**6. Conclusion**

PHA (final 200 mL) (31). Detailed experimental protocol is described elsewhere (31). The effects of mAbs (monospecific mAb TFL-033 and polyreactive mAb TFL-007) on untreated (no PHA) and PHA-treated T lymphocytes in these categories of T cells: CD4+/CD8-, CD4-/CD8 +, CD4 + /CD8 +, and CD4-/CD8- are presented in **Table 9**. There was a significant increase in numbers of CD4-/CD8+ T lymphoblasts among the PHA-treated T lymphoblasts under the influence of TFL-033 s at 1:30 and 1:150). Numbers of PHA-untreated T lymphoblasts increased for almost all mAbs, TFL-033 s at 1/30 and 1/150, TFL-034 s at 1/10 and 1/50, TFL-073 s at 1/50, TFL-074 s at 1/10 [35]. An increase in PHA-untreated T lymphoblasts clarifies the functional potential of HLA-E monospecific mAbs in augmenting CD4/ CD8+ T lymphoblasts. A significant increase in numbers of PHA-treated CD3+/CD4-/CD8+ lymphoblasts suggests that monospecific monoclonal mAbs, particularly TFL-003 confers the potential to augment cytotoxic T cells. Results prompt investigating humanized version TFL-003 on

*5.7.2.2 HLA-E expressed on cancer cells can directly bind to CD8+ T cells and NK cells*

with immunolocalization and overexpression of cell surface HLA-E [71, 91, 128–132]. HLA-E gene expression in some cancers [e.g., melanoma] is ranked 19th among overexpressed genes [133]. HLA-E overexpression and loss of HLA-Ia in

*Binding of HLA-E to the inhibitory receptors CD94 and NKG2A on both CD8+ CTLs and NKT cells. The structural configuration of the binding of HLA-E and the inhibitory receptors, leading to the arrest of the antitumor activity function of CD8+ and NKT cells. The interaction between HLA-E and the inhibitory receptors involves the binding of amino acids located on the α1 and α2 helices of HLA-E to specific amino acids on CD94 and NKG2A. The amino acid sequences on HLA-E recognized by the inhibitory receptors are unique and specific for HLA-E and they are also recognized by HLA-E monospecific mAbs. The binding involves H-bonding (H), van der Waal forces (vf), and salt linkages (salt) of the amino acids of HLA-E a1 and a2 helices and CD94 and NKG2A inhibitory receptors. (*Modified from Ravindranath et al. Monoclon Antib

Cancer cells lose their cell surface HLA-Ia alleles (HLA-A, HLA-B, and HLA-C) and upregulate the surface expression of HLA-Ib molecules (HLAE, HLA-F, and HLA-G) [57, 82, 119–128]. The upregulation of HLA-E gene expression is correlated

proliferation cytotoxic T-cells.

*Monoclonal Antibodies*

**Figure 10.**

**66**

Immunodiagn Immunother. 2015,34(3):135–53*).*

*and suppress their tumor-killing activity*

The anti-HLA-E mAbs TFL- 033, TFL-034, TFL-073, and TFL-074 due to their monospecificity are advantageous than the commercial anti-HLA-E mAbs for specific identification and localization of HLA-E on the surface of human cells, particularly in different cancer types. Our observations stress the need for characterization of monospecificity and epitope specificity of any mAb, after analyzing binding affinity on a multiplex solid matrix assays coated with the desired antigen (in question) and the closely related antigens and inhibition of the binding affinity using peptides sequences specific for the antigen in question. This is an important criterion to be followed for all clinical diagnostic and therapeutic antibodies. If specific epitopes are exposed to antigen located on the cell surface, it would be a more valuable diagnostic tool, than those binding to specific but cryptic epitopes.

The HLA-E monospecific antibodies (e.g., TFL-033) are capable of augmenting proliferation of non-activated CD8+ T cells and activated CD8+ T-lymphoblasts. TFL-033 binds to a unique epitope of HLA-E, a region that is involved in binding to inhibitory receptors (CD94 and NKG2A) present on CD3+/CD8+ T cells (Cytotoxic T cells) and CD3-/CD8+ NKT cells and NK cells. The binding of HLA-E to inhibitory receptors results in the suppression of anti-tumor cytotoxic functions of these immune cells. *Since TFL-033 can also upregulate anti-tumor cytotoxic T cell lymphoblasts and also capable of blocking the interaction between cancer-associated HLA-E and inhibitory receptors CD94/NKG2A, the mAb can be considered as a double-edged sword to eliminate cancer cells.* Therefore, TFL-033 could be a valuable therapeutic agent for passive immunotherapy of human cancer, provided the mAb is humanized.

