**3. Diagnostic and clinical relevance of non-classical HLA class Ib antigens**

Unlike classical HLA-Ia (HLA-A, HLA-B & HLA-C), non-classical HLA-Ib (HLA-E, HLA-F & HLA-G) genes and molecules are oligotrophic, with restricted and selective tissue distribution [18–20]. HLA-Ib molecules are expressed in a diverse array of cells including T and B lymphocytes, Natural Killer Cells, monocytes, macrophages, megakaryocytes, and organs i.e., lymph nodes, spleen, skin, salivary glands, thyroid, stomach, liver, kidney, urinary bladder, endometrial, and *Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E… DOI: http://dx.doi.org/10.5772/intechopen.95235*

trophoblasts. Their overexpression is reported on activated T cells bone marrow cells inflamed cells and tissues (e.g. synovial fibroblasts), tumor cells [21–24].

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 healthy and in cancer patients [40–42].

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

#### **4.1 Unique characteristics of 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 phosphor-

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

**3. Diagnostic and clinical relevance of non-classical HLA class Ib**

Unlike classical HLA-Ia (HLA-A, HLA-B & HLA-C), non-classical HLA-Ib (HLA-E, HLA-F & HLA-G) genes and molecules are oligotrophic, with restricted and selective tissue distribution [18–20]. HLA-Ib molecules are expressed in a diverse array of cells including T and B lymphocytes, Natural Killer Cells, monocytes, macrophages, megakaryocytes, and organs i.e., lymph nodes, spleen, skin, salivary glands, thyroid, stomach, liver, kidney, urinary bladder, endometrial, and

ylated and plays a role in signal transduction [11].

*The fate of HLA-I molecule after antigen presentation.*

as intravenous immunoglobulin (IVIg) [16, 17].

**antigens**

**46**

**Figure 3.**

*Monoclonal Antibodies*

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 peptides.

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

#### **4.2 HLA-E expression on cancer cells using mAb-based diagnostic assays: Limitations and reliability**

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


presented in **Figure 4** show that the commercial anti-HLA-E mAbs react with HLA-A, HLA-B and HLA-C in the following order: MEM-06 > MEM-02 > MEM-07 > MEM >08 >> > 3D12. That the mAbs are recognizing the epitopes shared with several HLA-Ia (HLA-A, HLA-B, HLA-C) antigens confirms that none of the above mAbs are specific for HLA-E. Therefore conclusions concerning the expression of HLA-E in human cancers require further validation with monospecific anti-HLA-E mAbs.

*Expression of HLA-E on human cancer cells (biopsies or cell lines) monitored with commercial mouse anti-*

*HLA-E mAbs (MEM-E/02, MEM-E/06, MEME/07. MEM-E/08, 3D12, 3H2679).*

Many Cancers 3D12 Sensi M, et al. Int Immunol. 21(3):257–268. 2009. [95]

**5. Anti-HLA-E mAbs: Characteristics, diagnostic and therapeutic**

microspheres with a magnetic core and polystyrene surface. The beads are

**5.1 The technology that clarifies monospecificity or polyreactivity of a mAb of**

Luminex multiplex assays are based on xMAP (Multi-Analyte Profiling) technology that enables simultaneous detection and quantitation of antibodies reacting to multiple proteins simultaneously, using detection mAbs [16, 17, 21, 35–39, 96–98]. The results are comparable to assays such as ELISA but with greater specificity, sensitivity and resolution. The technology employs superparamagnetic 6.5-micron

**potentials**

**NATURE OF HUMAN CANCER**

Ovarian cancer/ Cervical cancer

Cervical squamous and adenocarcinoma

Serous Ovarian Adenocarcinoma

Serous Ovarian Adenocarcinoma

Chronic Lymphocytic

Chronic Lymphocytic

Leukemia

Leukemia

**Table 3.**

**COMMERCIAL mAbs**

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

Breast cancer MEM-E/02 de Kruijf EM et al. J Immunol. 185:7452, 2010 [82] Breast cancer MEM-E/02 da Silva et al. Int J Breast Cancer. 2013:250435. 2013. [83]

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

Cervical cancer MEM-E/02 Gonçalves MA et al. Eur J Obstet Gynecol Reprod Biol.

Cervical cancer MEM-E/02 Spaans VM et al., J Transl Med. 10:184. 2012. [86]

Renal Cell Carcinoma MEM-E/02 Hanak L et al. Med Sci Monit. 15(12):CR638–43.2009. [90] Renal Cell Carcinoma MEM-E/02 Kren L et al., Diagnostic Pathology, **7**:58, 2012 [91] Thyroid cancer MEM-E/02 Zanetti et al. Int J Immunopathol Pharmacol. 26(4):889–96,

Hodgkin Lymphoma MEM-E/02 Kren L, et al., Pathology, Research and Practice 208: 45–49,

**REFERENCES**

141:70–4. 2008. [85]

[88]

2013. [92]

2012. [93]

2016. [94]

MEM-E/02 Gooden M et al.PNAS USA 108:10656, 2011. [84]

MEM-E/02 Ferns et al. J Immunother Cancer. 4:78, 2016. [87]

MEM-E/02 Zheng et al. Cancer Sci. 106(5): 522–528, 2015. [89]

MEM-E/02 Andersson et al. Oncoimmunology, 25;5(1):e1052213, 2015.

