**4. Targets and types of immunotherapy in solid tumors**

Immunotherapy is defined as an interaction with the immune system aiming to treat/cure cancer. Immunotherapy could be largely divided into passive and active.

#### **4.1. Active immunotherapy**

Active immunotherapy has recently undergone active clinical research. As tumors express multiple tumor-associated antigens or neonatigens, the immune system should respond by adaptive activation of T-lymphocytes against those potential targets as previously described in section 3.1.2. Any mechanism leading to activation of the immune system is considered as active immunotherapy. Active immunotherapy has been developed in order to induce and stimulate the individual's own immune response. An example of this still-developing branch of immunotherapy represents Sipuleucel-T, which is the first active cellular immunotherapy approved for clinical use by the American Food and Drug Agency (FDA) in the treatment of prostate cancer based on the data that a benefit in survival was observed in the group of asymptomatic or minimally symptomatic patients with Castrate-resistant prostate cancer (CRPC) [24]. It consists of autologous peripheral-blood mononuclear cells obtained by leucopheresis, cultured and activated ex vivo with a recombinant human fusion protein PA2024 consisting of prostatic acid phosphatase linked to granulocyte-macrophage colonystimulating factor (PAP-GM-CSF). GM-CSF stimulates the maturation process of APCs to mature DCs, while PAP peptides functions with MHC I and II. Upon reinfusion in the patient, an immune response against PAP-containing cells is triggered [24,25].

Another example of active immunotherapy is the cellular adoptive immunotherapy using transfusion of the patient's own T-lymphocytes previously stimulated ex vivo and currently tested in phase I/II trials. There is also a wealth of trials with autologous or donor dendritic cells, autologous tumor cell lysate, activated lymphocytes, or vaccines (DNA, peptide, recombinant viral vector vaccines, etc.) used as monotherapy or in combination with chemo‐ therapy or other passive immunotherapy options such as anti-CTLA 4 MAB, anti-PD-1 MAB, and anti-PD-L1 MAB [26-30].

#### **4.2. Passive immunotherapy**

There is a theory suggesting that cells and tissues are constantly monitored by an ever-alert immune system, and that such immune surveillance is responsible for recognizing and eliminating the vast majority of incipient cancer cells and thus nascent tumors. According to this logic, solid tumors that do appear have somehow managed to avoid detection by the immune system or have been able to limit the extent of immunological killing, thereby escaping eradication. This is a process called immunoediting—a tumor mechanism, exerting an extrinsic suppression; it occurs only after a malignant cancerous transformation has already occurred and the intrinsic tumor suppressor mechanisms have already failed. The cancer immunoedit‐ ing roughly consists of three sequential phases: elimination, equilibrium, and escape. In the first phase, the body immune system successfully detects cancer cells and eliminates them efficiently [18,19]. In the equilibrium phase the immune system obstructs tumor growth, but is unable to completely eradicate the tumor. This step is thought to be continuous in time and it could either progress to the last escape phase or reverse backwards, leading to complete tumor eradication by the immune system. If this second phase continues for a longer period of time, the immune system is incapable of tumor eradication and continuously interacts with the tumor, thus sculpturing or editing the tumor genetics [20-22]. In the last phase, the tumor growth is no longer controlled and blocked by the immune system and the tumor spreads and

produces clinically apparent diseases [23].

82 Immunopathology and Immunomodulation

**4.1. Active immunotherapy**

**4. Targets and types of immunotherapy in solid tumors**

cancer. Immunotherapy could be largely divided into passive and active.

an immune response against PAP-containing cells is triggered [24,25].