In contrast to monospecific mAbs, HLA-I polyreactive anti-HLA-E monoclonal Abs (TFL-006 and TFL-007) mimic not only HLA-I reactivity of IVIg but also performs several critical immunoregulatory functions of IVIg, better than IVIg *per se*. These functions include suppression of blastogenesis and proliferation of CD4+ T cells and CD8+ T cells, effective inhibition of production of anti-HLA-I and

HLA-II Abs. HLA-I polyreactive anti-HLA-E monoclonal Abs (TFL-006 and TFL-007) are capable of upregulating T-regs. T-regs acting alone is capable of suppressing CD4+ T cells, CD8+ T cells, and antibody.

**References**

San Diego, 2000, 400p.

[1] Marsh SGE, Parham P, Barber LD. The HLA. Facts Book. Academic Press.

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

the surfaces of activated B and T cells. J Exp Med 1991;174(5);1085–1095, DOI:

[9] Raine T, Brown D, Bowness P, Hill Gaston JS, Moffett A, Trowsdale J, et al. Consistent patterns of expression of HLA class I free heavy chains in healthy individuals and raised expression in spondyloarthropathy patients point to physiological and pathological roles. Rheumatology (Oxford) 2006;45(11);

[10] Cardoso EM, Esgalhado AJ, Patrao L, Santos M, Neves VP, Martinez J, et al. Distinctive CD8+ T cell and MHC class I

signatures in polycythemia vera patients. Ann. Hematol. 2018;97(9): 1563–1575, DOI: 10.1007/s00277-018-

[11] Santos SG, Powis SJ, Arosa FA. Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine

phosphorylation. J Biol Chem. 2004; 279 (51):53062–53070. DOI: 10.1074/jbc.

[12] Goodridge JP, Lee N, Burian A, Pyo CW, Tykodi SS, Warren EH, Yee C, Riddell SC, Geraghty DE. HLA-F and MHC-I open conformers cooperate in an MHC-I antigen cross-presentation pathway. J. Immunol. 2013;191:1567– 1577, DOI:10.4049/jimmunol.1300080.

[13] Brynjolfsson SF, Persson Berg L, Olsen Ekerhult T, Rimkute I, Wick MJ, Mårtensson IL, Grimsholm O. Long-Lived Plasma Cells in Mice and Men. Front Immunol. 2018;9:2673. DOI: 10.3389/fimmu.2018.02673.

[14] Morales-Buenrostro LE, Terasaki PI, Marino-Vázquez LA, Lee JH, El-Awar N, Alberú J. "Natural" human leukocyte

3332-7

M408794200.

10.1084/jem.174.5.1085.

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

1338–1344, DOI: 10.1093/ rheumatology/kel305

[2] Arosa FA, Esgalhado AJ, Padrão CA, Cardoso EM. Divide, Conquer, and Sense: CD8+CD28- T Cells in

Perspective. Front Immunol 2017;7:665, DOI:10.3389/fimmu. 2016.00665.

[3] Arosa FA, Santos SG, Powis SJ. Open conformers: the hidden face of MHC-I molecules. Trends Immunol 2007;28(3): 115–123, DOI:10.1016/j.it. 2007 01.002.

[4] Schnabl E, Stockinger H, Majdic O, Gaugitsch H, Lindley IJ, Maurer D, et al., (1990) Activated human T lymphocytes express MHC class I heavy

2microglobulin. J Exp Med, 1990;171(5):

[6] Benjamin RJ, Madrigal JA, Parham P. Peptide binding to empty HLA-B27 molecules of viable human cells. Nature, 1991;351(6321);74–77. DOI: 10.1038/

[7] Majdic O, Schnabl E, Stockinger H, Gadd S, Maurer D, Radaszkiewics T, et al. LA45, an activation-induced human lymphocyte antigen with strong homology to MHC class I molecules. In

[8] Madrigal JA, Belich MP, Benjamin RJ, Little AM, Hildebrand WH, Mann DL,

Leukocyte Typing IV. Oxford University Press, 1989, 511p.

et al., Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on

chains not associated with beta

[5] Schumacher TN, Heemels MT, Neefjes JJ, Kast WM, Melief CJ, Ploegh HL. (1990) Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro. Cell 1990;62(3);563– 567. DOI:10.1016/0092-8674(90)

1431–1442. DOI: 10.1084/

jem.171.5.1431.

90020-F

351074a0.

**69**
