Personalized Immunotherapy for Tumor Diseases and Beyond

Major Histocompatibility Complex (MHC) is a gene region, which is named human leucocyte antigen (HLA) in humans. The human leukocyte antigen (HLA) system is a highly polymorphic family of genes involved in immunity and responsible for identifying self-cells versus no self-cells. Although HLA typing is essential for solid organ and bone marrow transplantation, at present, MHC is going to study on cancer immunotherapy increasingly. In order to introduce MHC related to cancer immunotherapy, the chapter aims at focusing on several MHC issues related to cancer immunotherapy. For example, MHC research and development (R&D) in MHC class I molecular loss related to cancer immunotherapy; tumor immune escape related to nonclassical MHC I; T-cell epitope vaccines; as well as MHC issues in adoptive immune cell therapy and personalized immunotherapy. In each part for MHC related to immune responses for tumor disease, we also introduce clinical uses in a study on MHC issues for T-cell immunotherapy, MHC for T-cell vaccines, and MHC TCR reconstructions for tumor shared/specific antigen related TCR T-cell personalized immunotherapy.

Lymphocytes play important roles in body defense for a few diseases, such as tumor diseases, autoimmune diseases, allergic inflammation. The genomic profiles with their analyses for body defense of lymphocytes have been applied to identify and verify disease-associated and disease-specific biomarkers. The genomic profiles of lymphocytes also can provide more information to understand their functions and roles in the development of tumor diseases, although genomic profiles from lymphocytes are still not completed for different tumor diseases. The chapter first reviews subtypes/functions and signaling/pathways of different lymphocytes and then introduce genomic profiles with their networks on these types of lymphocytes to highlight the genomic profiles of lymphocytes in tumor diseases. The genomic profiles are going to produce clinical potentials of precision medicine such as tumor prediction, tumor prevention, and prognostic estimation and personalized therapy of lymphocytes so that, here, we will more focus on the study of genomic expression and network of personalized immunotherapy because the profiles of gene expression with their network in lymphocytes start a new chance to develop personalized immunotherapy soon.

The immune response is a dynamic reaction of a body, allowing to fight against tumor cells. However, an unbalanced host immune response is highlighted in tumor disease. Abnormal responses lead to autoimmune diseases, whereas low responses favor opportunistic infections and host with tumor cells. The conflicting situations make it difficult to arrange an appropriate immunoassay for immunotherapy. Before human genomics decode in 2004, testing the immune response with this profile of patients remains a challenge. This is due to individual variability so that it impedes immunoassay for personalized immunotherapy. After the fifteen years’ effort, immunoassays are committed to personalized immunotherapy of tumor diseases for precise prediction/prevention, immune targeting therapy, and personalized immunotherapy. Now, identifying new targets at the protein level, SNP at the DNA level, and mRNA expression at the RNA level may guide a new generation of immunoassay. The techniques to test the immune responses to tumor diseases are currently being studied, but they still have many influence factors such as technical standardization and technique selection, and interpretation, and therefore, the chapter gives a comprehensive insight into the immunoassay for personalized immunotherapy.

Immune cells and antibodies respectively play a role in the cell-mediated and humorous-mediated immune response to tumor cells, while tumor microenvironment (TME) and tumor cells in tumor tissue take many strategies to evade the host immune response by creating many immune-suppressive factors, and thus, we should understand the knowledge of TME before performing personalized immunotherapy. TME consists of tissues, cells, and signaling molecules in tumor tissue, affecting the immune response to tumor cells. Furthermore, TME will be quickly changed by inflammation, hypoxia, and tumor growth in vivo so that its highly dynamic alteration must be considered for treatment selection for personalized immunotherapy. All TME elements of tissues, cells, and molecule factors interact with each other, including those during the early period of tumor tissues and those in an aggressive period in tumor tissues. Before human genomics decode in 2004, studying TME components with their genomic profiles of patients is a rare possibility. After human genomics decoded with research and development (R&D) of their techniques, TME is going to be increasingly considered by personalized immunotherapy of tumor diseases. Now, identifying regulating TME cells and regulating molecules with their therapeutic agents is largely reported. A few reports have outlined some networks such as extracellular matrix (ECM) and pathways such as adenosine (ADO) and indole-2,- -dioxygenease (IDO) with their therapeutic agents that may guide a new generation of immunotherapy.

Currently, in the study of new anti-tumor therapies, the suppression of tumor growth through target checkpoints is a breakthrough in this treatment method. Now, this has gradually become the focus of in-depth research. By acting on specific molecular targets, tumor cells can be inhibited through information transmission in the human immune pathway, thereby inhibiting their growth and proliferation. Molecularly targeted checkpoint inhibitors can specifically kill tumors within the tumor microenvironment (TME), inhibiting the occurrence and development of tumors. On the other hand, they can target and inhibit other molecules, so that they can restore immune cell activity, and improve the body's anti-tumor immune function, namely the tumor immune microenvironment (TIME). At present, molecular target checkpoints that have been increasingly studied within TIME include PD-1, PD-L1, CTLA-4, TIM- 3, LAG-3, and Siglec-15. Corresponding molecular target inhibitors have been prepared for these molecular targets, and thus they have been increasingly applied to the clinic. Although these inhibitors have unavoidable adverse reactions and limitations in their scope of application in certain types of tumors, they still offer hope for the successful elimination of tumors.

