The development of antibodies specifically targeting these receptors and modulating their functions has provided a new perspective for immunotherapeutic approaches.

Under physiological stimulus, TCR binding causes activation of a complex signaling cascade culminating in downstream activation of the NF-kB, NFAT pathways and target gene transcription (Chan et al. 1995; Zhang et al. 1998; van Leeuwen and Samelson 1999; Tybulewicz et al. 2003). However, naive T cells require CD28 costimulation to maintain this cascade. When CD28 is activated, it potentiates the cascade through activation of PI3K and Sos resulting in stabilization of mRNA for NF-kB, NFAT (Pages et al. 1994). The importance of costimulation in T cell physiology is highlighted by CD28— /— mice which have dramatic reduction in the ability to maintain T cell activation (Lucas et al. 1995). Thus, CD28 provides the first checkpoint in T cell activation, sensing the expression of CD80/CD86 on an activated antigen presenting cell (APC). If the APC has not experienced the proper "danger signals" (e.g., cytokines, TLR, Fc Receptor stimulus), it will not optimally upregulate the expression of CD80/CD86. This need for CD28 costimulation is thought to reduce inadvertent activation of possibly self-reactive T cell clones in the periphery, which have escaped thymic deletion. Reinforcing this checkpoint is CTLA-4, which acts as a coinhibitory molecule. Antagonizing CD28 T cell costimulation, CTLA-4 binds with much greater affinity to CD80/CD86 and effectively shuts off TCR signaling (van der Merwe et al. 1997).

2 CTLA-4 Preclinical Data

CTLA-4 is a member of the CD28:B7 immunoglobulin superfamily. In contrast to CD28, CTLA-4 is normally expressed at low levels on the surface of naive effector T cells (Teff) and mainly exists in prepackaged vesicles inside the cytosol (Alegre et al. 1996). When the TCR stimulus to the naive T cell is too strong or lasts too long, CTLA-4 is recruited to the cell surface in a polar manner with release at the site of the immunological synapse (IS) (Linsley et al. 1996). Once at the IS, CTLA-4 can now compete with CD28 for CD80/CD86 (Chuang et al. 1999; Carreno et al. 2000; Cinek et al. 2000), effectively shutting off TCR signaling (van der Merwe et al. 1997). While CTLA-4 translocation to the cell membrane has been shown to depend on many events downstream of TCR signaling (reviewed in (Rudd et al. 2009)), it is not yet entirely clear how CTLA-4 mediates TCR signaling shut down upon binding to CD80/CD86. CTLA-4 does not have traditional immune tyrosine inhibitory motif (ITIM) domains, which are known to recruit regulatory proteins through binding of SH2 domains to phosphorylated tyrosine residues in the (I/V/L/ S)xYxx(L/V) motif (Blank et al. 2009). However, CTLA-4 does have other tyrosine motifs which have been shown to recruit phosphatases SHP-2, and PP2A, yet the functional significance of these interactions remain unclear. For instance, SHP-2 also associates with CD28 and has been shown to potentiate the TCR cascade (Gadina et al. 1998). Although PP2A may act as a negative regulator of the TCR cascade, whether or not it does this through its interactions with CTLA-4 is also not fully established (Chuang et al. 2000). Besides directing downstream signaling events, it is thought that CTLA-4 may function simply to sequester CD80/CD86 away from CD28 because of its higher affinity for these molecules. In fact, mutant CTLA-4 molecules with levels of surface expression that lack tyrosines or prolines in their cytoplasmic tail are still able to suppress CD80-mediated CD28 activation signals (Carreno et al. 2000; Cinek et al. 2000).

Once it has been triggered, CTLA-4 may destabilize components of the super molecular activation complex (SMAC) in the IS. In fact, CTLA-4 was shown to alter lipid raft formation, resulting in reduced phosphorylation of linker for the activation of T cells (LAT) after TCR and CD28 stimulation (Martin et al. 2001). This block in IS formation applies also to ZAP-70 microclusters which are needed to maintain calcium flux and downstream events in TCR signaling (Bunnell et al. 2002; Schneider et al. 2008). Thus, CTLA-4 requires initial TCR signaling for its membrane targeting, and subsequently shuts off these early activation events.

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