Personalized Immunotherapy for Tumor Diseases and Beyond

MedicineClevelandUSA

MHC and Cancer Immunotherapy

Li-Hua Jiang¹, *, Wei-Hua Yan²

¹ Departments of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China

² Medical Research Center, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai, 317000, China

Abstract

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.

Keywords: Dendritic Cell-Based Cancer Vaccine, Human Leucocyte Antigen (HLA), Major Histocompatibility Complex (MHC), NK Vells, T-Vells, TCR T-Cell Personalized Immunotherapy.

* Corresponding author Li-Hua Jiang: Departments of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China; E-mail:[email protected]

OVERVIEW OF MHC GENES AND MOLECULES

The major histocompatibility complex (MHC) is a common genetic region in vertebrates. It is called human leukocyte antigen (HLA) in humans and histocompatibility-2 (H2) in mice. MHC contains multiple genetic loci, which is highly polymorphic. It was first discovered because it played a decisive role in determining transplant rejection, that is, the degree of histocompatibility between the donor and recipient of organ tissue transplantation [1].

It was later uncovered that its main biological role of MHC class I and II molecule-encodes provide antigens that can be recognized by T lymphocytes [2], which are the center of the T cell immune response. Another interesting issue is that MHC class I molecules (including classical and nonclassical Class I molecules) can be used as targets for surface inhibitory receptor recognition by NK, playing an essential role in regulating immune cells' effect for tumor immunotherapy [3].

1. Characteristics of the HLA Gene Complex

The classical HLA gene system is located in the short arm of chromosome 6 (the 6p21 region) and has a length of 3600 kb as Fig. (1). It contains 224 gene loci, of which 128 are functional genes with product expression [4].

Fig. (1))

HLA gene complex.

The most important loci are the classical HLA class I, containing HLA-A, HLA-B and HLA-C genes encoding the classical HLA class I molecules alpha chains; and the classical HLA class II encoding the alpha and beta chains from genes of HLA-DRA, HLA-DRB, HLA-DQA, HLA-DQB, HLA-DPA, HLA-DPB genes. Molecules encoded by HLA-E, HLA-F, HLA-G, and MIC loci, which are located in nonclassical HLA class I loci, play an important role in immune regulation. Other genes are also involved in the body's immunity, such as HLA-DM, HLA-DO, TAP, and PSMB for re-antigen processing and presentation, complement genes, and TNF genes for inflammation.

Most of these genes have polymorphism or be highly polymorphic at each locus with a difference of one base to multiple base pairs, individually, so that clinically HLA polymorphisms have become a major obstacle to allografts, and often also become necessary factors to in immunotherapy of tumors [5].

2. HLA Molecule Structure

The classical HLA class I molecules alpha chain encoded from HLA-A, HLA-B, and HLA-C and β2 micro-globulin (β2M) encoded by the non-HLA gene from chromosome 15 as Fig. (2). Classical HLA class II molecules composed of the alpha chain encoded by the A gene of HLA class II and the beta chain encoded by the B gene, encoding from HLA-DR, HLA-DQ, and HLA-DP as Fig. (2).

Fig. (2))

HLA structure.

The critical difference between Class I molecules and Class II molecules is that the two ends of the peptide-binding groove are closed in Class I molecules so that Class I molecules generally can only accommodate peptides of 9 amino acids. In contrast, Class II molecules are open so that they can accommodate more peptides. For example, the peptide with a length of 16-18 amino acids with nine amino acids are bound in the groove [7]. The Ig-like region is responsible for maintaining the stability of the three-dimensional structure and selectively binds to CD4 or CD8 molecules on the surface of T-cells, thereby limiting the recognition pattern of T-cells. CD8+T-cells recognize antigens presented by MHC class I molecules, while CD4+T-cells recognize the antigen presented by MHC class II molecules.

3. Distribution and Function of HLA Molecules

Classical HLA class-I molecules are distributed on the surface of all nucleated cells, responsible for presenting the antigens produced inside the cells for recognition by CD8+T-cells. In contrast, HLA class II molecules are mainly distributed in antigen-presenting cells (APC), responsible for presenting antigens for recognition by CD4+T-cells.