Immunotherapy is defined as an interaction with the immune system aiming to treat/cure

Active immunotherapy has recently undergone active clinical research. As tumors express multiple tumor-associated antigens or neonatigens, the immune system should respond by adaptive activation of T-lymphocytes against those potential targets as previously described in section 3.1.2. Any mechanism leading to activation of the immune system is considered as active immunotherapy. Active immunotherapy has been developed in order to induce and stimulate the individual's own immune response. An example of this still-developing branch of immunotherapy represents Sipuleucel-T, which is the first active cellular immunotherapy approved for clinical use by the American Food and Drug Agency (FDA) in the treatment of prostate cancer based on the data that a benefit in survival was observed in the group of asymptomatic or minimally symptomatic patients with Castrate-resistant prostate cancer (CRPC) [24]. It consists of autologous peripheral-blood mononuclear cells obtained by leucopheresis, cultured and activated ex vivo with a recombinant human fusion protein PA2024 consisting of prostatic acid phosphatase linked to granulocyte-macrophage colonystimulating factor (PAP-GM-CSF). GM-CSF stimulates the maturation process of APCs to mature DCs, while PAP peptides functions with MHC I and II. Upon reinfusion in the patient,

At present, passive immunotherapy is still more commonly used as it refers to the delivery of previously synthesized agents that could be used by the immune system; typical examples are the use of non-specific immunomodulatory cytokines IFN-α, IL-2, or the specific MAB. Early clinical studies demonstrated that the use of immunomodulatory cytokines such as interferon alpha (IFN α) or interleukin 2 (IL-2) may induce antitumor immune-mediated effects as tumor regression in some solid malignancies [31,32]. Cytokines have been used as cancer immuno‐ therapy for long decades and they work either by exerting a direct antitumor effect or by indirectly enhancing the antitumor immune response [33]. Multiple in vitro studies have shown that TNF-α and IL-6 exert direct antitumor effect suppressing cancer cell growth and survival. However, clinical use of these cytokines in cancer patients has led to less successful results because of significant toxicity and the controversial influence of a single molecule such as TNF-α and IL-6. Although they are able to suppress tumor growth, they actually promote growth of other tumors; further on, IL-6 may also exert immunosuppression. Therefore, the use of the direct antitumor effect of cytokines remains exclusively an academic pursuit.

In contrast, other cytokines may enhance the antitumor immune response through a variety of different pathways and thus they are more widely used in the clinical practice. For example, IL-2 and IFN-α promote T-lymphocytes and NK cells growth and activation, while granulo‐ cyte-macrophage colony-stimulating factor (GM-CSF) acts on APCs, increasing the processes of antigen processing and presentation as well as the production of co-stimulatory cytokines. These cytokines are nowadays well-established cancer immunotherapies, e.g., IL-2 is used in the treatment of metastatic melanoma and metastatic renal cell carcinoma, and IFN-α is approved for the treatment of malignant melanoma [34,35]. There are reports in the literature where recombinant IL-2 has also been used in the treatment of other solid tumor malignancies, including neuroendocrine tumors [36]. This led to the introduction of immunotherapy as an anticancer treatment for metastatic renal cell carcinoma in 1992 and metastatic melanoma in 1998. Subsequently, immunotherapy with interferon was also approved in the adjuvant setting in patients with high-risk malignant melanoma as it was considered a beneficial approach [37,38]. Some other cytokines, such as IL-7, IL-11, IL-12, IL-15, IL-21, IFN-β, and IFN-γ, are also currently evaluated as cancer immunotherapies.

Another typical example of passive immunotherapy is the use of MAB. There are multiple MAB used in the treatment of solid malignancies such as the MAB against the Epidermal Growth Factor (EGFR antibody) *cetuximab* or the antibody targeting the Human Epidermal Receptor type 2 (HER 2) *trastuzumab*. These MAB specifically target their receptor at the cancer cell surface and by binding to it, they prevent the signal cascade, transmitted intracellularly, thus preventing further tumor growth or reproduction. MAB may also target soluble circula‐ tory factors that are important for the tumor such as the MAB *bevacizumab*, which targets the vascular endothelial growth factor (VEGF).

MAB may target not only tumor pathways. More recent research focused on the "communi‐ cation" between the host and the tumor, targeting the immune system as a mechanism and controlling this process. The PD-1/PD-L1 interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1 under healthy conditions is to downmodulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 is expressed by activated T cells, mediating immunosuppression (Figure 2).

**Figure 2.** Immunosuppression, mediated via PD-1/PD-L1 pathway. A) PD-1 is expressed by activated T cells; by bind‐ ing to PD-L1, it mediates T-lymphocyte suppression. B) The use of immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 MAB) leads to the interruption of this immunosuppression and potential cytotoxicity exerted by the T cells.

