Open access peer-reviewed chapter - ONLINE FIRST

Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E: Diagnostic and Therapeutic Relevance

By Mepur H. Ravindranath and Fatiha E.L. Hilali

Submitted: July 22nd 2020Reviewed: November 26th 2020Published: January 22nd 2021

DOI: 10.5772/intechopen.95235

Downloaded: 15

Abstract

A monoclonal antibody (mAb) binds to an antigen recognizing an epitope (a sequence of amino acids). A protein antigen may carry amino acid sequence unique to that antigen as well as sequences found in other proteins. Human leukocyte antigens (HLA), a family of proteins expressed by the Major Histocompatibility Complex gene family represent a special case, in that it displays a high degree of polymorphism. Every HLA molecule possesses both specific (private) epitopes and epitopes shared (public) with other HLA class Ia and class Ib molecules. HLA-E is overexpressed in cancer cells more than any other HLA Class I molecules. Therefore specific localization of HLA-E with mAbs is pivotal for developing targeted therapy against cancer. However, the commercially available mAbs for immunodiagnosis are polyreactive. We have developed anti-HLA-E mAbs and distinguished monospecific from polyreactive mAbs using Luminex multiplex single antigen bead (SAB) assay. HLA-E-binding of monospecific-mAbs was also inhibited by E-restricted epitopes. The amino acid sequences in the region of the epitopes bind to CD94/NKG2A receptors on CD8+ T cells and NK cells and block their antitumor functions. Monospecific-HLA-E mAbs recognizing the epitopes sequences can interfere with the binding to restore the anti-tumor efficacy of NK cells. Also, monospecific-mAbs augment the proliferation of CD4-/CD+ cytotoxic T-lymphocytes. Therefore, anti-HLA-E monospecific-mAb can serve as a double-edged sword for eliminating tumor cells.

Keywords

  • human leukocyte antigen (HLA)
  • epitope
  • monospecific
  • polyreactive
  • cytotoxic T-lymphocytes
  • inhibitory receptors
  • NK cells

1. Introduction

An in-depth understanding of amino acid sequences and conformations of primary antigens recognized by any monoclonal antibody (mAb) is a necessary prerequisite for clarifying the specificity and functional limitations of a mAb. A protein antigen may be glycosylated or can occur as a monomer or a dimer or a trimer. 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 diagnostic 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 immune cells.

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 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, 5, 6, 7, 8, 9, 10, 11, 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].

Figure 1.

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

HLA Class I
GeneABCEFG
Alleles6,2917,5826,2232564582
Proteins3,8964,8033,681110622

Table 1.

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

PROFILES OF HLA TYPING: HLA ISOFORMS AND THEIR ALLELES
HLA CLASSISOFORMSBROTHER*SISTER
IA*[11:02][33:01][01:01][11:02]
IB*[15:01][58:01][40:01][57:01]
IC*[15:02][15:02][03:04][06:02]
IIDRB1[04:03][13:02][07:01][11:01]
IIDRB3,4,5[3*03:01][4*01:01][3*02:02][4*01:01]
IIDQA[01:02][03:01][01:02][03:01]
IIDQB[03:01][06:09][02:02][03:01]
IIDPA[01:03][01:03][01:03][02:01]
IIDPB[02:01][03:01][01:07][01:11]

Table 2.

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

Mepur H. Ravindranath (brother) and his first sister.The alleles in bold letters refer to alleles shared by the brother and the sister.


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).

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 antibody-producing 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, 15, 16], as well as in the pooled and purified plasma also known as intravenous immunoglobulin (IVIg) [16, 17].

Figure 3.

The fate of HLA-I molecule after antigen presentation.

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, 19, 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 trophoblasts. Their overexpression is reported on activated T cells bone marrow cells inflamed cells and tissues (e.g. synovial fibroblasts), tumor cells [21, 22, 23, 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, 26, 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, 29, 30, 31, 32, 33, 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, 36, 37, 38, 39]. Soluble HLA-Ib is also found in the synovial fluid and the circulation of healthy and in cancer patients [40, 41, 42].

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

4.1 Unique characteristics of HLA-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, 45, 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, 47, 48, 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, 51, 52, 53, 54, 55, 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, 36, 37, 38, 39, 96, 97, 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.