The presentation of MHC molecules to antigens is a prerequisite for immune recognition of T-cells because the TCR antigen receptor only recognizes the linear epitope (epitope) presented by MHC, affinity short peptide cut off from the protein antigen. Therefore, the recognition of antigens by T-cells is deeply affected by MHC molecules, so-called MHC restriction [8].

MHC class I molecules also have an important function as ligands for some receptors on the surface of NK cells, including KIR2D/KIR3D belonging to the killer cell immunoglobulin-like receptor (KIR) family, killer cell lectin-like receptor (KLR), CD94/NKG2 family, regulating the killing activity of NK cells. The MHC class I molecules play a decisive role in the inhibitory receptors of NK cells because NK cells can recognize and kill tumor cells that lack own MHC class I molecules [3].

4. MHC Allele Detection

The typing of HLA based on the antigenic difference of HLA molecules is to use allotype antisera with complement-dependent micro-lymphocytic methods. Following research and development (R&D) of molecular biology techniques, the molecular typing method has, currently, replaced the serological method. According to the purposes of different laboratories, the technologies currently used are:

PCR-SSO (Sequence-specific oligonucleotide probes) can use PCR to amplify specific fragments of the HLA gene, fix the PCR amplification product on the hybrid membrane, and then use a series of labeled oligonucleotide oligo-nucleotide probes for hybridizing various HLA allele-specific DNA sequences, and finally, determine the type of allele according to the hybridization pattern of each specimen.

PCR-SSP (Sequence-specific primer assay), based on the DNA sequence differences of various HLA alleles, applies for a series of targeted primers to amplify various allele-specific fragments, and then to determine the HLA allele by electrophoresis based on PCR products.

Real-time PCR: the basic principle is like PCR-SSP, but it is more convenient and accurate because it does not require electrophoresis operations after PCR. After the first half of the method is the same as PCR-SSO; the PCR products are identified by Sanger sequencing for alleles.

Next-generation sequencing (NGS): After RNA-seq and DNA-seq are developed since 1995, most scientists working on screening techniques have often used genomic techniques to discover new candidates of neoantigens related to TCR recognizing MHC-peptide. Moreover, engineering T-cells with tumor antigen-specific TCR related to binding MHC can be used to generate TCR T-cells for personalized adoptive T-cell immunotherapy.

MHC CLASS I MOLECULAR LOSS AND CANCER IMMUNOTHERAPY

A variety of biological products have been used for cancer immunotherapy such as BCG, Polysaccharide K (PSK), IL-2, IFN, tumor vaccine, tumor infiltration lymphocytes (TIL). In recent years, novel therapies using antibodies that block immune checkpoint molecules involved in the regulation of T-cell has achieved good results. Unfortunately, only small proportions of patients benefit from these therapies [9]. These immune escaping indicated T-cells in tumor immune surveillance/escape, tumor occurrence and resistance to immunotherapy, for example, loss of tumor antigen, lack of co-stimulatory signals for T cell activation, the resistance of tumor cells to IFN, Resistance of tumor cells to apoptosis, immune negative regulatory cells and immunosuppressive factors in the tumor microenvironment (TME). Considering that the presentation of tumor antigen by MHC I is the premise of T cell immune recognition, the lack or down-regulation of MHC I expression on the tumor cell surface is an important strategy for immune escapes.

1. Loss of Class I HLA Expression in Tumor Tissues

Class I HLA molecules are normally expressed in nearly all nucleated cells, except for trophoblastic placental cells, central nervous system, and an exocrine portion of the pancreas. Hepatocytes and skeletal muscle cells have relatively low expression of Class I HLA proteins. Due to the characteristic of co-dominant expression for Class I HLA alleles, each cell can express six different molecules, including two types of A, B, and C, respectively. All the HLA molecules are thought to be expressed in tumor cells at the early stage of tumorigenesis. However, studies have shown that loss of Class I HLA expression on the tumor cell surface is very common in various types of tumor [10], such as biliary tract carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, colorectal carcinoma, endometrial cancer, esophageal carcinoma, gastric carcinoma, glioblastoma, head and neck carcinoma, hepatocellular carcinoma, lymphoma and leukemia, lung carcinoma, melanoma, neuroblastoma, ovarian cancer, pancreas carcinoma, prostate carcinoma, renal cell carcinoma, thyroid carcinoma, and uveal melanoma. The detection rate is estimated to be around 90% in many neoplasms [11].

