**2. Effect of immune checkpoint blockade on effector T cells**

#### **2.1 Effect of PD-1 blockade on effector T cells**

PD-1 is an important immune checkpoint expressed on activated T cells and known to regulate their functional activity. By binding to its ligands, PD-L1 or PD-L2, which are expressed on tumor cells and a variety of immune cells [3], PD-1 is able to inhibit downstream signaling of the T cells receptor (TCR) [4]. Hence, ICIs targeting the PD-1/PD-L1 axis are developed to modulate this negative feedback loop and restore T cells activity. Indeed, response to anti PD-1 drugs is characterized by the upregulation of genes associated with activation of effector T cells [5–7]. Moreover, the blocking of PD-1 has been linked to an expansion of CD8+ effector T cells within the tumor. Interestingly, this CD8+ T cells expansion was found to follow a specific gradient that decreases from the margins of the tumor into its center [5, 8, 9]. In addition, the noted expansion in CD8+ T cells was also found to coincide with an increase in CD8+ T cell clonality in the tumor microenvironment (TME) [8]. This is clearly indicative of the fact that CD8+ T cells expanded in the tumor upon blocking PD-1 are indeed tumor-reactive and stand as a consistent correlate of treatment benefit.

Another interesting aspect is the heterogeneity noted in the tumor infiltrating CD8+ T cells depending on their different phenotypes and functional states. One such subset is known to express high levels of PD-1 and co-express the following immune modulating proteins: the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), the lymphocyte-activation gene-3 (LAG-3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD39, which are all linked to T cells exhaustion [Tex] [10, 11]. Exhausted CD8+ T cells represent a dysfunctional cell state that develops due to chronic antigen exposition. However, exhausted CD8+ T cells in the TME have a higher potential for tumor antigens recognition and a higher clonal distribution than any other CD8+ T cells subset in the TME [12, 13].

**31**

sion of both CD4+

In few studies, CD8+

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors…*

Further, the Tex phenotype also defines an impaired capacity of producing effector cytokines such as IL-2, TNF-α and IFN-γ [12]. The inhibition of the PD-1/PD-L1 axis was initially suggested to reinvigorate the anti-tumoral immune response by reversing the state of these terminally exhausted tumor-reactive T cells. However, this function is challenged by the fact that this state of terminal exhaustion is characterized by a distinct epigenetic profile which limits its reversibility [14–16]. In support of this, recent evidence has shown that anti-PD-1 therapy can engage progenitor Tex subsets that co-express PD-1 and the lineage-determining transcription factor 1 (tcf-1), instead of terminally exhausted subsets [17, 18]. Similarly, certain studies on animal models also show that blocking PD-1 leads to the engaging

Interestingly, it has been shown that Tex subsets might promote the recruitment of other immune cells into the TME and consequently support the anti-tumoral immune response [12]. In addition, anti-PD-1 treatment was shown to engage memory-precursor like CD8+ T cell subsets, leading to their accumulation in the TME [21] and to a subsequent enhanced cytotoxicity towards cancer cells [22]. The on-treatment increase in memory CD8+ T cells was suggested to correlate with response to anti-PD-1 therapy in both preclinical models and clinical studies [9, 23]. blocking of PD-1 is known to cause an increase in the proliferation of CD8+ T cells very early during the course of treatment [24, 25]. This proliferative response can be observed in the periphery, where it peaks as early as 7 days following the introduction of anti-PD-1 therapy [24, 26]. In one of our studies on a gastric cancer patient undergoing anti-PD-1 therapy, we observed that the frequency of

/CD107+

has reported that the proliferative response of peripheral CD8+

cells that are shown to correlate with anti-PD-1 treatment outcome.