NATURE OF HUMAN CANCERCOMMERCIAL mAbsREFERENCES
Melanoma Cervical Cancer3D12Marín R et al. Immunogenetics. 54(11):767–75.2003 [57]
MelanomaMEM-E/02Derré L et al. J Immunol. 177:3100–7. 2006. [22]
Melanoma and other cancersMEM-E/07Allard M et al. PLoS One 6(6):e21118, 2011 [55]
MEM-E/08
Lip squamousal cell carcinomaMEM-E/02Goncalves et al. Human Immunol. 77(9): 785–790, 2016 [58]
Laryngeal carcinomaMEM-E/02Silva TG et al. Histol Histopathol. 26:1487–97. 2011 [59]
Vulvar intraepithelial carcinomaMEM-E/02van Esch EM et al. Int J Cancer. 135(4): 830–42, 2014 [60]
Penile CancerMEM-E/02Djajadiningrat et al. J Urol. 193(4):1245–51. 2015. [61]
GlioblastomasMEM-E/02Mittelbronn, M. et al., J. Neuroimmunol. 189: 50–58. 2007 [62]
GlioblastomasMEM-E/02Kren L et al. J Neuroimmunol. 220:131–5. 2010 [63]
GlioblastomasMEM-E/02Kren L et al. Neuropathology. 31: 129–34. 2011 [64]
Glioblastomas stem cells3D12Wolpert et al. J Neuroimmunol. 250(1–2):27–34 2012 [65]
Glioblastomas3D12Wischhusen J et al. J Neuropathol Exp Neurol. 64:523–8. 2005 [66]
Neuroblastoma3H2679Zhen et al. Oncotarget. 7(28): 44340–44349, 2016. [67]
Neuroblastoma3D12Morandi et al. J Immunol Res. 2016:7465741, 2016. [53]
Oral OsteosarcomaMEM-E/02Costa Arantes et al. Oral Surg Oral Med Oral Pathol Oral Radiol. 123(6):e188-e196. 2017. [68]
Intraoral mucoepidermoid carcinomaMEM-E/02Mosconi C Arch Oral Biol. 83:55–62, 2017. [69]
Rectal CancerMEM-E/02Reimers et al. BMC Cancer BMC Cancer. 14:486.1–12, 2014. [70]
Colorectal carcinomaMEM-E/08Levy et al. Int J Oncol. 32(3): 633–41. 2008 [71]
Colorectal carcinomaMEM-E/08Levy et al. Innate Immun. 15(2):91–100. 2009. [72]
Colorectal carcinomaMEM-E/02Benevolo M, et al. J Transl Med. 9:184. 2011. [73]
Colorectal carcinomaMEM-E/02?Bossard C et al. Int J Cancer. 131 (4): 855–863. 2012. [67]
Colorectal carcinomaMEM-E/02?Zhen et al., Med Oncol. 30(1):482. 2013. [74]
Colorectal carcinomaMEM-E/02Zeestraten et al. Br J Cancer. 110(2):459–68. 2014. [75]
Colorectal carcinomaMEM-E/02Guo et al. Cell Immunol. 293(1):10–6, 2015. [76]
Colorectal carcinoma3H2679Ozgul Ozdemir et al. Ann Diagn Pathol. 25:60–63, 2016 [77]
Colorectal carcinomaMEM-E/02Huang et al. Oncol Lett. 13(5):3379–3386, 2017. [78]
Colon carcinoma and leukemia (K562)MEM-E/06Stangl S et al. Cell Stress Chaperones. 13(2):221–30. 2008. [79]
Colon carcinomaMEM-E/02Zeestraten EC et al. Br J Cancer. 110(2): 459–68.2014. [75]
Hepatocellular carcinomaMEM-E/02Chen et al. Neoplasma. 58(5):371–376, 2011. [80]
Non-small cell Lung CarcinomaMEM-E/02Talebian-Yazdi et al. Oncotarget. 7(3):3477–3488, 2016. [81]
Breast cancerMEM-E/02de Kruijf EM et al. J Immunol. 185:7452, 2010 [82]
Breast cancerMEM-E/02da Silva et al. Int J Breast Cancer. 2013:250435. 2013. [83]
Ovarian cancer/ Cervical cancerMEM-E/02Gooden M et al.PNAS USA 108:10656, 2011. [84]
Cervical cancerMEM-E/02Gonçalves MA et al. Eur J Obstet Gynecol Reprod Biol. 141:70–4. 2008. [85]
Cervical cancerMEM-E/02Spaans VM et al., J Transl Med. 10:184. 2012. [86]
Cervical squamous and adenocarcinomaMEM-E/02Ferns et al. J Immunother Cancer. 4:78, 2016. [87]
Serous Ovarian AdenocarcinomaMEM-E/02Andersson et al. Oncoimmunology, 25;5(1):e1052213, 2015. [88]
Serous Ovarian AdenocarcinomaMEM-E/02Zheng et al. Cancer Sci. 106(5): 522–528, 2015. [89]
Renal Cell CarcinomaMEM-E/02Hanak L et al. Med Sci Monit. 15(12):CR638–43.2009. [90]
Renal Cell CarcinomaMEM-E/02Kren L et al., Diagnostic Pathology, 7:58, 2012 [91]
Thyroid cancerMEM-E/02Zanetti et al. Int J Immunopathol Pharmacol. 26(4):889–96, 2013. [92]
Hodgkin LymphomaMEM-E/02Kren L, et al., Pathology, Research and Practice 208: 45–49, 2012. [93]
Chronic Lymphocytic Leukemia3D12McWilliams et al., Oncoimmunology. 5(10):e1226720, 2016. [94]
Chronic Lymphocytic Leukemia3D12Wagner et al. Cancer, 23(5):814–823, 2017. [52]
Many Cancers3D12Sensi M, et al. Int Immunol. 21(3):257–268. 2009. [95]

Table 3.