2. Types of Class I HLA Deletion.

Various types of Class I HLA deletion exist in tumor tissues [11, 12]. Compared with complete deletion, the partial deletion of Class I HLA is found to have higher carcinogenicity. This is because the part of a class I HLA with high efficiency for presenting tumor antigen epitopes is lost, resulting in the failure of T cells to recognize tumor antigen efficiently.

Complete deletion of Class I HLA molecules. The mechanisms of formation include β2M deletion caused by loss of heterozygosity (LOH) on chromosome 15 and mutation of β2M gene on another chromosome 15, transcriptional down-regulation of antigen presentation machinery (APM), HLA-I or β2M genes, and hypermethylation of Class I HLA genes [11-16].

Deletion of haplotypes of HLA. Deletions occur as a set of HLA genes (type A, B, or C), which may be involved in the loss of an entire or part of chromosome 6 [11].

Down-regulated expression of HLA gene locus (type A, B, or C). As a result, tumor cells only express the other two gene loci of the four alleles, which can be caused by either decreased transcription level or expression of specific transcription factors. Cytokines secreted by TH1 cells, such as IFN-γ, can reverse the down-regulated expression [11, 12].

Deletion of HLA alleles. The deletion occurs at any 1 product from the 3 class I HLA gene loci of the six alleles, which may be caused by somatic mutation of the allele. It is undetectable in most cases since a specific monoclonal antibody against that allele product is required for the detection [11, 12].

Co-occurrence of above-mentioned II, III and IV. This type of deletion may lead to the expression of 1 allele to the minimum [15-17].

Unresponsive deletion to up-regulation of Class I HLA induced by IFN. This type may be caused by genetic mutations on molecules related to IFN-γ or IFN-α signal pathway [18, 19].

In summary, loss of Class I HLA expression can be caused through a variety of mechanisms, including abnormalities occurring at any step during synthesis and assembly of Class I HLA molecules. According to whether the deletion can be reversed through interacting with cytokines produced by immunotherapy, the mechanisms are divided into 2 classes [11, 20, 21]: Irreversible: including LOH, mutations or deletions on chromosome 6 and 15 (harboring α chain gene and β2M gene, respectively), defect of IFN signal transduction pathway (such as interruption of Jak-STAT pathway); Reversible: including down-regulated expression of HLA-A, B or C gene locus, heavy chain gene of Class I, β2M gene, APM genes resulting from abnormal transcription regulation, down-regulation of Class I or APM gene expression caused by hypermethylation, and inhibition upon post-transcriptional modification of Class I MHC mRNAs.

3. Expression of Class I HLA Molecules and Efficiency of Immunotherapy

Several scenarios are raised to predict the immunotherapy efficacy on class-I HLA expression. As for tumors with positive expression of Class I HLA molecules, the immunotherapy efficacy is influenced by other immune escape associated factors, such as tumor antigenicity, apostolicity of tumor cells, and co-stimulatory signals. Appropriate treatment strategies should be put forward towards those impact factors.

Treatment can recover the expression of Class I HLA molecules. In other words, the deletion belongs to the above-mentioned reversible type. Immunotherapy can lead to local release of cytokines produced from TH1 T-cells in the tumor microenvironment, such as IFN-α and IFN-γ, which reversely upregulate Class I HLA molecule expression on tumor cells and the tumor antigens presented by the class-I HLA molecules can re-activate specific T cells to recognize and kill tumor cells. Studies have shown a positive correlation between expression recovery of Class I HLA molecules and immunotherapy efficacy on tumors [11, 20, 21].