**2.2 Effect of CTLA-4 blockade on effector T cells**

and CD8+

T cells appeared to be of a greater importance than that of CD8+

tremelimumab, reports no association between the expansion of CD8+

antigen closely correlated with the patient clinical outcome [27]. Another study

be predictive of durable clinical benefit in patients with solid tumors receiving anti-PD-1 therapy [26]. Thus, these studies support the fact that the systemic CD8+ effector T cells response plays a key role in tumor management under anti-PD-1 treatment and suggest their importance as a predictive biomarker of response to PD-1 blockade. In **Figure 1** we illustrate the dynamics of intra-tumoral effector T

When T cells are engaged in an active immune response, the expression of the surface protein CTLA-4, a homolog of CD28 with high affinity to B7–1 and 2 ligands, will be upregulated. CTLA-4: B7–1/2 binding acts as a co-inhibitor of the TCR signal [28]. Thus, the primary role of the CTLA-4 checkpoint is to negatively regulate T cell activation especially during the priming phase upon binding to the B7 ligands expressed by antigen presenting cells (APC). In accordance with this, a major aspect of anti-CTLA-4 therapy is its ability to reinvigorate T cell proliferation and activation. Indeed, the effects of anti-CTLA-4 therapy are evident in several studies on mouse models. For example, CTLA-4 deficient mice were found to display rapidly lethal lymphoproliferation [29]. On the other hand, anti-CTLA-4 therapy led to the expan-

subsets are necessary in mediating tumor immune control [31], the increase in CD4+

tive response to immune checkpoint blockade therapy [7, 32]. However, a study on advanced melanoma patients undergoing treatment with the anti-CTLA-4 antibody

T cells inducing their self-regeneration or differentiation into

specific for the NY-ESO-1 cancer testis

effector T cells in the tumor [30]. Although both T cells

T cells expansion has been shown to translate into an effec-

T cells [30].

effector

/PD-1+

T cells could

cells that eventually develop functional states of exhaustion [19, 20].

*DOI: http://dx.doi.org/10.5772/intechopen.96494*

of PD-1+

effector tcf-1<sup>−</sup>

/tcf-1+

/CD8+

peripheral cytotoxic T cells CD8+

#### *Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors… DOI: http://dx.doi.org/10.5772/intechopen.96494*

Further, the Tex phenotype also defines an impaired capacity of producing effector cytokines such as IL-2, TNF-α and IFN-γ [12]. The inhibition of the PD-1/PD-L1 axis was initially suggested to reinvigorate the anti-tumoral immune response by reversing the state of these terminally exhausted tumor-reactive T cells. However, this function is challenged by the fact that this state of terminal exhaustion is characterized by a distinct epigenetic profile which limits its reversibility [14–16]. In support of this, recent evidence has shown that anti-PD-1 therapy can engage progenitor Tex subsets that co-express PD-1 and the lineage-determining transcription factor 1 (tcf-1), instead of terminally exhausted subsets [17, 18]. Similarly, certain studies on animal models also show that blocking PD-1 leads to the engaging of PD-1+ /tcf-1+ /CD8+ T cells inducing their self-regeneration or differentiation into effector tcf-1<sup>−</sup> cells that eventually develop functional states of exhaustion [19, 20]. Interestingly, it has been shown that Tex subsets might promote the recruitment of other immune cells into the TME and consequently support the anti-tumoral immune response [12]. In addition, anti-PD-1 treatment was shown to engage memory-precursor like CD8+ T cell subsets, leading to their accumulation in the TME [21] and to a subsequent enhanced cytotoxicity towards cancer cells [22]. The on-treatment increase in memory CD8+ T cells was suggested to correlate with response to anti-PD-1 therapy in both preclinical models and clinical studies [9, 23].

blocking of PD-1 is known to cause an increase in the proliferation of CD8+ T cells very early during the course of treatment [24, 25]. This proliferative response can be observed in the periphery, where it peaks as early as 7 days following the introduction of anti-PD-1 therapy [24, 26]. In one of our studies on a gastric cancer patient undergoing anti-PD-1 therapy, we observed that the frequency of peripheral cytotoxic T cells CD8+ /CD107+ specific for the NY-ESO-1 cancer testis antigen closely correlated with the patient clinical outcome [27]. Another study has reported that the proliferative response of peripheral CD8+ /PD-1+ T cells could be predictive of durable clinical benefit in patients with solid tumors receiving anti-PD-1 therapy [26]. Thus, these studies support the fact that the systemic CD8+ effector T cells response plays a key role in tumor management under anti-PD-1 treatment and suggest their importance as a predictive biomarker of response to PD-1 blockade. In **Figure 1** we illustrate the dynamics of intra-tumoral effector T cells that are shown to correlate with anti-PD-1 treatment outcome.