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).

Figure 4.

HLA-IA-polyreactivity of the commercial anti-HLA-E mAbs indicates that these mAbs cannot be considered monospecific or specific for HLA-E. The mAbs were tested at a dilution of 1/300. These mAbs were used to conclude on the expression of HLA-E on human cancers.

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

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

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, 36, 37, 38, 39, 96, 97, 98]. The results are comparable to assays such as ELISA but with greater specificity, sensitivity and resolution. The technology employs superparamagnetic 6.5-micron microspheres with a magnetic core and polystyrene surface. The beads are internally dyed with precise proportions of red and infrared fluorophores. The Luminex xMAP detection systems identifies differing proportions of the red and infrared fluorophores that result in 100 unique spectral signature microspheres. The antigens are individually attached to polystyrene microspheres by a process of simple chemical coupling. The conjugation of a mAb to one or more of the antigen-coated beads allows it to be evaluated for the mono- or polyreactivity of mAb [96, 97, 98]. Figure 5 illustrates the SAB Assay used for determining the monospecificity or polyreactivity of mAbs as well as evaluating the strength of the antibodies measured as mean fluorescent intensity (MFI) at specified dilution. The assay is also used to measure antibody specificity by peptide inhibition assays, to define the epitope-specificity of a mAb. Commercial HLA class I or II beadsets are commercially available as LABScreen (One Lambda Inc., now merged with Thermofisher Inc) and LIFECODES (Immucore Inc)]. The both beadsets together is useful to distinguish CCs from OCs of HLA-I molecules, using a mAb (HLA-I mAb, TFL-006) (See Table 7 in [99]).

Figure 5.

Luminex single antigen bead assay is used to determine the monospecificity or polyreactivity of the mAbs as well as to determine the strength of the antibodies measured as mean fluorescent intensity (MFI) at specified dilution. The assay is also used to measure the antibody strength titrimetrically. Using peptide inhibition assay epitope affinity or specificity of a mAb can be studied to determine monospecificity or polyreactivity of the mAb. Using a mAb (e.g., HLA-I mAb, TFL-006) recognizing the most commonly shared epitope of an HLA-I (or HLA-II) in an open conformer, the commercial beads can be distinguished as those containing open conformers or closed conformers.

5.2 Development of mAbs against HLA-E

Following guidelines of the National Research Council’s Committee on Methods of Producing Monoclonal Antibodies [35, 98, 100], 235 anti-HLA-E mAbs were generated immunizing mice with recombinant HCs of HLA-ER107 (Immune Monitoring Lab, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA) (10 mg/ml in MES buffer). In a separate mouse model, HLA-EG107 (heavy-chain only) was used as an immunogen. The β2m-free HC of HLA-E (50 μΜ in 100 mL of PBS (pH 7.4) mixed with 100 mL of TiterMaxVR Gold adjuvant (CytRx, San Diego, CA) were injected into the mouse footpad and intraperitoneum. Three immunizations were given at 12-day intervals. The B cell clones were cultured in RPMI 1640 medium w/L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, cat. no. R8758), 15% fetal calf serum, 0.29 mg/ml L-glutamine, Pen-Strep (Gemini-Bio, MEd Supply Partners, Atlanta, GA, cat. no. 400–110) and 1 mM sodium pyruvate (Sigma, cat. no. S8636). Several clones were grown using Hybridoma Fusion and Cloning Supplement (HFCS) (Roche Applied Science, Indianapolis, IN, cat. no. 11363735001). The purified-mAbs from HLA-E hybridoma culture supernatants and ascites of hybridoma immunized in BALB/c mice were examined for HLA-I reactivity using Luminex SAB Assay.

5.3 Characterizing the diversity of anti-HLA-E mAbs using single antigen bead (SAB) assay

The HLA-I reactivity of the mAbs was examined by their dose-dependent binding to microbeads coated with 31 HLA-A, 50 HLA-B, and 16 HLA-C antigens and with recombinant single alleles of HLA-E, -F, and -G [35, 98, 100]. The HLA-Ia microbeads have built-in control beads: positive beads coated with human IgG and negative beads coated with serum albumin (human or bovine). For HLA-Ib, the control beads (both positive and negative) were added separately. PE-conjugated anti-human IgG-detection mAbs were used for immunolocalization of mAb bound to HLA antigens coated on beads [35, 36, 37, 96, 97, 98, 99, 100]. Table 4 summarizes the diverse types of mAbs observed after immunizing with heavy chains of HLA-E. Group 1 consists of mAbs that are only bound to HLA-E. Anti-HLA-E mAbs were also characterized for their IgG subclasses, using monoclonal IgG specific for the Fc portion of the subclasses