Treatment cannot recover the expression of Class I HLA molecules since the deletion belongs to the above-mentioned irreversible type. Immunotherapy is generally ineffective on tumors with such type of deletion, and the effort in the future is to find a way of increasing the expression of Class I HLA molecules. For instance, blocking antibodies for immune checkpoint usually have no effect on patients with mutated β2M and LOH on chromosome 15, which can cause inefficient antigen presentation [11, 22, 23].

Expression of Class I HLA molecules is restorable, while the treatment itself has no impact on its expression. In order to have a better clinical effect in such a situation, other methods are required to enhance the expression of Class I HLA molecules. Therefore, it is imperative to identify the types of Class I HLA deletion for the prediction of therapeutic efficiency.

4. The Relation Between Selective Pressure of Immunocyte and Changes of Class-I HLA Expression Pattern in Tumor Cells

Tumor cells are evolved from normal cells containing a positive expression of Class I HLA molecules. Loss of class-I HLA molecules occurs during the progressive development of neoplasms. Studies have shown that deletion is a gradual process with different cellular clones and various expression levels of class-I HLA molecules. AT the 12th International Conference on HLA, Garrido et al. proposed that tumor tissue could be classified into three types based on the expression of class-I HLA molecules, including positive, heterozygosity, and negative types. It is commonly believed that these three types of tumors reflect the transformation process of class-I HLA molecule expression from positive to negative. Tumor cells display positive expression at the early stage of tumor formation, and then heterozygous state with the gradual appearance of negative clones. Pure negative state indicates a late stage of tumorigenesis. Tumor cell clones with positive expression of class-I HLA molecules can be recognized and destroyed by activated anti-tumor T-cells. However, tumor cell clones of negative expression gradually form through a mechanism such as mutation and escape from recognition and elimination. As a result, only clones with negative expression of class-I HLA molecules finally remain in tumor tissues under such selective pressure [11, 24, 25].

Many kinds of researches have demonstrated a positive correlation between expression of class-I HLA molecules and the infiltration of immune cells, such as T-cells and M1 cells in tumor tissues. During the development of tumorigenesis, tumor cells gradually lose class-I HLA molecules accompany structure change of tumor tissue. In a positive and heterozygous state, there are lymphocytes and macrophages infiltrated in the tumor tissue due to the ability of class-I HLA molecule expressed cells to activate T-cells. However, in a negative expression state, the tumor tissues have deficient infiltration of immune cells, and tumor cells with class-I HLA molecules locate at tumor surrounding stroma region, forming an obvious boundary from negatively expressed tumor cells in the center of the lesion. Meanwhile, many fibroblasts emerge in peritumoral tissues, which mix with various immune cells to form a granulomatous structure of tumor tissue surrounded by non-neoplastic tissues. Such kind of granulomatous structure can be observed in many tumors, which continue to progress due to the inactive state of surrounding immune cells [15, 25, 26].

Although studies are still limited, comparison of class-I HLA molecule expression between metastatic and primary tumor tissues can inspire additional perspectives: 1) metastases with either expressed or not expressed class-I HLA molecules can derive from both primary tumor with positive and negative expression of class-I HLA molecules; 2) the type of deletion in metastatic and primary tumors can be same or different; 3) metastases can have a complete positive expression of class-I HLA molecules even if the primary lesion is negative. These interesting phenomena suggest that selective pressure posed by T-cells and NK cells plays an important role in the expression state of class-I HLA molecules in metastases and primary tumors [11, 27, 28].

There is also a relationship between the expression level of class-I HLA molecules in metastases and the immune status of the body. In the immunosuppressive state, metastasis can be induced, and then an expression of class-I HLA molecules can be restored as well. Studies in mouse models revealed that among multiple tumor cell clones, H-2 positive clones have higher immunogenicity and metastatic ability in comparison to H-2 negative clones. More significantly, sarcoma cell clones with reversible deletion of class-I HLA molecules after induction of IFN could enter a state of so-called immune quiescence in mice with normal immune function. However, metastases with positive expressed class-I HLA molecules were formed after T-cell deficiency was induced in mice. Immune quiescence refers to a static condition that tumor cells are neither progressive nor destroyed by the immune system. The status of equilibrium between tumor cells and the body's immune system can be maintained for a long period of time without any clinical symptoms unless the body immunity is decreased [11, 29-31].