### **2.2 Effect of CTLA-4 blockade on effector T cells**

When T cells are engaged in an active immune response, the expression of the surface protein CTLA-4, a homolog of CD28 with high affinity to B7–1 and 2 ligands, will be upregulated. CTLA-4: B7–1/2 binding acts as a co-inhibitor of the TCR signal [28]. Thus, the primary role of the CTLA-4 checkpoint is to negatively regulate T cell activation especially during the priming phase upon binding to the B7 ligands expressed by antigen presenting cells (APC). In accordance with this, a major aspect of anti-CTLA-4 therapy is its ability to reinvigorate T cell proliferation and activation. Indeed, the effects of anti-CTLA-4 therapy are evident in several studies on mouse models. For example, CTLA-4 deficient mice were found to display rapidly lethal lymphoproliferation [29]. On the other hand, anti-CTLA-4 therapy led to the expansion of both CD4+ and CD8+ effector T cells in the tumor [30]. Although both T cells subsets are necessary in mediating tumor immune control [31], the increase in CD4+ T cells appeared to be of a greater importance than that of CD8+ T cells [30].

In few studies, CD8+ T cells expansion has been shown to translate into an effective response to immune checkpoint blockade therapy [7, 32]. However, a study on advanced melanoma patients undergoing treatment with the anti-CTLA-4 antibody tremelimumab, reports no association between the expansion of CD8+ effector

*Advances in Precision Medicine Oncology*

exploration in validation trials.

ing parameter [2].

tic response.

mechanisms of action of ICIs, allowing the identification of a number of novel candidate dynamic biomarkers predictive of ICI treatment response meriting further

Tumor biopsy of tissue from primary or metastatic site is a major mainstay of treatment decisions as the molecular features and histology can reveal the complex cancer landscape. However, tissue biopsy has various limitations such as high heterogeneity, invasive nature of tissue sampling and skilled expertise/techniques required for analyzing and reporting making it a difficult specimen especially for treatment monitoring purpose [1]. Therefore, emphasis on utility of liquid biopsies as prognostic and predictive soluble biomarkers especially in cancer immunotherapy is gaining a lot of attention. The main advantages of blood-based specimens are that these are easy to extract and analyze with limited skilled expertise or techniques. Furthermore, biomarkers in the blood can represent dynamic alterations of the evolving cancer in response to treatment and can help in longitudinal monitoring. In addition to this, these can also be utilized for risk prediction of immune-related adverse events (irAE) which is an important and critical monitor-

In this chapter, we attempt to discuss in relevant details the purpose and role of immune modulatory molecules and of the different serum soluble biomarkers in various human and animal models with an aim to show insight on to their mechanisms of action and resistance, thus conveying information predictive of therapeu-

PD-1 is an important immune checkpoint expressed on activated T cells and known to regulate their functional activity. By binding to its ligands, PD-L1 or PD-L2, which are expressed on tumor cells and a variety of immune cells [3], PD-1 is able to inhibit downstream signaling of the T cells receptor (TCR) [4]. Hence, ICIs targeting the PD-1/PD-L1 axis are developed to modulate this negative feedback loop and restore T cells activity. Indeed, response to anti PD-1 drugs is characterized by the upregulation of genes associated with activation of effector T cells [5–7]. Moreover, the blocking of PD-1 has been linked to an expansion of CD8+ effector T cells within the tumor. Interestingly, this CD8+ T cells expansion was found to follow a specific gradient that decreases from the margins of the tumor into its center [5, 8, 9]. In addition, the noted expansion in CD8+ T cells was also found to coincide with an increase in CD8+ T cell clonality in the tumor microenvironment (TME) [8]. This is clearly indicative of the fact that CD8+ T cells expanded in the tumor upon blocking PD-1 are indeed tumor-reactive and stand as

Another interesting aspect is the heterogeneity noted in the tumor infiltrating CD8+ T cells depending on their different phenotypes and functional states. One such subset is known to express high levels of PD-1 and co-express the following immune modulating proteins: the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), the lymphocyte-activation gene-3 (LAG-3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD39, which are all linked to T cells exhaustion [Tex] [10, 11]. Exhausted CD8+ T cells represent a dysfunctional cell state that develops due to chronic antigen exposition. However, exhausted CD8+ T cells in the TME have a higher potential for tumor antigens recognition and a higher clonal distribution than any other CD8+ T cells subset in the TME [12, 13].

