**Abstract**

The rational design of immunotherapeutic agents has advanced with a fundamental understanding that both innate and adaptive immunity play important roles in cancer surveillance and tumor destruction; given that oncogenesis occurs and cancer progresses through the growth of tumor cells with low immunogenicity in an increasingly immunosuppressive tumor microenvironment. Checkpoint inhibitors in the form of monoclonal antibodies that block cancer's ability to deactivate and evade the immune system have been widely indicated for a variety of tumor types. Through targeting the biological mechanisms and pathways that cancer cells use to interact with and suppress the immune system, immunotherapeutic agents have achieved success in inhibiting tumor growth while eliciting lesser toxicities, compared to treatments with standard chemotherapy. Development of "precise" bio-active tumor-targeted gene vectors, biotechnologies, and reagents has also advanced. This chapter presents ongoing clinical research involving immune checkpoint inhibitors, while addressing the clinical potential for tumor-targeted gene blockade in combination with tumor-targeted cytokine delivery, in patients with advanced metastatic disease, providing strategic clinical approaches to precision cancer immunotherapy.

**Keywords:** PD-1 inhibitor, CTLA4 inhibitor, DeltaRex-G, DeltaVax, NK cells, checkpoint inhibitors, cell cycle control, GMCSF

#### **1. Introduction**

The human immune system is an intricate network of cell types and signaling pathways that act in a concerted effort to ensure that when an immune response is elicited, it is directly proportional to the severity of the attack. Although this network exists to protect the body from foreign invasion, an overactive immune response can lead to immunopathogenesis and autoimmunity, thus it is crucial that there are mechanisms set in place to ensure this system remains tightly regulated [1]. The immune system achieves this strict regulation by engaging a complex system of checkpoint control pathways. These checkpoints act as metaphorical gateways that require a specific key, in the form of a protein or a small molecule, in order to initiate tightly regulated signaling pathways that prevent over-reactivity of an immune response through the binding of specific cell surface receptors. This process is known as peripheral tolerance [2]. Certain checkpoint pathways,

including those involving transmembrane protein receptors cytotoxic t-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), play pivotal inhibitory roles in T-cell activation. Specifically, the CTLA-4 checkpoint is designed to inhibit T-cells from becoming autoreactive during the beginning stages of T-cell activation, while the PD-1 checkpoint is part of a family of costimulatory receptors that, when bound to its ligand, inhibits T-cell proliferation [2].

Tumor cells exploit the process of peripheral tolerance as a way to evade immunological surveillance by mimicking inhibitory receptors that are normally expressed on the surface of antigen presenting cells [3]. Expressing these inhibitory receptors allows cancer cells to effectively downregulate an immune response by deactivating the T-cells they come into contact with. The development of genetically engineered immune checkpoint inhibitors (ICIs) to treat malignancies therefore has the potential to revive pre-existing immune responses that would have otherwise been suppressed by the cancer [4]. Immunotherapies have been developed over the past decade using monoclonal antibodies as checkpoint inhibitors, binding the inhibitory receptor on T-cells and blocking tumor cells from binding to these sites.

The first immune checkpoint inhibition therapies to enter clinical trials for patients with advanced cancer were two fully human CTLA-4 blocking antibodies, ipilimumab and tremelimumab. Clinical activity of the CTLA-4 blockade was most significant in advanced melanoma patients, leading to a 15% response rate that, for some patients, persisted for over 10 years after discontinuing therapy [5]. In 2010, a large Phase III trial was published showing ipilimumab to have significantly improved overall survival rates in patients with metastatic melanoma, compared to treatment with standard gp100, a synthetic peptide cancer vaccine, alone [6]. Ipilimumab has since been FDA approved in combination for the treatment of advanced renal cell carcinoma, microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), and malignant pleural mesothelioma.

The PD-1 checkpoint pathway was the next to be targeted with antibody therapy. Similar to ipilimumab, the first nivolumab trials were also shown to be most efficacious in melanoma patients, although it is now approved not only for the treatment of melanoma, but also of non-small cell lung cancer (NSCLC), small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, MSI-H or dMMR metastatic colorectal cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. A study assessing the efficacy of anti-CTLA-4 and anti-PD-1 combined therapy in melanoma patients showed even more significant results, with 53% of patients achieving an objective response, and ≥ 80% tumor reduction was reported in all patients [7].

Thus far, the only two immune checkpoint inhibitors that have been successfully brought to market are those that involve the PD-1/PD-L1 checkpoint and CTLA-4 checkpoint. These targets are within the adaptive immune system, but scientists are looking at the potential anti-tumor effects of exploring checkpoint targets within the innate immune system. Another target currently being investigated involves immune checkpoint inhibition within natural killer (NK) cell-mediated immunity. Cancer cells frequently downregulate their MHC expression, rendering T-cell mediated immunotherapy insufficient for killing these tumor cells. NK cell-mediated treatment can, in theory, compensate for this. As a first line of defense within the immune surveillance system, NK cells are quicker to become activated and will indiscriminately induce apoptosis in any cell lacking MHC-receptors.