mAbs formed after immunizing HLA-E
HLA Class IaHLA Class Ib
HLA-AHLA-BHLA-CHLA-EHLA-FHLA-G
Group 1(−)(−)(−)(+)(−)(−)24 TFL-monospecific anti-HLA-E mAbs
Group 2(−)(−)(−)(+)(+)(−)TFL-anti-HLA-E/F mAbs
Group 3(−)(−)(−)(+)(−)(+)TFL-anti-HLA-E/G mAbs
Group 4(−)(−)(−)(+)(+)(+)TFL-anti-HLA-Ib sepecific mAbs
Group 5(+)(+)(+)(+)(−)(−)Reactivity of the mAbs 3D12, MEM-E/02 & MEM-E/07 & TFL series
Group 6(+)(+)(+)(+)(+)(−)Reactivity of the mAb MEM-E/06 & TFL-series
Group 7(+)(+)(+)(+)(−)(+)Reactivity of the mAb MEM-E/08 & TFL series
Group 8(+)(+)(+)(+)(+)(+)Reactivity of the mAb TFL-006, TFL-007 & other TFL mAbs

Table 4.

The diverse HLA-E monospecific and polyreactive mAbs generated after immunizing mice with a recombinant heavy chain of HLA-ER107 & HLA-EG107.

Fluorophore intensity was measured in a specialized flow cytometer (Luminex) together with microbead identifiers, and the fluorescence measurement classified by the bead identifier. Fluorescent intensity generated by Luminex Multiplex Flow Cytometry (LABScan 100) was analyzed using the same computer software and protocols. For each analysis, at least 100 beads were counted. The “trimmed mean” is obtained by trimming a percentage of the high and low ends of distribution and finding the mean of the remaining distribution. Trimmed mean fluorescence intensity (MFI) for the SAB reactions are obtained from output (CSV) file generated by flow analyzer, and it was adjusted for background signal using the formula (sample #N bead – sample negative control bead) [35, 36, 37, 96, 97, 98, 99, 100]. The MFI was compared with the negative control mean and the standard deviation of MFI recorded. The purpose of MFI is to define the affinity of mAbs to HLAs and the intensity or strength of the mAbs.

5.4 The diversity anti-HLA-E mAbs

Of the 235 hybidomas generated, mAbs secreted by 214 hybridomas were reactive to HLA-E. These mAbs included both monospecific [35, 98] and polyreactive (with other HLA-Ia and HLA-Ib molecules) [98, 101]. Table 5, A presents category 1 correspond to monospecific mAbs reacting restrictively to mAbs with HLA-E and failing to recognize HLA-F, HLA-G, HLA-A, HLA-B, and HLA-C. Category 2 refers to HLA-Ib specific anti-HLA-E mAbs (Table 5, B). Category 3 presents anti-HLA-E mAbs reactive with several HLA-Ia molecules (HLA-A, HLA-B, and HLA-C) but not reactive to HLA-F and HLA-G (Table 5, C). Category 4 presents mAbs recognizing both HLA-Ib and HLA-Ia molecules (Table 5, D).