**2. Effect of immune checkpoint blockade on effector T cells**

**2.1 Effect of PD-1 blockade on effector T cells**

a consistent correlate of treatment benefit.

**30**

**Figure 1.** *Effect of immune checkpoint inhibition on effector T cells.*

T cells and successful anti-tumor response [33] despite similar CD8+ T cells activation profiles observed in lesions that responded to anti-CTLA-4 therapy and those that did not respond to this therapy [33]. These findings, possibly explained by the action of immunosuppressive elements in the TME, highlight that adequate CD8+ effector T cells function is necessary but not sufficient for complete suppression of tumor growth under immunotherapy [34]. Another interesting effect noted upon CTLA-4 blockade is the enhanced expansion of memory CD8+ T cell [35] which will help to promote long-term tumor control post-therapy. Moreover, this expansion of memory CD8+ T cells is considered as an indicator of treatment benefit in a few clinical studies [36, 37].

CD4<sup>+</sup> T cells expanding in the tumors of mouse models under anti-CTLA-4 treatment are found to exhibit a Th1-like effector phenotype with a noticeable expression of ICOS, a known marker of follicular T helper cells [30]. Interestingly, this phenotype of CD4+ T cell was observed in mice after genetic knock-down of negative co-inhibitory molecules such as CTLA-4 [38]. Moreover, ICOS-deficient mice showed an impaired anti-tumor T cell response to anti-CTLA-4 therapy [39]. In addition, a tumor microenvironment that is rich in CD4+ Th1 effector T cells infiltration was shown to be critical to develop response to anti-CTLA-4 therapy in castration resistant prostate cancer [40]. Furthermore, a higher expression of Th1 associated genes was observed in melanoma tumors of patients responding to ipilimumab compared to non-responders, supporting the functional relevance of a Th1- response in CTLA-4 inhibitor treatment benefit [41]. Interestingly, while the presence of CD4+ Th1 cells is predictive of response to anti-CTLA-4, a peripheral blood profile rich in Th17 cells was reported to be rather predictive of autoimmune toxicity under anti-CTLA-4 therapy [42]. Moreover, several clinical studies have reported an increase of ICOS+ /CD4+ T cells in the tumor and peripheral blood of patients treated with anti-CTLA-4 [43–49]. Additionally, an increased proliferation of both peripheral CD4+ and CD8+ T cells is observed as early as 3 weeks after the first dose of anti-CTLA-4 treatment [50–52]. Such a response may partly be due to the bulk expansion in the periphery of specific T cells against known tumor antigens in anti-CTLA-4 treated patients [53–55]. Of note, an increase in absolute

**33**

induced Foxp3<sup>+</sup>

tive effector Tregs (Foxp-3hi/CD45−

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors…*

lymphocyte count (ALC) was shown to correlate with enhanced overall survival

The distribution of the TCR repertoire may be described by different metrics. For example, TCR richness invoke the number of unique T cell clones while its evenness refers to the frequency of their distribution. Some studies reported an increase in the richness of the TCR repertoire under anti-CTLA-4 therapy [60, 61]. On the contrary, the evenness of the TCR repertoire under anti-CTLA-4 therapy is comparatively less impacted [60, 62, 63]. This increase in richness of the TCR repertoire under the effect of anti-CTLA-4 therapy is indicative of unleashed T-cell priming possibly allowing for enhanced tumor immune control through the promotion of new anti-tumor T cells responses [64]. However, in a study on metastatic melanoma and prostate cancer, it has been shown that enhanced clinical outcomes under CTLA-4 blockade are associated with less clonotype loss and on-treatment stability of existing high-frequency TCR clonotypes [61]. These findings suggest that response to anti-CTLA-4 treatment occurs despite the remodeling of the peripheral TCR repertoire rather than as a result of it. In **Figure 1** we illustrate the dynamics of intra-tumoral effector T cells that are