Similar to the immune system, a checkpoint control system is also used to control the distinct phases of the cell division cycle in order to regulate cellular proliferation. Unrestrained cell division is a fundamental characteristic of oncogenesis, therefore

**5**

*Immune and Cell Cycle Checkpoint Inhibitors for Cancer Immunotherapy*

cell cycle checkpoint control is vital in preventing the development of cancer. The mechanism of action in this case of checkpoint control is site-specific protein phosphorylation executed largely by cyclin-dependent proline-directed protein kinases. For example, Cyclin D1 and CDK4/6 are downstream of growth-initiating signaling pathways which lead to cellular proliferation. Palbociclib, an inhibitor of cyclindependent kinases CDK4/CDK6 is approved for the treatment of HR-positive, HER2-negative advanced or metastatic breast cancer in combination with an

aromatase inhibitor as initial endocrine based therapy in postmenopausal women or fulvestrant in women with disease progression following endocrine therapy [8]. Another example of an executive cell cycle regulatory protein is the cyclin G1 protein, product of the CCNG1 proto-oncogene: (i) identified as the prime molecular driver of "Cell Competence" (to proliferate), (ii) needed for quiescent cells to enter the G1 phase, subject to oncogene-addiction as a molecular survival factor [9]. Tumor-targeted gene therapy involving CCNG1 blockade was tested in a number of clinical trials over a decade ago, and has recently been revived for clinical use, upon analysis of long-term cancer-free survival data, as the first clinically validated tumor-targeted gene therapy vector of this kind. This genetic medicine, known as DeltaRex-G (Former names: Mx-dnG1, dnG1, Rexin-G), is a "retroviral expression vector displaying a cryptic/designer collagen-binding motif on its gp70 surface envelope, designed specifically for targeting abnormal (anaplastic) Signature (SIG) proteins in the tumor microenvironment and encoding a dominant-negative mutant construct (dnG1) of human CCNG1 (Cyclin G1)oncogene/survival factor [10]. Once administered intravenously, the DeltaRex-G nanoparticles (~100 nm) accumulate in cancerous lesions, where the transgene is expressed, using the tumor cell's replication machinery to translate a mutant, cytocidal protein that is specifically designed to block the Cyclin G1 pathways of cell competence and survival function,

Herein, we discuss the current landscape of immune and cell cycle checkpoint inhibition by presenting a selected number of ongoing and past clinical research for advanced malignancies at the Cancer Center of Southern California (CCSC)/ Sarcoma Oncology Research Center (SORC) in Santa Monica, California, in context

Ongoing clinical research is either investigator-initiated or company sponsored.

In the case of investigator-initiated research, CCSC/SORC serves as the sponsor, conceives and designs the clinical protocol, and manages the entire clinical trial with or without funding by a pharmaceutical company, the FDA or the NIH. Company-sponsored research is developed, monitored, and funded by a pharma-

*2.1.1 SAINT: An Expanded Phase II Study Using Safe Amounts of Ipilimumab, Nivolumab, and Trabectedin as First-Line Treatment of Advanced Soft Tissue Sarcoma (NCT03138161). Erlinda M. Gordon, Principal Investigator*

Soft tissue sarcomas comprise a rare, heterogenous category of malignancies originating from connective tissue, blood vessels or lymphatic tissue [11].

*DOI: http://dx.doi.org/10.5772/intechopen.96664*

leading to active cancer cell death via apoptosis.

and collaboration.

ceutical company.

**2. Ongoing clinical research**

**2.1 Investigator initiated research**

*2.1.1.1 Background & rationale*

#### *Immune and Cell Cycle Checkpoint Inhibitors for Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.96664*

*Advances in Precision Medicine Oncology*

bound to its ligand, inhibits T-cell proliferation [2].

reduction was reported in all patients [7].

including those involving transmembrane protein receptors cytotoxic t-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), play pivotal inhibitory roles in T-cell activation. Specifically, the CTLA-4 checkpoint is designed to inhibit T-cells from becoming autoreactive during the beginning stages of T-cell activation, while the PD-1 checkpoint is part of a family of costimulatory receptors that, when

