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Immunologic correlates in the course of treatment with immunomodulating antibodies Benjamin Weide, Anna-Maria Di Giacomo, Ester Fonsatti, Laurence Zitvogel

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Cite this article as: Benjamin Weide, Anna-Maria Di Giacomo, Ester Fonsatti, Laurence Zitvogel, Immunologic correlates in the course of treatment with immunomodulating antibodies, Semin Oncol, http://dx.doi.org/10.1053/j. seminoncol.2015.02.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunologic correlates in the course of treatment with immunomodulating antibodies Benjamin Weide1, Anna-Maria Di Giacomo2, Ester Fonsatti2, Laurence Zitvogel3-6 1

Division of Dermatooncology, Department of Dermatology, University Medical Center Tübingen, Germany

2

Division of Medical Oncology and Immunotherapy, University Hospital of Siena, Italy

3

Gustave Roussy Cancer Campus, Villejuif, France;

4

INSERM U1015, Villejuif, France

5

Université Paris Sud-XI, Faculté de Médecine, Le Kremlin Bicêtre, France;

6

Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 507, Villejuif, France

Corresponding author: Benjamin Weide Dept. of Dermatology, University Medical Center Tübingen Liebermeisterstr. 25, 72076 Tübingen, Germany Tel. +49 70712984555, Fax.:+49 7071 295265 , [email protected]

Financial disclosure and conflict of interest statements: BW: received research grants, travel grants and honoraria from Bristol-Myers Squibb. AMD: served as speaker to Bristol-Myers Squibb, Roche-Genentech. EF: No conflicts of interest to declare LZ: served as an administrator of Transgene, consulting fees from GSK and MedImuune, scientific collaboration and support from Lytix pharmaceuticals, Transgene and Invectys.

Abstract Monoclonal antibodies targeting immune checkpoints like CTLA-4 or PD-1 have come of age in the treatment of metastatic melanoma and further approvals are expected for other malignancies like lung or renal cell cancer as well. However, the majority of patients still does not experience clinical benefit upon these therapies. Moreover, immune-related side effects and the costs of these therapies prompt the search for their precise mode of action and for biomarker discovery. Here, we describe different classes of immunologic correlates such as pharmacodynamic changes observed in all treated patients, correlates with response during treatment (surrogate markers) or at the time-point of tumor assessment, as well as predictive markers for response and for immune-related adverse events. This review gives an overview over available data about correlates analyzed in the serum, in immune cell subsets in the peripheral blood or in tumor-infiltrating lymphocytes. We will discuss how to prospectively validate and integrate these parameters for routine assessment of patients in daily clinical practice and give an outlook on promising future directions of biomarker research.

Introduction Despite these breakthroughs, the mode of action of immune-checkpoint blockade remains incompletely understood. CTLA-4, PD-1 and PD-L1 antibodies (Ab) have no known direct toxic or growth-inhibiting effect on tumor cells but act indirectly through immune cells, which induce immunological rejection of cancer cells in a subset of treated patients (1). All immune checkpoint blocking Ab finally provide activating signals to T cells by blocking suppressive interaction between CTLA-4 or PD-1 on T cells and their ligands (PD-L1, PD-L2, CD80 and CD86) on tumor cells or immune-response regulating cells, e.g. dendritic cells and myeloid-derived suppressor cells (MDSCs). Fingerprints of these treatments can therefore be expected in T cells upon immune-checkpoint blockade. In addition to assumed immune correlates in cells expressing the molecular targets of immune-checkpoint blockers, secondary correlates might also be observed. The immunologic consequences after activation of CD4+ T cells are complex and hardly predictable, and CTLA-4 or PD-1 blockade in these cells might lead to completely different

