**2. Nature and characteristics of human leukocyte antigens**

Human Leukocyte antigens (HLA) are a subgroup of the Major Histocompatibility Complex (MHC) gene family. The genes that encode the HLA class-I and class-II antigens are located on the short arm of human chromosome 6 [1]. Three constituent regions of the HLA gene complex are illustrated in **Figure 1**. Class, I genes are those encoding the heavy chains (HC) or α chains, of the six class I isoforms HLA-A, -B, -C, -E, -F, and -G. Extensive polymorphism of the glycosylated heavy chains of these HLA molecules are presented in **Table 1**. We carry a pair of alleles that represent each isoform derived from their mother and father (**Table 2**). Understanding HLA profiles of a patient is necessary when administering mAbs targeting a particular HLA molecule, for amino acid sequences of target HLA may cross-react with other HLA alleles of the patient. Native HLA-I

proteins are expressed on the cell surface as hetero-dimers, in combination with β2 microglobulin (β2-m) (**Figure 2A**). The gene encoding β2-m is situated on human chromosome 15. The hetero-dimers may also carry a peptide to form a trimer (**Figure 2B**), which is designated as "Closed Conformers (CCs)" [2]. Under the influence of cytokines (e.g. IFN- ɣ) and other activating factors (e.g. T-cell

*(A) Conformational structure of HLA class I. the native HLA-I proteins are expressed on the cell surface as hetero-dimers, the heavy chain in combination with β2-microglobulin (β2-m). (B) the hetero-dimer on the cell surface may carry a short peptide to generate trimeric structure, designated as "closed conformer"(CC).*

**HLA Class I Gene** *A B C E FG* **Alleles 6,291 7,582 6,223 256 45 82 Proteins 3,896 4,803 3,681 110 6 22**

*Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E…*

*Numbers of HLA alleles (as of September 2020) and their proteins. See updated information at* https://www.

**PROFILES OF HLA TYPING: HLA ISOFORMS AND THEIR ALLELES**

03:01] **[4**\*

II DQA **[01:02] [03:01] [01:02] [03:01]** II DQB [03:01] [06:09] [02:02] **[03:01]** II DPA **[01:03] [01:03] [01:03] [02:01]** II DPB [02:01] [03:01] [01:07] [01:11]

**01:01]** [3\*

02:02] **[4**\*

**01:01]**

**HLA CLASS ISOFORMS BROTHER\* SISTER** I A\* **[11:02]** [33:01] [01:01] **[11:02]** I B\* [15:01] [58:01] [40:01] [57:01] I C\* [15:02] [15:02] [03:04] [06:02] II DRB1 [04:03] [13:02] [07:01] [11:01]

**Table 1.**

*\**

**Table 2.**

**Figure 2.**

**45**

ebi.ac.uk/ipd/imgt/hla/stats.html*.*

II DRB3,4,5 [3\*

*DOI: http://dx.doi.org/10.5772/intechopen.95235*

*Mepur H. Ravindranath (brother) and his first sister.*

*The alleles in bold letters refer to alleles shared by the brother and the sister.*

*Pair of HLA alleles representing each of the commonly typed HLA isoforms.*

**Figure 1.** *Profile of the HLA gene complex on chromosome 6. All regions contain additional genes.*

*Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E… DOI: http://dx.doi.org/10.5772/intechopen.95235*


**Table 1.**

In this regard, human leukocyte antigen (HLA) classes are a structurally identical complex family of glycosylated homo- or hetero-dimeric proteins. They are expressed on cell surface complexed with an exogenous or endogenous peptide, as trimers. Defining the monospecificity of mAb raised against one family member of HLA is challenging. Often anti-HLA mAbs are polyreactive in that they bind to sequences common to all family member antigens, which are also known as "public epitopes". It is difficult to identify mAbs binding to unique sequences or private epitopes. Identifying such monospecific mAbs are critical for defining specific functions of antigens. Although sensitive and specific assay protocols are available to define the monospecificity of mAbs, many commercial mAbs, apparently specific for a unique HLA antigen, remain without defining their monospecificity. This review aims to distinguish monospecific mAbs that recognize private epitopes from polyreactive mAbs that bind to public epitopes of one of the HLA class Ib molecules, namely HLA-E, commonly overexpressed on human cancers. A pool of mouse mAbs was developed at Terasaki Foundation Laboratory (TFL) after immunizing with HLA-E. After validating the monospecificity of anti-HLA-E mAbs, their diag-