PD-1 functions in peripheral tissues where T cells encounter immunosuppressive PD-1 ligands PD-L1 and PD-L2 that are expressed by tumor cells, stromal cells, or both [39-42]. Inhibition of the interaction between PD-1 and PD-L1 enhances T cell responses in vitro and mediates preclinical antitumor activity [39,43]. PD-L1 leads to inhibition of the T-lymphocyte prolifer‐ ation, survival and effector functions (cytotoxicity, cytokine release), inducing apoptosis of tumor-specific T cells, and promoting the differentiation of CD4+ T cells into regulatory T cells. The blockade of PD-1/PD-L1 results in a potent and durable tumor regression and prolonged stabilization in patients with advanced malignancies [44]. Therefore, inhibition of PD-L1 binding to PD-1 represents an attractive strategy to restore tumor-specific T cell immunity.

The mechanism by which PD-1 down modulates T cell responses is similar to, but distinct from, that of CTLA-4 as both molecules regulate an overlapping set of signaling protein. PD-1 was shown to be expressed on T cells, B cells, monocytes, and natural killer T cells, following their activation [45,46]. PD-L1 and PD-L2 are expressed in a variety of cell types, including non-hematopoietic tissues, as well as in various malignancies. PD-L1 is expressed at low levels on non-hematopoietic tissues, whereas PD-L2 protein is only detectably expressed on antigenpresenting cells in lymphoid tissue or chronic inflammatory environments. PD-L2 controls immune T cell activation in lymphoid organs, whereas PD-L1 serves to protect healthy tissues from unwarranted T-cell immune-mediated damage.

Although healthy organs express little (if any) PD-L1, many cancers express abundant levels of this T cell inhibitor. High expression of PD-L1 on tumor cells (and to a lesser extent, PD-L2) has been found to correlate with poor prognosis and survival in various cancers, including RCC [47], pancreatic carcinoma [48], hepatocellular carcinoma [49], and ovarian carcinoma [50]. Furthermore, PD-1 has been suggested to regulate tumor-specific T cell expansion in melanoma patients [51].

The observed correlation of clinical prognosis with PD-L expression in multiple cancers suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.

Over the past several decades, these observations have resulted in intensive efforts to develop immunotherapeutic approaches as cancer treatment options. Such agents include immunecheckpoint-pathway inhibitors such as anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) antibody (ipilimumab), anti-programmed death 1 (anti-PD-1) inhibitor (pembrolizumab, nivolumab, pidilizumab), anti-PD-L1 inhibitors (MPDL3280A, BMS-936559, MEDI4736), etc. (Table 1).

So far, passive immunotherapy has had limited success in the treatment of solid tumors, except in the treatment of malignant melanoma and renal cell cancer (RCC) [52-55]. The therapeutic options for advanced disease in RCC comprise of tyrosine-kinase inhibitors, m-TOR inhibitors, IL-2, antiangiogenic VEGF inhibitors, and IFNα. Spontaneous remissions and durable responses have been largely described as a result of this non-specific immune response. The prognosis of those patients unfortunately remains poor with a 5-year overall survival below 5% [56]. Thus, new options appear on the horizon involving the new specific targeted immu‐ notherapies, focusing on the blockade of T cell regulation and functions, as well as activation of the dendritic cells (a form of active immunotherapy, described below). There are also phase I/II trials, studying the potential benefit of cellular adoptive immunotherapy using transfusion of stimulated patient's own T-lymphocytes. This adoptive T-lymphocyte therapy consists of infusion of ex vivo isolated, activated, or expanded tumor-specific T-lymphocytes [57]. There are different types of adoptive therapy, including TILs, engineered T-cells, expressing a specific cancer-related receptor (TCRs) or chimeric antigen receptor (CAR). Each of these approaches has its own advantages and disadvantages.