Specific T-cells, TCR T-cells, and CAR-T-cells require to establish some techniques of molecular biology to support them, including screening tumor-associated antigen (TAA)/tumor specific antigen (TSA) and mutant proteins/peptides; constructing an expression system; packaging an expression vector. The molecular biology technique is a very important performance in targeting neoantigens for tumorspecific T-cells of adoptive T-cell immunotherapy. Since tumor cells often accumulate hundreds of mutations and harbor several immunogenic neoantigens, the repertoire of mutant protein or neoantigen from patient tumor cells might need to screen and discover the antigens for engineering specific T-cells, TCR T-cells, and CAR T-cells. In order to understand the procedures for T-cell adoptive immunotherapy based on molecular biology techniques for mutant proteins and neoantigens from an individual patient, in this chapter, we focus on streamlining of screening tumor antigens (TAA or TSA) and mutant proteins (proteins or peptides), constructing an expression and packaging system with the expression. Moreover, because the three T-cells are distinct from development and clinical application, we first introduce their research and Development (R&D). These methodologies are increasingly supporting clinical oncologists to apply to T-cell immunotherapy. The chapter aims to present fundamental of molecular biology for adoptive T-cell immunotherapy of clinical patients.

Tumor-associated antigen (TAA) or tumor-specific antigen (TSA) is essential for the target of tumor-specific T-cells such as tumor-infiltrating T cells (TIL), specific T-cells, TCR T-cells and CAR-T-cells for adoptive T-cell immunotherapy. The tumor cells often accumulate hundreds of mutations and harbor several immunogenic neoantigens, and thus, the repertoire of mutation or neoantigen from patient tumor cells might need the screen to uncover for engineering these T-cells. To understand the Tcell screening and determining tumor antigen-based on primary tumor cells from an individual patient, this chapter, we focus on streamlining the process of ex vivo T-cell culture and primary tumor cell culture, T-cell cloning for tumor neoantigen-specific T cells, allowing the patient to the benefit of downstream T-cell targets. Because T-cell engineering cultures are very important methods for TIL, TCR and CAR T-cells, moreover, because using primary tumor cells isolation and cultures is very important for screening and identifying tumor antigen of patients, we first introduce primary cell culture techniques, including those developed from two-dimensional (2-D) tumor cell cultures, three-dimensional (3-D) tumor cell culture and multiple dimensional tumor cell culture (4-D cultures). These methodologies are increasingly supporting clinical oncologists to apply to tumor therapeutic agents and Ag targets for patients in the clinical laboratory. Besides, we also conclude some growth factors for T-cell cloning cultures. The chapter aims to present a foundation to adoptive T cell immunotherapy of clinical patients.

Epitope discovery of tumor antigen and mutant proteins has enabled a better application of T-cell immunotherapy. Genomic profiles analyzed by genomic expression and single nucleotide polymorphisms (SNP) by genome-wide association studies (GWAS) are an essential fundamental to screen and define T-cell therapeutic targets. To determine tumor antigens or mutant proteins related to T-cell targets with their TCR or CAR reconstruction, we will introduce the SNP technique related to primary tumor cells for personalized T-cell immunotherapy, including global and local SNP detection of the therapeutic targets. Moreover, the use of mRNA genomic expression can discover gene expression signature and further uncover tumorassociated antigen (TAA) or tumor-specific antigen (TSA) for T-cell immunotherapy. Accompany with the ongoing development of next-generation sequencing, epitope discovery of tumor neoantigen and mutant proteins will be irreplaceable for a novel generation of T-cell adoptive immunotherapy. System biology, which is a mathematical modeling of complex biological systems,can integrate data of SNP signature and genomic expression signature. Thus, a new bioinformatics platform with the analysis of GWAS and genomic expression profile along with system modeling is an essential fundamental for T-cell adoptive immunotherapy.

Lymphocytes play vital roles in surveillance of the formation and development of tumors as well as control of tumor disease. Employing the immune cells to recognize and destroy tumor cells is a central task of anticancer immunotherapy. Since 1987 cultured tumor-infiltrating lymphocytes (TIL) from the site of tumor tissue have been discovered more than 100-fold to kill tumor cells to compare cultured T-cell from peripheral blood, we have been studying TIL anti-tumor mechanism and clinical feasibility of immune-cell immunotherapy, especially functionally inducing TILs for immunotherapy purpose for more than two decades. At present, to make it a clinically feasible treatment, there have been increased reports in optimizing those procedures. Several standard protocols of laboratory performance and clinical treatments have been quickly developed in cancer immunotherapy. With using this standard protocol, cytotoxic T-cells are infused into cancer patients with cytokine help in recognizing, targeting, and destroying tumor cells. In the chapter, we review some of the significant successes of adoptive T-cell immunotherapy (AIT or ACT) and the significant obstacles that have been overcome to optimize ACT. Here, we also more focus on the study of research and development of T-cell inducing, culture and proliferation for adoptive immunotherapy, and eventually introduce clinical knowledge of lymphocytes application including feasible and affordable to treat patients.