Nature of mAbsmAb specificitynumber of mAbsExamples of TFL mAbsAntigen (heavy chain only) tested on beadsSubclassHLA-EHLA-FHLA-GHLA-AHLA-BHLA-C
Reactivity in MFI
AAntigen immunized: β2-microglobulin-free heavy chain of HLA-ER107
HLA-E Monospecific mAbs (Category 1)HLA-E16TFL-145, TFL-33, TFL, 34, TFL-73, TFL-74HLA-ERIgG14 K–22 K00000
3TFL-001HLA-ERIgG2a0.9 K - 4 K00000
TFL-016
TFL-013
Antigen immunized: β2-microglobulin-free heavy chain of HLA-EG107
5TFL-185HLA-EGIgG119 K00000
TFL-184
TFL-186
TFL-226
TFL-254
BAntigen immunized: β2-microglobulin-free heavy chain of HLA-ER107
HLA-IB polyreactive and HLA-IA and non-reactive HLA-E mAbs (Category 2)HLA-Ib specific mAbs1TFL-050HLA-ERIgG2b4 K3 K2 K000
Antigen immunized: β2-microglobulin-free heavy chain of HLA-EG107
3TFL-208, TFL-209, TFL-223,HLA-EG HLA-ERIgG121 K8 K20 K000
4TFL-164HLA-EGIgG2b14 K–15 K8 K–9 K24 K–25 K000
TFL-165
TFL-162
TFL-161
E + G+1TFL-191HLA-EGNK1 K01 K000
E + F+1TFL-228HLA-EGIgG119 K1 K0000
CAntigen immunized: β2-microglobulin-free heavy chain of HLA-ER107
HLA-IA Polyreactive HLA-E mAbs (Categroy 3)E + B + C+31TFL-059HLA-EG HLA-ERIgG1 (n = 12) IgG2A (n = 9) IgG2b (n = 9) IgG3 (n = 1)8 K–20 K0001 K–17 K1 K–7 K
TFL-143
TFL-158
TFL-076
TFL-159
E + A + B + C+68TFL-119HLA-EG HLA-ERIgG1 (n = 27) IgG2A (n = 23) IgG2b (n = 17) IgG3 (n = 1)11 k–22 k001 K–4 K1 K–24 K1 K–13 K
TFL-142
TFL-153
TFL-118
TFL-133
TFL-141
TFL-095
EG + B+3TFL-173HLA-EGIgG112 K0001 K0
TFL-174
TFL-175
E + B+1TFL-219HLA-EG/RIgG1210002 K0
EG + A + B + C+6TFL-167HLA-EGIgG115 K-25001 K–9 K1 K 20 K1 K–20 K
TFL-170
TFL-169
TFL-166
TFL-168
TFL-205
E + A + B + C+35TFL-243HLA-EG/RIgG1 (n = 22) IgG2A (n = 6) IgG2b (n = 6) IgG3 (n = 1?)13 K–26 K001 K–9 K1 K–24 K1 K–20 K
TFL-246
TFL-244
TFL-245
TFL-172
TFL-171
Nature of mAbsImmunogen usedmAb specificitynumber of mAbsExamples of TFL mAbsSubclassHLA-EHLA-FHLA-GHLA-AHLA-BHLA-C
Reactivity in MFI
ER107EG107
D. Category 4. HLA- IA and IB polyreactive anti-HLA-E mAbs. (n = 36)
HLA = IA Polyreactive HLA-IB mAbs (Category 4)HLA-EGE+/F+/G+4TFL-232IgG313–22212 to 1011 to 211 to 131 to 201 to 20
TFL-177IgG1 (n = 3)0
TFL-176
TFL-1980
E+/G+16TFL-236IgG1 (n = 14)18–22 (n = 13)0018–22 (n = 11)1 to 91 to 251 to 24
TFL-238
TFL-256IgG322271
TFL-229IgG2b302218
E+/F+10TFL-210IgG1 (n = 10)18–2117–195 to 1101 to 151 to 201 to 22
TFL-211
TFL-212
TFL-235
HLA-ERE+/F+/G+3TFL-049IgG2b15–228 To 122 to 71 to 101 to 101 to 17
TFL-006IgG2a (n = 2)
TFL-007
E+/G+2TFL-103IgG1 (n = 2)17,18041 to 61 to 111 to 11
TFL-104
E+/F+1TFL-063IgG2b22302-Jan1 tp 73 to 8

Table 5.

Different categories of mAbs (n = 212) formed after immunizing mice with HLA-E open conformer (β2-microglobulin-free heavy chain) of HLA-ER107 or HLA-EG107.

mAbs in Bold are highly polyreactive,

5.5 Unique (private) and common (public) epitopes of HLA-E

The international immunogenetics project (http://www.ebi.ac.uk; orhttp://www.ebi.ac.uk/ipd/imgt/hla/intro.html) updates HLA genes and sequence alleles yearly. We have compared the entire amino acid sequences of HLA-E (Figure 6) with 511 alleles of HLA-A, 846 alleles of HLA-B, 275 alleles of HLA-C, 2 alleles of HLA-F, and 2 alleles of HLA-G sequences(see Table 1). Amino acid sequences unique to HLA-E (private epitopes) and common amino acid sequences (public epitopes) can be identified by comparing the amino acid sequences of HLA-E with thousands of HLA-Ia and Ib antigens (Table 6). Anti-HLA-E mAbs could bind to HLA-E restricted (monospecific) or HLA-I amino acid sequences. Several HLA-E sequences are shared with HLA-A loci or HLA-C loci or specific alleles such as A*3306 or B*8201. Table 7 shows HLA-E restricted amino acid sequences found in α1 and α2 helices, which were used for peptide inhibition assays. Figure 7A illustrates locations of private and public epitopes. Figure 7B shows allele-specific amino acid sequences in α1 & α2 helical groove and Figure 7C shows shared peptide amino acid sequences.

Figure 6.

Amino acid sequence of HLA-ER107. Two sets of serial numbers provide one to include leader sequence and another after deleting leader sequence. Sequences in the boxes refer to either specific (private) or shared (public) epitopes. The box with bold letters was used to test for peptide inhibition in our experiments using TFL-monospecific mAbs.

Comparison of the amino acid sequences of HLA-E with other HLA-I antigens
HLA alleles
HLA-E peptide sequencesNumber of amino acidsClassical HLA-IaNon-classical HLA-IbSpecificity
ABCwFG
47PRAPWMEQE55910000A*3306 restricted
59EYWDRETR65850000A-restricted
65RSARDTA71600000E-monospecific
90AGSHTLQW9781104800Multispecific
108RFLRGYE1237240000A-restricted
115QFAYDGKDY123911047500Multispecific
117AYDGKDY12374918312712130Highly Multispecific
126LNEDLRSWTA135102392192612130Multispecific
137DTAAQI14260824248030Multispecific
137DTAAQIS14370524030Multispecific
143SEQKSNDASE1521000000E-monospecific
157RAYLED162601000B*8201-restricted
163TCVEWL1686282206200030Multispecific
182EPPKTHVT1908001900C-restricted

Table 6.