consequences in different CD4+ T cell subsets, such as regulatory T cells (Tregs) versus Th1 cells. These secondary consequences may explain for instance observed changes of humoral immune responses upon immune-checkpoint blockade. Immunologic correlates represent potential biomarkers that might help to address unsolved problems arising for patient treatment by these agents. A large fraction of treated patients does not benefit from the treatment. While a minority of patients exhibits a complete, long-lasting response, others do not respond but are still at risk for side effects. No reliable laboratory parameters or clinical characteristics predicting efficacy or toxicity in an individual patient have been identified thus far, which are used in daily clinical routine. Many candidates were described in the literature, but due a lack of predictive power, validation, and/or feasibility, none is considered for treatment decisions thus far. It is clear that the identification of such biomarkers would substantially improve the risk/benefit assessment of an individual patient and would be an important step towards stratified treatment decisions (2). In this review, we focus on different classes of immunologic correlates: (i) pharmacodynamic changes observed in all treated patients; (ii) correlates with response at the time-point of tumor assessment; (iii) correlates during treatment associated with later response (surrogate markers); (iv) predictive markers analyzed before start of treatment indicating a high chance of clinical benefit or a high risk for the occurrence of immune-related adverse events. These correlates can be found in the serum or among immune cell subsets, which usually are analyzed in the peripheral blood or in tumor tissues (tumor infiltrating lymphocytes - TILs).

1. Immune correlates related to CTLA-4 antibodies The exact mode of action of CTLA-4-Ab is incompletely understood thus far, but it is generally assumed that this agent restores proper T cell activation by preventing inactivation through CTLA-4 signaling and indirectly ensuring activation via CD28-engagement. Preclinical studies using Ab against the mouse CTLA-4 to explore the mechanisms of the tumoricidal activity revealed several important findings that prompted investigators to probe them in humans. Hence, a role for CD4+ and CD8+ T lymphocytes (but

not natural killer cells), of the ratio between effector T cells and Tregs in tumor beds, and of signaling through ICOS, IL2R as well as FcγR and IFNγR was reported in mouse transplantable tumor models (35). As a subset of T cells (e.g. T cells after initial activation or Tregs) express CTLA-4, these can be regarded as important cellular targets of CTLA-4 Ab. Many pharmacodynamic observations are in agreement with this proposed mode of action. We will give an overview of available biomarker studies investigating pharmacodynamic, surrogate and predictive immune parameters in blood, serum and tumor lesions upon treatment with CTLA-4 Ab.

1A Circulating immune cells Lymphocytes An increase in the absolute lymphocyte count (ALC) upon treatment with ipilimumab was observed in several studies (6, 7) that was ascribed to T cell proliferative capacities. Both, CD4+ as well as CD8+ T cells were found to be increased in the circulation during and after CTLA-4 blockade (8). Indeed, T cells express the proliferation marker Ki67 on CD4+ and CD8+ cells at both 3- and 6-month post-ipilimumab (9). The induction of lymphocyte proliferation could also be demonstrated in-vivo by imaging techniques (10). Only one study reported no changes in T cell frequencies during ipilimumab (11). Ipilimumab appeared to release the arrested cell cycle in CD4+ and CD8+ T cells as evidenced by consistent up-regulation of CDC2 and other cell cycle-related genes (9). Signaling pathways downstream of the T cell receptor (TCR) and cytokine receptors were also influenced, as shown with the increased phosphorylation of p38, of STAT1 and STAT3, with a concomitant decrease in the activation of Lck, ERK1/2 and STAT5 (12). The ALC represents a pharmacodynamic parameter of interest for its potential use as a surrogate marker of response (6, 7, 13-18). Berman et al. reported that an increase in ALC during the first weeks of treatment was associated with clinical activity in patients treated with ipilimumab in phase II trials. Interestingly, changes were more pronounced if patients were treated at higher doses (6). The same group reported that mean ALC increased significantly over time in patients who received ipilimumab in combination with chemotherapy and peptide vaccination. In contrast, no significant

increase in ALC in pts who received vaccine alone or BRAF-inhibitor monotherapy. In patients participating in the MDX010-20 trial, those with a greater positive rate of change in ALC from baseline to week 7 tended to have longer overall survival (OS). A similar association was found between OS and ALC ≥ 1000/µl after two doses of ipilimumab. However, in the MDX010-20 trial, the survival benefit upon ipilimumab was not limited to patients with increasing ALC during the first weeks of treatment (7). Further phenotypic characterization of the ipilimumab-induced changes in lymphocytes revealed that naïve (defined as CCR7+CD45RA+) CD4+ and CD8+ T cells were significantly reduced 6 months after ipilimumab, with a corresponding rise in CD8+ central memory and CD4+ effector memory T cells (9). CTLA-4 blockade may also affect innate lymphocytes. Invariant central memory NKT cells, which express CD8 and exhibit a polyfunctional activation pattern increased post-ipilimumab and correlated with clinical responses in a trial combining tremelimumab and DC vaccines (19).