For several decades, clinical scientists and physicians have been studying gene-modified T-cells to treat tumor patients, called T-cell based gene therapy. However, T-cell based gene therapy has efficacy questions and side-effects so that these techniques limited to the clinical application quickly. Therefore the possibility of creating cell-based gene therapy is just like a dream for tumor patients. With the recent development of CRISPR technology and genomic decoding, it is becoming increasingly possible to engineer cells by gene-modification for patients with tumor diseases. Gene-editing systems based on CRISPR, as well as transcription activatorlike effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for the new generation tool of gene therapy. However, each of these systems to effectively apply for patients, including safe delivery and gene modification technologies, is still unknown. This chapter briefly introduces the history of gene therapy, the principle of gene editing with their non-viral and viral delivery methods. The chapter aims to discuss the latest developments in gene-editing technology and discuss their application to adoptive T cell immunotherapy.

T-cells play an essential role in the cell-mediated immune response to tumor cells, while tumor cells in tumor sites take many strategies to evade the host immune response, including creating many immune-suppressive factors from tumor microenvironment (TME) or decreasing expression of immunogenicity of target antigens. To resolve the evasion of tumor cells from T-cells attacking, some strategies such as genetically modified T-cells altering the specificity of the T-cell receptor (TCR) or introducing antibody-like recognition of chimeric antigen receptors (CARs) have made significant advances. The modified TCR T-cells or CAR T-cells have been administered to cure B-cell lymphoma or B-lymphocyte leukemia in clinical trials successfully. We have been going to study the specificity and safety of T-cell adoptive immunotherapy for more than 30 years so that our experiences to apply for genetically modified T-cell more focus on the specificity and safety of these therapies. Moreover, the strategies using genetically modified T-cell immunotherapy need face challenges for immunogenicity from different types of tumors. The chapter will introduce T-cell specific affinity between T-cell and tumor cells such as TCR and CAR T-cells, discuss challenges from the selection of antigen targets, and address safety issues to clinical development. All in all, T-cell adoptive immunology regarding TCR and CAR T-cell improves the clinical application.

When primary-cells, including non-genetically modified and genetically modified T-cells to produce a special substance, are infused into patients, these performances would be defined as cell therapy. An excellent cell performance with its optimal proliferation for cell therapy should maintain its functional feature and efficacy in vivo with ethical acceptance and safe application. Because the efficacy of cell therapy maybe will be decreased in vivo special microenvironment after infusion, moreover, because cell therapy with these genetically modified T-cells would be faced by a safe challenge in clinics, a functional induction/inhibition of some genes’ expressions used in T-cell growth without genetic modification has been increasingly studied. Here, T-cell therapy based on system biology for an induction/inhibition of special function and maintaining a special function in vivo microenvironment is called as functional cell therapy. Nowadays, following research and development (R&D) of T-cell proliferatively engineering techniques and system modeling by this computational simulation performance, the novel techniques of T-cell culture based on genomic analysis and supported by system biology will be increasingly studied for adoptive T-cell therapy so that oncologists can safely and effectively utilize the new strategy for personalized immunotherapy.

A biobank is a resource for keeping blood and tissue samples, which is going to play an increasing role for personalized immunotherapy, called precision immunotherapy. Moreover, large profits from biobanks are their futures of patients' personalized immunotherapy. A new generation of immunotherapy often relies on a person's tissues/cells/molecules/data so that personalized immunotherapy starts to deposit the patient's specimens and clinical information with genomics data. If tumor patients can save their specimens such as tumor tissues or blood before treatment, furthermore, if patients can achieve some genomic data for future treatment, the specimens saved in biobanks and the genomics data observed from their tissues can contribute clinical physicians and clinical scientists to develop a new generation of treatment, for example, patients respond to immunotherapy predicted by individualized measurement and undergoing personalized T-cell immunotherapy. This chapter introduces biobank, one of the most up-to-date personalized immunotherapies, which enable conducting research and development (R&D) for professional collection of clinical specimens and clinical data. The chapter aims at describing the concept of biobank and future potential to treat patients. It also includes sample preservation protocols and data management as well as online service of samples and clinical data. Thus, some standard operating procedures apply for personalized immunotherapy of diagnostic and treatment procedures. Finally, ethics for sampling, clinical information, and genomics data are mentioned to support the biobank chapter.