#### **4.3. Immunotherapy as cancer prevention**

cell surface and by binding to it, they prevent the signal cascade, transmitted intracellularly, thus preventing further tumor growth or reproduction. MAB may also target soluble circula‐ tory factors that are important for the tumor such as the MAB *bevacizumab*, which targets the

MAB may target not only tumor pathways. More recent research focused on the "communi‐ cation" between the host and the tumor, targeting the immune system as a mechanism and controlling this process. The PD-1/PD-L1 interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1 under healthy conditions is to downmodulate unwanted or excessive immune responses, including autoimmune reactions.

**Figure 2.** Immunosuppression, mediated via PD-1/PD-L1 pathway. A) PD-1 is expressed by activated T cells; by bind‐ ing to PD-L1, it mediates T-lymphocyte suppression. B) The use of immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 MAB) leads to the interruption of this immunosuppression and potential cytotoxicity exerted by the T cells.

PD-1 functions in peripheral tissues where T cells encounter immunosuppressive PD-1 ligands PD-L1 and PD-L2 that are expressed by tumor cells, stromal cells, or both [39-42]. Inhibition of the interaction between PD-1 and PD-L1 enhances T cell responses in vitro and mediates preclinical antitumor activity [39,43]. PD-L1 leads to inhibition of the T-lymphocyte prolifer‐ ation, survival and effector functions (cytotoxicity, cytokine release), inducing apoptosis of

The blockade of PD-1/PD-L1 results in a potent and durable tumor regression and prolonged stabilization in patients with advanced malignancies [44]. Therefore, inhibition of PD-L1 binding to PD-1 represents an attractive strategy to restore tumor-specific T cell immunity.

The mechanism by which PD-1 down modulates T cell responses is similar to, but distinct from, that of CTLA-4 as both molecules regulate an overlapping set of signaling protein. PD-1 was shown to be expressed on T cells, B cells, monocytes, and natural killer T cells, following

T cells into regulatory T cells.

tumor-specific T cells, and promoting the differentiation of CD4+

PD-1 is expressed by activated T cells, mediating immunosuppression (Figure 2).

vascular endothelial growth factor (VEGF).

84 Immunopathology and Immunomodulation

Tumor cells express neoantigens that are expressed as a consequence of the malignant transformation of the host cell. The expression of neoantigens could also be the result of a


This is not an entirely comprehensive list of all trials that have been listed in www.clinicaltrials.com. (Source www.clinicaltrials.com)

Abbreviations:

CTLA-4: Cytotoxic T-lymphocyte-associated protein 4

PD-1: Programmed death 1

PD-L1: Programmed death 1 ligand

MAB: Monoclonal antibody

MEL: Melanoma

CRPC: Castrate-resistant prostate cancer

RCC: Renal cell carcinoma

NSCLC: Non-small cell lung cancer

CRC: Colorectal cancer

SCCHN: Squamous cell carcinoma of the head and neck

**Table 1.** Immune-checkpoint-pathway inhibitors and their targets in currently running clinical trials.

viral or (more rarely) bacterial infection that induced and provoked this malignant transfor‐ mation and thus the idea to vaccinate against those pathogens and prevent the associated cancer. More than 15% of all cancers are considered to be related to infectious agents [58]. Infection with human papilloma viruses (HPVs) is associated in about 30% of those cases (5% of all cancers) and hepatitis B and C viruses together with *Helicobacter pylori* (*H. Pylori*) account for 60% more of all infectious-agent-related cancers. It is logical that the success of convention‐ al antimicrobial vaccines could encourage potential cancer vaccine prevention research. This approach has proven its efficacy in hepatitis B-induced hepatocellular carcinoma [59]. In carcinoma of the uterine cervix, it is a well-known fact that about 70% of them are caused by HPV types 16 and 18, and it is expected that HPV-vaccination could decrease the incidence not only of cervical cancer [60,61], but also of head-and-neck squamous cell carcinoma [62]. The mechanism, by which HPV induces malignant transformation, is by provoking the synthesis of two oncogene products, encoded by the virus, which degradate the tumor suppressor protein p53 and block other tumor suppressor proteins cells in the premalig‐ nant dysplasia cells, as well as the cell in the in situ and the invasive carcinomas. The recombinant vaccination against HPV leads to secretion of specific antibodies, protecting the non-infected organisms from HPV-infections, and the subsequent development of HPVinfection-related cancer sites [63]. No significant effect was demonstrated in already HPVinfected individuals [64].