EXPRESSION LEVELS OF NONCLASSICAL MHC CLASS I AND TUMOR IMMUNE ESCAPE

1. The HLA Class I Antigen Expression and Cancer

The HLA class I antigens play essential roles in immune response, for example, HLA can present peptide to T-cells and serve for ligands for a panel of receptors expressed on immune cells. Based on the tissue expression pattern, genetic polymorphism, and molecular function, HLA class I antigens can be grouped as the classical (HLA Ia for HLA-A, -B, and -C) and nonclassical antigens (HLA Ib for HLA-E, -F and -G). Unlike highly polymorphic and ubiquitously expressed HLA Ia antigens on nucleated cells, HLA Ib molecules have features such as limited tissue localization, low genetic diversity, limited peptide repertoire, and distinct functional profiles [32]. In the context of malignancies, alternation of HLA Ia antigen expression abnormalities can escape from host anti-tumor immune responses while induction of HLA Ib antigen expression on cancer cells can be involved in HLA Ib antigens to bind immunoinhibitory receptors such as immunoglobulin-like transcripts (ILT)2, ILT4 and NK receptor group 2 (NKG2) [33].

2. Roles of HLA Ib Antigens in Immune Modulation

The nonclassical HLA I antigens HLA-E, HLA-F, and HLA-G, were identified during the late 1980s. Contrary to the high polymorphic of HLA Ia antigens, only 43, 44, and 69 alleles with 11, 6, and 19 different proteins for HLA-E, HLA-F, and HLA-G, respectively (http://hla.alleles.org/nomenclature/stats.html). During the past three decades, the biological function and related clinical significance of HLA-E and HLA-G have been widely investigated in various physiological and pathological conditions [33].

HLA-E binds to the inhibitory receptors CD94/NKG2A and the activating receptor CD94/NKG2C. However, HLA-E preferentially binds to the inhibitory receptor CD94/NKG2A with much higher avidity than that of the activating receptor CD94/NKG2C. Consequently, HLA-E interacts with CD94/NKG2A can inhibit the activation and proliferation of NK cells and impair survival of the CD8+ tumor-infiltrating T lymphocytes in the tumor microenvironment [34].

HLA-G is the most intensively investigated molecule among the HLA Ib family, and seven HLA-G isoforms (HLA-G1~ HLA-G7) generated by its primary transcripts alternative splicing have been identified. Isoforms HLA-G1~HLA-G4 are membrane-bound while HLA-G5~HLA-G7 is soluble. Different HLA-G isoforms are distinguished by the number of extracellular immunoglobulin-like domains and by the transmembrane residues they have or not. HLA-G1 is encoded by the full-length HLA-G mRNA, and with three extracellular immunoglobulin-like domains (α1, α2, and α3), other isoforms may lack the α2, α3 domain or both, respectively. HLA-G can render comprehensive immune suppressive function by binding to receptors such as ILT2 and ILT4. ILT2 can be expressed on B-cell, T cells, NK cells, DCs, myeloid-derived suppressive cells (MDSCs), and monocytes, while ILT4 is expressed on DCs and monocytes, neutrophils and MDSCs. HLA-G/ILT2/4 inhibitory signal pathway can impair immune cell proliferation, differentiation, cytotoxicity, cytokine secretion, and chemotaxis. Moreover, HLA-G/ILT2/4 interaction can induce regulatory cells and promote the generation of MDSCs or polarization of M1 type macrophage to the M2 type. ILT2/4 recognizes HLA-G in its extra cellar α3 domain. However, the binding specificity of ILT2 and ILT4 is different according to the structure of the HLA-G molecule, that ILT4 recognizes both HLA-G associated with β2M and free HLA-G heavy chains, whereas ILT2 only recognizes HLA-G associated with β2M [35]. Given immune-suppressive functions resulted from the HLA-E and HLA-G and the interaction of their receptors in cancer immunology, HLA-G/ILT2/4 and HLA-E/NKG2A signaling pathway have been proposed as new immune checkpoints as the well established cytotoxic T lymphocyte-associated protein 4 (CTLA-4)/B7 and programmed cell death protein-1 (PD-1)/PD-L1. Antibodies target to these immune checkpoints can restore or re-activate T-cell and NK cell anti-tumor responses. Several approaches based on the HLA-G/ILT2/4 and HLA-E/NKG2A signaling pathway are currently under development for cancer immunotherapy [36, 37].