**3. Effect of immune checkpoint blockade on immune suppressive T cells**

Regulatory T cells (Tregs), an immunosuppressive subset of T cells, are known to be closely involved in the regulation of the immune responses to cancer [65–67]. The tumor-infiltrating subsets of Tregs are characterized by their high surface expression of PD-1 [68–70] and the PD-1/PD-L1 axis is known to modulate Tregs function via cell-intrinsic pathways. For example, the blocking of PD-1 in animal models reduced the immunosuppressive function of Tregs and declined their expression in the TME [71, 72]. Moreover, studies on murine models have shown

that anti-PD-1 therapy is associated with an increase rather than a decrease in Tregs infiltration in the TME [24, 75]. The proliferation of Tregs under anti-PD-1 therapy could be explained by a treatment-induced reversal of the exhausted state of PD-1Hi in the TME [76, 77]. The expansion of Tregs upon blockade of PD-1/PD-L1 axis has been observed at the tumor level as well as in the peripheral blood. One particular study showed that patients with gastric adenocarcinoma responding to anti-PD-1 displayed an on-treatment decrease in intra-tumoral Tregs, whereas non-responders had post-treatment tumor biopsies exhibiting an infiltration of highly prolifera-

CD4+

changes in intra-tumoral T reg infiltration could be a relevant dynamic parameter to account for in predicting anti-PD-1/PD-L1 treatment response. The predictive insight provided by the on-treatment dynamics of circulating Tregs under PD-1/ PD-L1 blockade has also been investigated. In a study conducted on melanoma patients exposed to nivolumab, an expansion of circulating Tregs under therapy was shown to positively correlate with treatment benefit [79]. These observations suggest that Tregs dynamics under anti-Pd-1 therapy may contribute predictive insight into treatment benefit when monitored both in the TME and in the periphery.

regulated by anti-PD-1 therapy has been recently identified. These cells known as 4PD-1Hi express high levels of PD-1 while lacking Foxp-3 expression [80]. Also, these 4PD-1Hi cells were shown to accumulate in the tumor and to inhibit T cells effector

Tregs (iTregs) [73, 74]. Conversely, certain preclinical studies show

Th1 effector T cells into

) [78]. This suggests that on-treatment

T cell subset found to be

*DOI: http://dx.doi.org/10.5772/intechopen.96494*

and response to ipilimumab in several studies [56–59].

shown to correlate with anti-CTLA-4 treatment outcome.

**3.1 Effect of PD-1 blockade on immune suppressive T cells**

that PD-1/PD-L1 pathway mediates the conversion of CD4+

Interestingly, another immunosuppressive CD4+

*Evolving Dynamic Biomarkers for Prediction of Immune Responses to Checkpoint Inhibitors… DOI: http://dx.doi.org/10.5772/intechopen.96494*

lymphocyte count (ALC) was shown to correlate with enhanced overall survival and response to ipilimumab in several studies [56–59].

The distribution of the TCR repertoire may be described by different metrics. For example, TCR richness invoke the number of unique T cell clones while its evenness refers to the frequency of their distribution. Some studies reported an increase in the richness of the TCR repertoire under anti-CTLA-4 therapy [60, 61]. On the contrary, the evenness of the TCR repertoire under anti-CTLA-4 therapy is comparatively less impacted [60, 62, 63]. This increase in richness of the TCR repertoire under the effect of anti-CTLA-4 therapy is indicative of unleashed T-cell priming possibly allowing for enhanced tumor immune control through the promotion of new anti-tumor T cells responses [64]. However, in a study on metastatic melanoma and prostate cancer, it has been shown that enhanced clinical outcomes under CTLA-4 blockade are associated with less clonotype loss and on-treatment stability of existing high-frequency TCR clonotypes [61]. These findings suggest that response to anti-CTLA-4 treatment occurs despite the remodeling of the peripheral TCR repertoire rather than as a result of it. In **Figure 1** we illustrate the dynamics of intra-tumoral effector T cells that are shown to correlate with anti-CTLA-4 treatment outcome.