Identifying HLA-E specific epitope or amino acid sequences: Peptide sequences specific and shared between HLA-E and HLA class Ia alleles: Monospecific (HLA-E restricted) versus polyreactive epitopes.

Peptide [# 1] specific for HLA-EPeptide [# 2] specific for HLA-E
HLA Class Ibα1α1α1α1α1α1HLA Class Ibα2α2α2α2α2α2α2α2α2
656667686970143144145146147148149150151
E*01010101RSARDTE*01010101SEQKSNDAS
G*01010101RNTKAHG*01010101SKRKCEAAN
F*01010101GYAKANF*01010101TQRFYEAEE
A*110101RNVKAQA*110101TKRKWEAAH
B*1401QICKTNB*1401TQRKWEAAR
B*350101QIFKTNB*350101TQRKWEAAR
B*40060101QISKTNB*40060101TQRKWEAAR
B*530101QIFKTNB*530101TQRKWEAAR
B*5801RNMKASB*5801TQRKWEAAR
CW*050101QKYKRQCW*050101TQRKWEAAR
CW*080101QKYKRQCW*080101TQRKWEAAR
CW*1802QKYKRQCW*1802TQRKWEAAR
Qa-1(murine eq:HLA-E)WKARDMQa-1(murine eq:HLA-E)SKHKSEAVD

Table 7.

Identifying HLA-E specific epitope or amino acid sequences: Comparing the two HLA-E restricted sequences with other HLA-I amino acid sequences at the same position.

Figure 7.

Diagrammatic illustration of the structure of HLA-E, closed (intact trimer) and open conformers and specific (private) and shared (public) epitopes. (A) Illustrates the locations of allele-specific sequence (private epitope) and shared peptide (public epitopes) sequence. HLA-E with β2-microglobulin (in blue) showing (B) the allele-specific amino acid sequences (private epitopes) in α1 & α2 helical groove and (C) shared peptide amino acid sequences (public epitopes).

Peptide inhibition analyses were performed to confirm the monospecificity of HLA-E mAbs. Various concentrations of HLA-E-restricted peptides (serially diluted from the initial concentration of 100 μL to 100 μL) were added to the mAbs (7 μL). The mAbs were further diluted with 14 μL PBS-BSA (pH 7.0; final dilution 1/1200), and then exposed to 2 mL of beads. The two different HLA-E-restricted peptides, RSARDTA and SEQKSNDASE were synthesized and purified by GenScript Corporation (Piscataway, NJ). The assay was performed in triplicate. Dosimetric peptide inhibition analysis was performed for mAb TFL-033. Before dosimetric peptide inhibition, the mAb TFL-033 was dosimetrically titrated to assess their strength (MFI), and protein-G purified culture supernatants and ascites compared. Then, concentrated Protein-G purified from ascites is titrated and the protein content is measured. Titrimetric inhibition was done with ascites protein-G concentrate. A summary of the peptide inhibition experiments is presented in Figure 8. Results confirm that TFL-003 binding to HLA-E can be inhibited dosimetrically using two HLA-E-restricted epitopes. The level of inhibition differed between the two epitopes.

Figure 8.

Dosimetric inhibition of purified culture supernatants of TFL-033 with two HLA-E-restricted peptides, 65RSARDTA71 and 143SEQKSNDASE152, at concentrations ranging from 4.4 to 0.27 mg/well. Although both peptides showed inhibition, the α2 helical peptide SEQKSNDASE showed better dosimetric inhibition than the other peptide. Peptide concentration and peptide content (μG/well) in parenthesis are shown. Pair-sample or equal-variant t-tests were carried out in this investigation using a graphic website (www.originlab.com). (Source: U. S. Patent No 10,656,158 B2 (U.S. patent application No. 13/507,537) issued on May 19, 2020, to Dr. Mepur H. Ravindranath) see also Int J cancer. 2014;134(7):1558–70. DOI: 10.1002/ijc.28484.

5.6 Diagnostic potential of HLA-E monospecific mAbs

Immunolocalization of HLA-E on human melanoma cancer tissues was performed using culture supernatants (s) or ascites (a) of TFL monospecific mAbs (TFL-033, TFL-034, TFL-074, and TFL-216), and staining is compared with commercial anti-HLA-E mAb (MEM-E/02) [35, 98]. Titration of Protein-G purified culture supernatants and ascites concentrates of different anti-HLA-E monospecific mAbs are shown in Table 8. As revealed in Figure 4, the MEM-02 cross-reacts with several HLA class Ia alleles. Although it stains melanoma tissues, due to the paucity of HLA-E specificity, specific localization of HLA-E was confirmed with monospecific anti-HLA-E mAbs (Figure 9A). Similarly, immune-localization of HLA-E on human gastric diffused carcinoma paraffin tissue sections was observed after staining with the diluted ascites of monospecific mAb TFL-033a and MEM-E/02. The reliability of HLA-E tissue localization with monospecific immunostaining of human gastric adenocarcinoma (A, B) with TFL-033 and MEM-E/02 with that obtained for gastric diffuse carcinoma (C, D) control, stained without primary mAbs. MEM-E/02 failed to stain any cells while TFL-033a showed intense and widely distributed staining indicating the overexpression of intact HLA-E (Figure 9C). Immunostaining was performed on human breast ductal adenocarcinoma with TFL monospecific-mAbs and results obtained using monospecific anti-HLA-E mAb TFL-216, generated by immunizing HLA-EG, is presented in Figure 9D.