Specific T cells CTLA-4 blockade induces strong TCR-repertoire diversification as demonstrated in a study applying next generation sequencing to compare circulating T cells pre- and post-ipilimumab in patients with MM and prostate cancers. The authors suggest that anti-CTLA-4 Ab influence both the threshold of T cell activation as well as the preferential expansion of high avidity T cell clones, mostly relevant for the clinical benefit. Interestingly, the variation of the TCR-repertoire was not restricted to tumor-targeting T cells but also comprised an increase in anti-viral immune responses post-ipilimumab (20). Indeed, the prognostic impact of melanoma-specific T cells (21, 22) and the efficacy of the adoptive transfer of tumor-infiltrating or TCR–transduced lymphocytes (23, 24) in MM point to the relevance of specific T cell clones in the course of this disease. There is accumulating evidence that ipilimumab increases the frequency of preexisting melanoma-specific T cells. Tarhini et al. detected spontaneous CD4+ and CD8+ T cell responses targeting NY-ESO-1, Melan-A and gp100 in baseline blood samples. Such T cell responses were significantly potentiated after neoadjuvant therapy with ipilimumab. There was an association between the frequencies of specific T cell precursors and the time to progression at 6

months (25). Liakou et al. demonstrated an increase of NY-ESO-1-targeting CD4+ T cells upon ipilimumab in patients with bladder cancer (26). T cell responses targeting NY-ESO-1 were also found to be increased in frequency and functionality during ipilimumab in further studies. NY-ESO-1-seropositive patients with associated CD8+ T cells experienced more frequent clinical benefit (77%) than those with undetectable CD8+ T-cell response (clinical benefit in 14% of patients) and a significant survival advantage (27). This study as well as other studies conducted at the Sloan Kettering Institute (New York) suggested that combined humoral and T cell responses directed against NY-ESO-1 may have predictive value for ipilimumab treatment (27-29). More controversial findings about the prognostic significance of specific T cells were reported in trials combining ipilimumab with vaccines. No consistent change in specific T cells elicited by a multi-peptide vaccine targeting tyrosinase, Melan-A and gp100 was observed in combination with ipilimumab (11). An increase in Melan-A specific T cells, as observed using tetramer-staining by flow cytometry, was reported after six months of therapy while results of functional assays and T cells targeting gp100 and tyrosinase remained inconclusive (11). No accumulation of vaccine-specific CD8+ T cells was reported in two other trials combining peptide-based vaccines and CTLA-4 blockade (30, 31). Specific immune responses were not analyzed in the MDX020-10 trial, but those patients receiving ipilimumab in combination with a peptide vaccine had no survival advantage compared to ipilimumab monotherapy in this randomized controlled trial (32).

T cell activation markers Changes in the expression of different T cell activation markers were reported in patients during CTLA-4 blockade. Increased cell surface expression of HLA-DR was observed in both CD4+ and CD8+ T cells (11, 33). Similar results were reported for CD4+HLA-DR+ cells in other studies (30, 31). CD278 or ICOS (Inducible T-cell COStimulator) is a CD28-superfamily costimulatory molecule that is expressed on activated T cells. ICOS is upregulated on a fraction of T cells upon ipilimumab therapy and most of these T cells producing Th1 cytokines (3). This effect was reported in MM for CD4+ T cells (33)