3D12 McWilliams et al., Oncoimmunology. 5(10):e1226720,

3D12 Wagner et al. Cancer, 23(5):814–823, 2017. [52]

**MHC**

**49**

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


#### **Table 3.**

**NATURE OF HUMAN CANCER**

*Monoclonal Antibodies*

Cancer

cancers

Melanoma Cervical

Melanoma and other

Lip squamousal cell carcinoma

Vulvar intraepithelial

Glioblastomas stem

cells

Intraoral mucoepidermoid carcinoma

Colon carcinoma and leukemia (K562)

Hepatocellular carcinoma

**48**

Non-small cell Lung Carcinoma

carcinoma

**COMMERCIAL mAbs**

MEM-E/08

Melanoma MEM-E/02 Derré L et al. J Immunol. 177:3100–7. 2006. [22]

Laryngeal carcinoma MEM-E/02 Silva TG et al. Histol Histopathol. 26:1487–97. 2011 [59]

Penile Cancer MEM-E/02 Djajadiningrat et al. J Urol. 193(4):1245–51. 2015. [61] Glioblastomas MEM-E/02 Mittelbronn, M. et al., J. Neuroimmunol. 189: 50–58. 2007

Glioblastomas MEM-E/02 Kren L et al. J Neuroimmunol. 220:131–5. 2010 [63] Glioblastomas MEM-E/02 Kren L et al. Neuropathology. 31: 129–34. 2011 [64]

Glioblastomas 3D12 Wischhusen J et al. J Neuropathol Exp Neurol. 64:523–8.

Neuroblastoma 3H2679 Zhen et al. Oncotarget. 7(28): 44340–44349, 2016. [67] Neuroblastoma 3D12 Morandi et al. J Immunol Res. 2016:7465741, 2016. [53] Oral Osteosarcoma MEM-E/02 Costa Arantes et al. Oral Surg Oral Med Oral Pathol Oral

Rectal Cancer MEM-E/02 Reimers et al. BMC Cancer BMC Cancer. 14:486.1–12, 2014.

Colon carcinoma MEM-E/02 Zeestraten EC et al. Br J Cancer. 110(2): 459–68.2014. [75]

Colorectal carcinoma MEM-E/08 Levy et al. Int J Oncol. 32(3): 633–41. 2008 [71] Colorectal carcinoma MEM-E/08 Levy et al. Innate Immun. 15(2):91–100. 2009. [72] Colorectal carcinoma MEM-E/02 Benevolo M, et al. J Transl Med. 9:184. 2011. [73] Colorectal carcinoma MEM-E/02? Bossard C et al. Int J Cancer. 131 (4): 855–863. 2012. [67] Colorectal carcinoma MEM-E/02? Zhen et al., Med Oncol. 30(1):482. 2013. [74] Colorectal carcinoma MEM-E/02 Zeestraten et al. Br J Cancer. 110(2):459–68. 2014. [75] Colorectal carcinoma MEM-E/02 Guo et al. Cell Immunol. 293(1):10–6, 2015. [76] Colorectal carcinoma 3H2679 Ozgul Ozdemir et al. Ann Diagn Pathol. 25:60–63, 2016 [77] Colorectal carcinoma MEM-E/02 Huang et al. Oncol Lett. 13(5):3379–3386, 2017. [78]

**REFERENCES**

[58]

[62]

2005 [66]

Radiol. 123(6):e188-e196. 2017. [68]

[70]

3D12 Marín R et al. Immunogenetics. 54(11):767–75.2003 [57]

MEM-E/07 Allard M et al. PLoS One 6(6):e21118, 2011 [55]

MEM-E/02 Goncalves et al. Human Immunol. 77(9): 785–790, 2016

MEM-E/02 van Esch EM et al. Int J Cancer. 135(4): 830–42, 2014 [60]

3D12 Wolpert et al. J Neuroimmunol. 250(1–2):27–34 2012 [65]

MEM-E/02 Mosconi C Arch Oral Biol. 83:55–62, 2017. [69]

MEM-E/06 Stangl S et al. Cell Stress Chaperones. 13(2):221–30. 2008.

MEM-E/02 Chen et al. Neoplasma. 58(5):371–376, 2011. [80]

MEM-E/02 Talebian-Yazdi et al. Oncotarget. 7(3):3477–3488, 2016.

[79]

[81]

*Expression of HLA-E on human cancer cells (biopsies or cell lines) monitored with commercial mouse anti-HLA-E mAbs (MEM-E/02, MEM-E/06, MEME/07. MEM-E/08, 3D12, 3H2679).*

presented in **Figure 4** show that the commercial anti-HLA-E mAbs react with HLA-A, HLA-B and HLA-C in the following order: MEM-06 > MEM-02 > MEM-07 > MEM >08 >> > 3D12. That the mAbs are recognizing the epitopes shared with several HLA-Ia (HLA-A, HLA-B, HLA-C) antigens confirms that none of the above mAbs are specific for HLA-E. Therefore conclusions concerning the expression of HLA-E in human cancers require further validation with monospecific anti-HLA-E mAbs.