SampleDilutionTFL-033TFL-034TFL-073TFL-074
Culture SupernatantNeat112731160177818493
Protein-G purified Culture supernatant(1:10)4424273019742507
Protein-G purified Culture supernatant Concentrate(1:10)119531036477088467
(1:20)9423814668617500
(1:40)8167634753245883
(1:80)6203462237924176
(1:160)4139137926832438
(1:320)28626261454943
(1:640)1434198590474
(1:1280)69498275220
Protein-G purified Ascites Concentrate (Eluate # 2)(1:50)17898
(1:100)16246
(1:200)14004
(1:400)12520

Table 8.

Titration of protein-G purified culture supernatant and ascites concentrates of different HLA-E monospecific mAbs. These concentrates were used for immunolocalization, peptide inhibition studies as well as for their effects on T-lymphoblasts.

Figure 9.

Immunolocalization of HLA-E in cancer tissues with culture supernatants (s) or ascites (A) of TFL monospecific mAbs compared with staining by MEM-E/02, an HLA-E mAb that shows cross-reactivity to HLA class Ia alleles. (A) Human melanoma paraffin tissue sections stained with the culture supernatants of TFL monospecific MAbs and MEM-E/02. (B) Human gastric cancer (diffused carcinoma) paraffin tissue sections stained with the diluted ascites of monospecific MAb TFL-033a and MEM-E/02. (C). Immunostaining of human gastric adenocarcinoma (A, B) and gastric diffuse carcinoma (C, D) control, stained without primary mAbs. Note the differences in staining between the two antibodies; MEM-E/02 failed to stain any cells while TFL-033a showed intense and widely distributed staining indicative of overexpression of intact HLA-E. (D) Human breast ductal adenocarcinoma stained with monospecific anti-HLA-E mAb TFL-216 generated by immunizing HLA-EG. (source: U. S. Patent No 10,656,158 B2 (U.S. patent application No. 13/507,537) issued on May 19, 2020, to Dr. Mepur H. Ravindranath) see also Int J cancer. 2014;134(7):1558–70. DOI: 10.1002/ijc.28484.

Detailed immunodiagnostic analyses were performed using a tissue microarray (TMA) of normal gastric mucosal and primary gastric cancer tissues [98]. Three tissue microarrays (TMAs; US Biomax, Rockville, MD) were carefully selected. The tissue sections of all TMA were 1.5 mm in diameter and 5 μm thick. In TMA of normal gastric mucosa and of primary gastric cancer, which contained 30 adenocarcinomas, 40 diffuse carcinomas and ten normal gastric mucosae were immunostained. TMA array included: well-differentiated, moderately differentiated, poorly differentiated, and undifferentiated cancer. In addition, TMA also included Stages I to IV of metastatic gastric cancer with 5 peritoneal, 3 liver, 27 lymph node metastases. TMA was immunostained with TFL-033 mAbs (culture supernatants and ascites), controls were stained without primary mAbs [98]. The diagnostic potential of HLA-E-monospecific mAb TFL-033 for different kinds and stages of gastric cancer is illustrated in Figure 4a in International Journal of Cancer [98]. The observations confirm that specific identification and localization of MHC antigens, stringently require monospecific mAbs. The conclusion is highly reliable compared to the use of polyreactive commercial mAbs (MEM-E/02) [36, 98], presented in Figure 4. Importantly, characterizations of monospecificity should include (1) multiantigen coated solid matrix assays, e.g., Luminex multiplex SAB assay; (2) titrimetric inhibition with the private epitope of the antigen. Only such monospecific mAbs are reliable for diagnosis and therapeutic purposes.