or CD8+ T cells, mainly at later time-points after start of treatment (9, 34), and was also observed in patients with other solid tumors (26, 35, 36). The upregulation of ICOS on CD8+ T cells seemed to be dose-dependent (36). In a study of Di Giacomo et al. significant increases in the number of circulating CD4+ICOS+ and CD8+ICOS+ T cells were observed after start of ipilimumab compared to baseline. A fivefold increase in both ICOS+ T cell subsets at weeks 7 and 12 defined disease control. Moreover, better survival benefit was observed in patients exhibiting the most dramatic rise of ICOS expressing T cells at week 7 (34). Similar findings were reported by Carthon et al. in patients with localized urothelial carcinoma of the bladder (36). In this study, a persistent increase in CD4+ICOS+ cells at weeks 7 or 12 compared to baseline was reported as a surrogate marker for outcome upon ipilimumab. Of eight patients with persistent increase in CD4+ICOShi expression, seven had evidence of disease stabilization at week 24 in contrast to none of the six patients without persistent increase in this cell subset (p=0.004). A statistically significant difference in OS according to persistent versus non-persistent increase was also observed (36). Noteworthy, similar results with tremelimumab in malignant mesothelioma was recently reported by Calabrò et al. (37) and are discussed in this issue of Seminars in Oncology. The transcription factor EOMES, another activation marker of T cells pathognomonic of Th1/Tc1 cells, was also identified as a potential biomarker for ipilimumab. Increased frequencies of EOMES+CD8+ or GranzymeB+EOMES+CD8+ or decreased frequencies of Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse after surgery. At baseline, low proportions of Ki67+EOMES+CD8+ T cells were also associated with relapse in patients receiving ipilimumab (p≤0.001) (9). The frequency of IL-17 releasing CD4+ T cells after in-vitro stimulation (Th-17 inducibility) increased in surgically resected patients after adjuvant ipilimumab in a study of Sarnaik et al. A higher change in Th17 inducibility from baseline to 6 months was positively associated with freedom from relapse (11).

Regulatory T cells Ipilimumab acts not only on effector T cells but also on T cell subsets with immunosuppressive properties. Tregs have been shown to play a critical role in immune tolerance against tumors in both mouse systems

and humans (38, 39) and are directly targeted by ipilimumab due to the constitutive expression of CTLA-4 on their cell surface. In a pioneering work using tremelimumab in MM, Ménard et al. reported that CTLA4 blockade does not modulate the immunosuppressive effects of Tregs on T and NK cells but rather render effector T cells resistant to the inhibitory activity of Tregs (40). Ipilimumab-induced expansion of Tregs has been a controversial notion in the literature. A decrease of circulating CD25+CD4+ Tregs was observed by Attia et al. upon ipilimumab (31). In other studies, the frequency of Tregs cells did not change considerably during CTLA-4 blockade in patients with localized urothelial carcinoma of the bladder (36) or MM (33, 41). No change in the suppressive function of Tregs was observed in a trial of adjuvant ipilimumab combined with a peptide vaccine (11). But even if there is no consistent effect on the frequency of Tregs in the peripheral blood, Simeone et al. found a correlation between an decrease of FoxP3+Tregs and response in melanoma patients treated with ipilimumab (18). In sharp contrast, a significant increase in the percentage of circulating Tregs was found to be associated with improved progression free survival in another study (25). CTLA4+CD4+ T cells in the circulation were reported to be a dominant predictor for survival after GVAX/ipilimumab therapy in prostate cancer (42).