Tumor cells exploit the process of peripheral tolerance as a way to evade immunological surveillance by mimicking inhibitory receptors that are normally expressed on the surface of antigen presenting cells [3]. Expressing these inhibitory receptors allows cancer cells to effectively downregulate an immune response by deactivating the T-cells they come into contact with. The development of genetically engineered immune checkpoint inhibitors (ICIs) to treat malignancies therefore has the potential to revive pre-existing immune responses that would have otherwise been suppressed by the cancer [4]. Immunotherapies have been developed over the past decade using monoclonal antibodies as checkpoint inhibitors, binding the inhibitory receptor on T-cells and blocking tumor cells from binding to these sites. The first immune checkpoint inhibition therapies to enter clinical trials for patients with advanced cancer were two fully human CTLA-4 blocking antibodies, ipilimumab and tremelimumab. Clinical activity of the CTLA-4 blockade was most significant in advanced melanoma patients, leading to a 15% response rate that, for some patients, persisted for over 10 years after discontinuing therapy [5]. In 2010, a large Phase III trial was published showing ipilimumab to have significantly improved overall survival rates in patients with metastatic melanoma, compared to treatment with standard gp100, a synthetic peptide cancer vaccine, alone [6]. Ipilimumab has since been FDA approved in combination for the treatment of advanced renal cell carcinoma, microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), and malignant pleural mesothelioma.

The PD-1 checkpoint pathway was the next to be targeted with antibody therapy. Similar to ipilimumab, the first nivolumab trials were also shown to be most efficacious in melanoma patients, although it is now approved not only for the treatment of melanoma, but also of non-small cell lung cancer (NSCLC), small cell lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, MSI-H or dMMR metastatic colorectal cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. A study assessing the efficacy of anti-CTLA-4 and anti-PD-1 combined therapy in melanoma patients showed even more significant results, with 53% of patients achieving an objective response, and ≥ 80% tumor

Thus far, the only two immune checkpoint inhibitors that have been successfully brought to market are those that involve the PD-1/PD-L1 checkpoint and CTLA-4 checkpoint. These targets are within the adaptive immune system, but scientists are looking at the potential anti-tumor effects of exploring checkpoint targets within the innate immune system. Another target currently being investigated involves immune checkpoint inhibition within natural killer (NK) cell-mediated immunity. Cancer cells frequently downregulate their MHC expression, rendering T-cell mediated immunotherapy insufficient for killing these tumor cells. NK cell-mediated treatment can, in theory, compensate for this. As a first line of defense within the immune surveillance system, NK cells are quicker to become activated and will

Similar to the immune system, a checkpoint control system is also used to control the distinct phases of the cell division cycle in order to regulate cellular proliferation. Unrestrained cell division is a fundamental characteristic of oncogenesis, therefore

indiscriminately induce apoptosis in any cell lacking MHC-receptors.

**4**

cell cycle checkpoint control is vital in preventing the development of cancer. The mechanism of action in this case of checkpoint control is site-specific protein phosphorylation executed largely by cyclin-dependent proline-directed protein kinases. For example, Cyclin D1 and CDK4/6 are downstream of growth-initiating signaling pathways which lead to cellular proliferation. Palbociclib, an inhibitor of cyclindependent kinases CDK4/CDK6 is approved for the treatment of HR-positive, HER2-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women or fulvestrant in women with disease progression following endocrine therapy [8].

Another example of an executive cell cycle regulatory protein is the cyclin G1 protein, product of the CCNG1 proto-oncogene: (i) identified as the prime molecular driver of "Cell Competence" (to proliferate), (ii) needed for quiescent cells to enter the G1 phase, subject to oncogene-addiction as a molecular survival factor [9]. Tumor-targeted gene therapy involving CCNG1 blockade was tested in a number of clinical trials over a decade ago, and has recently been revived for clinical use, upon analysis of long-term cancer-free survival data, as the first clinically validated tumor-targeted gene therapy vector of this kind. This genetic medicine, known as DeltaRex-G (Former names: Mx-dnG1, dnG1, Rexin-G), is a "retroviral expression vector displaying a cryptic/designer collagen-binding motif on its gp70 surface envelope, designed specifically for targeting abnormal (anaplastic) Signature (SIG) proteins in the tumor microenvironment and encoding a dominant-negative mutant construct (dnG1) of human CCNG1 (Cyclin G1)oncogene/survival factor [10]. Once administered intravenously, the DeltaRex-G nanoparticles (~100 nm) accumulate in cancerous lesions, where the transgene is expressed, using the tumor cell's replication machinery to translate a mutant, cytocidal protein that is specifically designed to block the Cyclin G1 pathways of cell competence and survival function, leading to active cancer cell death via apoptosis.

Herein, we discuss the current landscape of immune and cell cycle checkpoint inhibition by presenting a selected number of ongoing and past clinical research for advanced malignancies at the Cancer Center of Southern California (CCSC)/ Sarcoma Oncology Research Center (SORC) in Santa Monica, California, in context and collaboration.