#### **4.4. Immunotherapy as anticancer treatment**

**Target Drug name Biological**

*Ipilimumab (BMS-734016)*

86 Immunopathology and Immunomodulation

*Nivolumab (BMS-936558)*

*Pembrolizumab Lambrolizumab (MK-3475)*

> *Pidilizumab (CT-011)*

*MEDI4736*

CTLA-4: Cytotoxic T-lymphocyte-associated protein 4

SCCHN: Squamous cell carcinoma of the head and neck

CTLA-4

PD-1

PD-L1

www.clinicaltrials.com)

PD-1: Programmed death 1

RCC: Renal cell carcinoma

CRC: Colorectal cancer

PD-L1: Programmed death 1 ligand MAB: Monoclonal antibody

CRPC: Castrate-resistant prostate cancer

NSCLC: Non-small cell lung cancer

Abbreviations:

MEL: Melanoma

**description**

Fully human IgG4 MAB

Humanized IgG4 MAB

Humanized IgG1

MAB

*AMP-224* IgG1 fusion

*BMS-936559* Fully human IgG4

*MPDL3280A* MAB

MAB Pancreatic tumors

*Tremelimumab* MAB MEL MEL -

CRC, HCC, prostate

NSCLC, MEL, CRC, RCC, ovarian, pancreatic, breast cancer

NSCLC, MEL, CRC, ovarian, pancreatic, breast cancer

*Medimmune-AZ* IgG4 MAB SCCHN MEL NSCLC

This is not an entirely comprehensive list of all trials that have been listed in www.clinicaltrials.com. (Source

**Table 1.** Immune-checkpoint-pathway inhibitors and their targets in currently running clinical trials.

**Phase of the trial by tumor site Phase I Phase II Phase III**

> Ovarian Gastric

carcinoma

RCC, bladder carcinoma

CRPC Esophageal

cancer RCC, CRC

MAB - MEL -

protein Solid malignancies - -

NSCLC CRPC

MEL, NSCLC

MEL NSCLC RCC

NSCLC


There are 271 trials that are recruiting patients as assessed on 23 Apr 2015 at www.clinicaltri‐ als.com. They include different DNA-vaccines, dendritic cell vaccines, peptide vaccines, allogeneic GM-CSF-secreting vaccines, recombinant vaccines, vaccines, targeting different auto-antigens as targets, etc. They are carried in patients with various solid malignancies, predominantly in melanoma, renal cell carcinoma, non-small cell carcinoma and other solid tumors. The immunotherapy approach was also implemented in the treatment of neuroendo‐ crine tumors, e.g., vaccination with tumor lysate-pulsed DCs that induced a significant antitumor immune response in a neuroendocrine carcinoma of the pancreas [65].

A common example of the vaccine's use as treatment is the Bacillus Calmette-Guerin (BCG), which represents an attenuated mycobacterium, originally developed as anti-tuberculosis vaccine. It was only subsequently proven in 1976 that its immunostimulation characteristics led to antitumor effects in preventing recurrence in patients who underwent transurethral resection of superficial non-muscle invasive bladder carcinoma and carcinoma in situ when used as local repeated intravesical instillations [66]. Besides the non-specific immune activa‐ tion, there is a theory suggesting that its anticancer effect might be attributed to specific BCG internalization in the tumor cells by the integrins and fibronectins of the tumor cells [67,68], provoking antigen-specific adaptive immune response as well [69-71].

A list of some of the more important clinical trials using cancer vaccines as therapeutic options are listed in Table 2.