Neutrophils and Eosinophils One study reported the ratio between absolute neutrophils and lymphocytes (N/L ratio) as a surrogate marker. Patients with an N/L ratio lower than the median values at weeks 7 and 10 had significantly improved OS compared with those with N/L ratios greater than the median value at these time points (34). The eosinophil count was analyzed in two studies which reported a predictive (43) or surrogate function (16) for outcome upon ipilimumab. A baseline absolute eosinophil count (AEC) ≥ 100/µl was significantly associated with improved OS (p=0.002) in the study of Schindler et al. with 6-, 12- and 18month survival rates of 79%, 60% and 48% compared to rates of 48%, 37% and 19% for patients with baseline AEC below 100/µl. Baseline relative eosinophil counts (REC) of 1.75% or higher showed an even stronger impact on OS (p 2 fold the normal range correlated with clinical failure of CTLA-4 blockade (15). Hence, LDH levels represent a meaningful prognostic marker, and potentially a negative predictive marker of response to CTLA-4 blockade in MM. Similarly, one study recently reported a deleterious impact of high baseline serum levels of VEGF, another tumor cell related serum factor, on the chance to experience a clinical response after ipilimumab (81). In addition to the immune status and tumor cell markers, the genetic background of patients also seems to influence the outcome of patients treated by immune checkpoint blockade. Genetic variation in the CTLA4 protein could influence response to CTLA-4 blockade therapy in MM. Breunis et al. investigated common single-nucleotide polymorphisms (SNPs) residing in the ctla4 gene in 152 Caucasian melanoma patients who received CTLA-4 blockade. Three SNPs were associated with response to therapy (79). However, Hamid et al. did not cooroborate these findings (59). None of the identified biomarker candidates is broadly used in daily clinical care and influences treatment decisions thus far due the lack of prospective validation studies in large multicenter cohorts. Moreover, further investigation of such markers is hampered by the lack of clear definitions and agreements about the methodology (for instance regarding the immunohistochemical analysis of PD-L1 expression in tumor tissue) or by the need for special laboratory equipment and highly trained personal to perform more sophisticated laboratory analyses (for instance flowcytometric analyses). While the FDA approval of ipilimumab will facilitate the prospective validation of candidate parameters, one can expect a retrospective exploitation of biobanks constituted during large Phase III trials, which is not yet reported. Moreover, national and international collaborations represent a prerequisite for successful validation studies to achieve the inclusion of relevant numbers of patients in validation studies.

In the future, the assessment of the TCR repertoire diversity by next generation sequencing (NGS) could be used to track the presence of high avidity T cell clones, and low avidity ones that may reach the threshold of activation during CTLA-4 blockade. Larger sample sizes and prospective validation of the “clonotype maintenance” theory will clarify whether these technological advances will reach the armamentarium to qualify as immunomonitoring tools for broad clinical usage. Recent data strongly suggest that neo-antigens resulting from specific tumor mutations may indicate the most immunogenic sequences that are clinically relevant as demonstrated for successful treatment by adoptive TIL transfer (82, 83). The individual mutations can nowadays be identified in a feasible way by NGS. Coupled with tools to analyze the frequency of specific T cell clones (e.g. detection by flowcytometry after staining with tetramers), whole-exome sequencing of tumor cells will allow precise assessment of mutation-specific T cells in the blood and tumor, respectively. These cells can be regarded as future biomarker candidates, not only for adoptive T cell transfer, but also in the scope of immune checkpoint blockade. Given the functional significance of FcγR signaling mediated through IgG1 (and to a lesser extent IgG2) Ab directed against CTLA-4 (4), future prospects should also evaluate the prognostic or predictive impact of macrophage densities in TILs in the scope of immune checkpoint blockade as a promising field for future biomarker discovery. Considering the emerging gut microbiome-immunity-tumor axis, it is conceivable that changes in gut microbiota could impact on local and systemic immunity and eventually affect clinical responses to such immunomodulators inducing some degree of colitogenic side effects (84-86). Blockades of the PD-1/PD-L1 and the CTLA-4/CD86 axis operate through different mechanisms as previously described (87, 88). It is therefore not surprising that some of the hallmark criteria of response to CTLA-4 blockade may not be shared with the other Ab as elaborated by Robert et al. for the TCR diversity (89). While PD-L1 expression by tumor-invading leukocytes or cancer cell themselves appears instrumental to enrich cohorts of patients with high chance for clinical responses upon PD-1 or PD-L1 Ab, this notion might be challenged by the capacity of IFN-γ-producing T cells to progressively infiltrate

lesions, eventually up-regulating iDO or PD-L1 locally. Therefore, selection of cancer patients for treatment with PD-1 Ab based on constitutive PD-L1 expression remains questionable. Finally, an important consideration is the future of combination therapies. It is conceivable that the mode of action of a combination of two immune checkpoint blockers will differ from one another, leading to novel hallmarks of clinical responses and biomarkers predicting benefit.

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