nostic and therapeutic potentials have been evaluated. These include (i) immunolocalization of cell surface expression HLA-E on human cancers, (ii) upregulation of CD8+ cytotoxic T lymphocytes, and (iii) restoration of antitumor activity of CD8+ T cells, NKT cells, and NK cells by preventing binding of HLA-E expressed on cancer cells to the inhibitory receptors (CD94/NKG2A) on the

**2. Nature and characteristics of human leukocyte antigens**

*Profile of the HLA gene complex on chromosome 6. All regions contain additional genes.*

Human Leukocyte antigens (HLA) are a subgroup of the Major Histocompatibility Complex (MHC) gene family. The genes that encode the HLA class-I and class-II antigens are located on the short arm of human chromosome 6 [1]. Three constituent regions of the HLA gene complex are illustrated in **Figure 1**. Class, I genes are those encoding the heavy chains (HC) or α chains, of the six class I isoforms HLA-A, -B, -C, -E, -F, and -G. Extensive polymorphism of the

glycosylated heavy chains of these HLA molecules are presented in **Table 1**. We carry a pair of alleles that represent each isoform derived from their mother and father (**Table 2**). Understanding HLA profiles of a patient is necessary when administering mAbs targeting a particular HLA molecule, for amino acid sequences of target HLA may cross-react with other HLA alleles of the patient. Native HLA-I

immune cells.

*Monoclonal Antibodies*

**Figure 1.**

**44**

*Numbers of HLA alleles (as of September 2020) and their proteins. See updated information at* https://www. ebi.ac.uk/ipd/imgt/hla/stats.html*.*


*\* Mepur H. Ravindranath (brother) and his first sister.*

*The alleles in bold letters refer to alleles shared by the brother and the sister.*

#### **Table 2.**

*Pair of HLA alleles representing each of the commonly typed HLA isoforms.*

#### **Figure 2.**

*(A) Conformational structure of HLA class I. the native HLA-I proteins are expressed on the cell surface as hetero-dimers, the heavy chain in combination with β2-microglobulin (β2-m). (B) the hetero-dimer on the cell surface may carry a short peptide to generate trimeric structure, designated as "closed conformer"(CC).*

proteins are expressed on the cell surface as hetero-dimers, in combination with β2 microglobulin (β2-m) (**Figure 2A**). The gene encoding β2-m is situated on human chromosome 15. The hetero-dimers may also carry a peptide to form a trimer (**Figure 2B**), which is designated as "Closed Conformers (CCs)" [2]. Under the influence of cytokines (e.g. IFN- ɣ) and other activating factors (e.g. T-cell

trophoblasts. Their overexpression is reported on activated T cells bone marrow cells inflamed cells and tissues (e.g. synovial fibroblasts), tumor cells

The HLA-Ib molecules are capable of interacting with cell-surface receptors present on specific immune-cell subsets, inducing activation or inhibition of signaling cascades within such specific immune cells as NK cells, macrophages, and dendritic cells [25–27]. Their interaction with different immunomodulatory (activating and/or inhibiting) cell-surface receptors on NK cells and macrophages signify their role in innate immunity; these receptors include CD94/NKG2, Ig-like transcript 2 (ILT2), Ig-like transcript 4 (ILT4), KIR2DL4, and CD160. These interactions are a component of innate immunity [27]; e.g., HLA-Ib is expressed during pregnancy, playing a major role in tolerance shown towards the fetus and placenta [28–34]. HLA-Ib molecules also generate a pool of antibodies *in vivo*, which may include monospecific or polyreactive (cross-reactive with other HLA-I molecule [16, 35–39]. Soluble HLA-Ib is also found in the synovial fluid and the circulation of

*Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E…*

Although several alleles of HLA-E (**Table 1**) exist, only two are extensively distributed among different ethnic groups [43]. The alleles differ by a single amino acid at position 107 [44–46]; Arginine in HLA-ER107 (HLAE\*01:01) is replaced by glycine in HLA-EG107 (HLA-E\*03:01) [45]. Such amino acid substitution influence thermal stability, which results in a more stable expression of cell surface HLA-E\*01:03 compared to HLA-E\*01:01 [44], including half-life of the molecule. HLA-E\*01:01 and HLA-E\*03:01 bind to different restricted sets of

HLA-E present peptides derived from HLA-Ia signal sequences (leader peptides), heat-shock protein (Hsp-60), human cytomegalovirus, Hepatitis C virus, Human Immunodeficiency Virus, Epstein Barr virus, Influenza virus, *Salmonella enteric* and *Mycobacterium* glycoproteins to T-lymphocytes [46–49]. The binding of HLA-E to the leader peptides of HLA-Ia stabilizes the HLA-E and enables migration to the cell surface [49]. When HLA-E does not reach the cell surface of a tumor cell, the cell is susceptible to lysis by NK cells. The crystallographic analyses of HLA-E structure reveals the molecular mechanisms underlying this function of HLA-E [24]. Importantly, tumor-associated HLA-E can be shed into the tumor microenvi-

ronment and circulation as soluble HLA-E (sHLA-E) [23, 50–56].

**4.2 HLA-E expression on cancer cells using mAb-based diagnostic assays:**

The literature (**Table 3**) on HLA-E expression on human cancers based on the commercially available diagnostic anti-HLA-E mAbs tests, reveals that none of the diagnostic mAbs were tested for their unique or monospecificity for HLA-E. If the mAb is not specific for the unique epitopes of antigen and if it binds to public epitopes or epitopes shared by a family of antigens, then data is unjustified to conclude the expression HLA-E. Principally this criterion is valid for any diagnostic or therapeutic antibody. We have undertaken efforts to examine, using Luminex multiplex SAB assay, the specificity of commercial anti-HLA-E mAbs employed in the 47 clinical studies (**Table 3**). Summary of the results [16, 21, 35–39, 96–98] is

[21–24].

peptides.

**47**

healthy and in cancer patients [40–42].

*DOI: http://dx.doi.org/10.5772/intechopen.95235*

**4.1 Unique characteristics of HLA-E**

**Limitations and reliability**

**4. Human leukocyte antigen-E (HLA-E)**

antibodies) or during inflammation, infection and tumorigenesis, the surface of metabolically active cells express only monomeric HLA heavy chains, called "Open Conformers (OCs) [3]. The examples include human T-lymphocytes activated *in vitro* and *in vivo,* as well as by EBV-transformed B-cells, CD19+ B-cells, CD8+ T cells, CD56+ NK-cells, CD14+ monocytes, extravillous trophoblasts and monocytes, dendritic cells (DCs), B-cell lines (RAJI, NALM6), and the myeloid cell line (KG-1A) [4–12]. The kinetics of conformational alterations in the naturally-occurring HLA-I OCs after activation has been investigated in healthy human T-cells [11]. The cytoplasmic c-terminal tail of naturally-occurring HLA-I OCs is tyrosine phosphorylated and plays a role in signal transduction [11].

HLA-I on antigen-presenting cells presents endogenous (intracellular) peptides. Importantly, viral peptides that have been broken by the proteasome are transferred to the endoplasmic reticulum (ER) via transporters (TAP). In ER, peptides are processed with OCs of HLA-I and exported to the cell surface as a trimer for presentation to T-cell receptors of CD8+ T-cells. This strategy kills the cell, thus preventing viral replication. After antigen presentation, the HLA-I is degraded (**Figure 3**). Ultimately, such degradation results in exposing the cryptic epitopes on the OCs to an individual's own immune system. Antibodies formed against the cryptic epitopes eliminate the degraded HLA from the circulation. The antibodyproducing cells may remain hidden and silent for long periods. They are referred to as "long-lived B cells" [13]. Evidently, anti-HLA antibodies occur in normal and healthy individuals [14–16], as well as in the pooled and purified plasma also known as intravenous immunoglobulin (IVIg) [16, 17].