LFA-3

**Vaccine class**

Tumor cell

DC / APCs

Peptides/ proteins

**Name and target of**

88 Immunopathology and Immunomodulation

Pancreatic tumor cell vaccine

Nelipepimut-S

MAGE-A3

**the vaccine Biological description**

Algenpantucel-L Allogeneic human pancreatic

SL-701 Multivalent glioma-associated

Ovapuldencel-T Autologous PBMCs in GM-CSF -

AGS-003 Autologous DCs transfected with

DCVAC/Pca Autologous DCs pulsed with

DCVax-L Autologous DCs pulsed with

CVac Autologous DCs pulsed with

ICT-107 Autologous DCs pulsed with

MelCancerVac Autologous DCs pulsed with

L-BLP25 (Tecemotide) Liposome-encapsulated synthetic

Rindopepimut hEGFR variant III specific peptide

POL-103A Protein antigens from 3 melanoma

IMA901 Synthetic vaccine consisting of 10

MAGE-A3 ASCI MAGE-A3 antigen-specific cancer

GV1001 hTERT peptide MEL,

HER2/*neu* peptide combined with

MAGE-A3 combined with GM-

GM-CSF gene-transfected tumor cell vaccine

**Phase of the trial by tumor site Phase I Phase II Phase III**

> Ovarian, peritoneal carcinoma

> > HCC

prostate, CRC

Pancreas adenocarcinom a


NSCLC, pancreatic

NSCLC

MEL Pancreas

cancer vaccine RCC, prostate MEL, NSCLC

antigen vaccine - GBM -

tumor and CD40L RNAs - - RCC

killed prostate cancer line LNCap - Prostate Prostate

MUC1-mannan fusion protein - Ovarian -

allogeneic melanoma cell lysate - CRC, NSCLC -

peptide derived from MUC-1 - Rectal, NSCLC,

pancreatic

conjugated to KLH - GBM GBM

different TUMAPs - - RCC

immunotherapeutic - NSCLC NSCLC

CSF - Bladder MEL

cell lines with alum adjuvant - - MEL

GM-CSF - - Breast

tumor lysate antigen - - GBM

antigens - GBM -

TUMAPs: Tumor-associated peptides

**Table 2.** Therapeutic use of cancer vaccines in clinical development in solid malignancies. This is not an entirely comprehensive list of all trials that have been listed in www.clinicaltrials.com. (Source www.clinicaltrials.com).

#### **4.5. Predictive and prognostic biomarkers for immunotherapy**

Research is ongoing in order to identify potential biomarkers for cancer immunotherapy. In order to optimize this process, we shall recently be in great demand of predictive/prognostic factors, justifying the selection of patients, who would be the best candidates for such novel, expensive, and potentially toxic treatments. PD-L1-positive cancers are associated with poorer prognoses than PD-1 negative. A correlation of PD-L1 expression and response rate was demonstrated in patients with the highest levels of PD-L1 expression and PD-L1-positive TILs [72]. The potential role of PD-L1 as well as TILs as a biomarkers remain to be elucidated.

The presence or absence of TILs also remains to be clarified. There are data that the immune system plays an important role in the process of recurrence of solid tumors. There is a multicenter study over 603 patients with colorectal cancer that showed the importance of the adaptive immune response and the presence/absence of T-lymphocytes in the resected tumor was a factor that correlated more accurately with clinical outcomes than the current parameters considered as gold standards for prognosis, histopathologically determined tumor stage (T) and nodal status (N), yielding a place for TILs as a potential prognostic marker in colorectal cancer [73] and potentially in other localizations of malignant tumors. It has also been proven for patients with large early-stage cervical cancer [74], muscle-invasive urothelial bladder carcinoma [75], and breast cancer [76]. All these findings suggest that assessment and consid‐ eration of the local intratumoral immune response in the primary tumor may have prognostic value and should be evaluated in the process of treatment decision taking.

#### **5. Adverse effects of immunotherapy**

Adverse events (AE) are graded using NCI Common Terminology Criteria for Adverse Events Version 4.0. Their management is important as the population of treated patients frequently consists of patients with disseminated disease or patients who have been previously treated with multiple treatment lines. Most frequent drug-related AEs with potential immune-related mechanism are hepatitis, pneumonitis, infusion reactions, colitis, arthralgia, and rash, necessitating sometimes the use of corticosteroids [77]. Fatigue, decreased appetite, nausea, dyspnea, diarrhea or constipation, vomiting, pyrexia, vitiligo, and headache are also described as immune-related AEs.