HLA-F is the least investigated molecule among HLA Ib antigens until recent data addressed that HLA-F can interact with either activating or inhibitory receptors on immune cells, depending on the conformation of HLA-F molecule. ILT2 and ILT4 can bind HLA-F/β2m/peptide complex through a docking strategy that precludes HLA-F open conformer recognition. However, KIRs (3DL1, 3DL2, 3DS1, and 2DS4) can bind HLA-F open conformer, where KIR3DS1 is of the highest binding affinity for HLA-F open conformer [38, 39]. These important findings Therefore, it is imperative to identifyprovide new evidence that HLA-F functions as an important immune regulatory molecule in human physiological and pathological conditions have been emerging. Though relative information of HLA-F/KIRs in cancer immunology is limited, the biological significance of HLA-F/KIR3DS1 in the viral infectious diseases has been highlighted in recent studies. For example, HLA-F open conformer /KIR3DS1 interaction can activate NK cell function in the control of viruses such as HIV and HCV replication [39, 40]. However, the inhibitory pathway between HLA-F/β2m/peptide complex/ILTs and HLA-F open conformer/KIR3DL1 remains to be investigated.

3. Clinical Relevance of HLA Ib Expression in Cancers

Cancer cells harness different strategies to escape surveillance from host both adaptive and innate anti-tumor immune responses. Abnormal expression and impaired function of HLA antigens on tumor cells frequently occurred in cancer immune evasion. In most cases, accompany with HLA Ia antigens down-regulated high-regulation of HLA-E, -F and -G have been detected in a wide variety of cancers associated with the progression and unfavorable clinical outcome of tumor diseases [41].

HLA-E expression has been found in many types of cancers, such as breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, lung cancer, melanoma, renal cancer, thyroid cancer, and vulvar squamous cell carcinoma. Among these studies, increased HLA-E expression in cancer lesions has been observed to be related to the cancer cell metastasis, recruitment of regulatory immune cells, or associated with a worse prognosis in various cancer patients [33]. HLA-F expression has been observed in breast cancer, bladder cancer, oesophageal squamous cell cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal cancer, neuroblastoma, and lung cancer. Among these studies, HLA-F expression in gastric adenocarcinoma has observed to be significantly correlated with the depth of invasion, nodal involvement, lymphatic and venous invasions, and with a worse prognosis [42]. In patients with HLA-F positive had a worse survival than those with HLA-F negative. In another study, upregulated HLA-F expression (lesion vs. normal tissue) was found to have significantly worse survival than those with HLA-F unchanged and downregulated in patients with oesophageal squamous cell carcinoma [43].

HLA-G expression in cancers was firstly reported in 1998. Later, HLA-G expression has been analyzed and evaluated worldwide in thousands of malignant samples with various types of cancers such as breast cancer, colorectal cancer (CRC), cervical cancer, endometrial cancer, oesophageal squamous cell carcinoma (ESCC), Ewing sarcoma, gastric cancer, glioblastoma, HCC, lung cancer, lymphoma, nasopharyngeal carcinoma, oral squamous cell carcinoma, ovarian cancer, pancreatic adenocarcinoma, thyroid cancer, and vulvar squamous cell carcinoma. Aberrant HLA-G expression in cancers has been found to be associated with advanced tumor stage, metastasis, and worse prognosis