5.7 Differences in the immunoregulatory potentials of HLA-E monospecific versus polyreactive mAbs

5.7.1 Potential of polyclonal anti-HLA-E mAbs in immune regulation

Immunoregulatory properties of both monospecific (TFL-033) and polyreactive (TFL-006 & TFL-007) anti-HLA-E mAbs were examined for their ability to suppress or activate CD3/CD4+, CD3/CD8+ T cells, T-regs, and CD3+/CD19/20+ B cells. The results show that the polyreactive anti-HLA-E mAbs (TFL-006/TFL-007) are immunosuppressive comparable to IVIg, used in immunotherapy of several diseases [16, 17]. Indeed the anti-HLA antibody profile of IVIg from different sources showed both HLA-Ia and HLA-Ib reactivities [16, 17]. IVIg preparations were reported to suppress CD4+ T cells [102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113], CD20+ B cells [108, 109, 110, 111, 112, 113] and expand CD4 + CD25+ T-regs [114, 115]. The polyreactive anti-HLA-E mAbs performed the major immunoregulatory functions better than IVIg [101, 116, 117, 118]. These functions are (1) suppression of CD19+ B lymphocyte blastogenesis, proliferation, and suppression of production of anti-HLA-I and anti-HLA-II IgG Abs, (2) suppression of blastogenesis and proliferation of CD4+ as well as CD8+ T lymphocytes, and (3) expansion of CD4+. CD25+ and FoxP3+ T-regs. The monospecific mAbs, when used as controls failed to perform these functions. Peptide inhibition analyses revealed that mAbs TFL-006 and TFL-007 bind to shared amino acid sequences of HLA-I molecules (117AYDGKDYLT125, 126LNEDLRSWTAV136, and 137DTAAQI142) (Figure 7C). Possibly such binding affinity of polyreactive but not monospecific mAbs contributes to the unique immunoregulatory functions mimicking IVIg [101, 118].

5.7.2 Therapeutic potential of anti-HLA-E monospecific mAbs

In contrast to polyreactive anti-HLA-E mAb, monospecific mAbs (TFL-033) recognized HLA-E- specific amino acid sequences (65RSARDT70 and154AESADNSKQES144) on the α1 and α2 helices (Figure 7B).

5.7.2.1 Monospecific mAbs promote the proliferation of CD8+ T lymphocytes

To test whether monospecific anti-HLA-E mAbs suppress proliferation of the CD3 +, CD4+, or CD8+ T cells, human T lymphocytes (both CD4+ and CD8+) isolated from whole blood of a normal male donor with Ficol Hypaque (31) were treated either with phytohaemagglutinin (PHA, EY Laboratories, San Mateo, CA) at a final concentration of 2.25 mL/mL or not exposed to PHA (31). The mAbs (monospecific mAbs TFL-033, TFL-034, TFL-073, TFL-074, and TFL-216, polyreactive mAb TFL007, and negative control antibodies) were separately added to cells in culture within 2 hours after adding 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 proliferation cytotoxic T-cells.

Presence or absence of CD4/CD8CD3+ NAÏVE T-CELLSCD3+ LYMPHOBLASTS
No PHAWith PHANo PHAWith PHA
CD4+/CD8-CD4-/CD8+CD4+/CD8-CD4-/CD8+CD4+/CD8-CD4-/CD8+CD4+/CD8+CD4-/CD8-CD4+/CD8-CD4-/CD8+CD4+/CD8+CD4-/CD8-
No mAb [n = 5]
Mean306354712494751976514152867325128289
SD149869937331435151151264384
2-tail p [<]<0.00010.0010NS0
mAb TFL-033 (IgG1) [n = 3]
[1/30]
Mean31857551170536223163153991129505152412
SD18014658124027801386231620
2-tail p [<]NSNSNS0.009NS0.015NS0.0050.0100.016NS0.014
[1/150]
Mean32386811149508252120205681266572157412
SD14642122301713980311416
2-tail p [<]NSNSNSNS0.0470.0010.020NS0.0010.003NS0.001
mAb TFL-007 (Polyreactivec anti-HLA-E, IgG2a) [n = 3]
[1/10]
Mean287645111834441646314552676317100222
SD1367219263323177925429
2-tail p [<]NSNSNSNSNSNSNSNS0.027NSNSNS
[1/50]
Mean3,0886671,07549123010719380892443122339
SD651655482371742618821
2-tail p [<]NS0.0180.013NSNSNS0.0190.006NSNSNSNS

Table 9.

TFL-033 promotes T-lymphoblast proliferation of CD8+ naïve T cells and T-Lymphoblasts in the absence or the presence of PHA. The proliferation of CD4+ T lymphoblasts occurs only after PHA activation.

5.7.2.2 HLA-E expressed on cancer cells can directly bind to CD8+ T cells and NK cells and suppress their tumor-killing activity

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, 120, 121, 122, 123, 124, 125, 126, 127, 128]. The upregulation of HLA-E gene expression is correlated with immunolocalization and overexpression of cell surface HLA-E [71, 91, 128, 129, 130, 131, 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 cancer cells are correlated with disease progression and poor prognosis [60, 82, 130, 134]. Disease progression is attributed to the suppression of the tumor-killing activity of CD8+ cytotoxic T lymphocytes (CTLs) and NKT cells.

Cell surface and soluble HLA-E are capable of binding to the inhibitory receptors CD94 and NKG2A on both CTLs (CD3+/CD8+), NK cells (CD2+, CD7+, CD11b+, CD11c+, CD90+, perforin+, & granzyme A+) and NKT cells (plus CD8+) [25, 27, 135, 136]. These cells are capable of destroying tumor cells. These cells interact with MHC-I ligands (HLA-E) on tumor cells through inhibitory receptors. The binding of above mentioned immune cells to HLA-E overexpressed on tumor cells cell surface may explain why the cancer patients failed to respond to NK cell therapies.

Interaction between HLA-E and inhibitory receptors involves the binding of HLA-E specific amino acids located on α1 and α2 helices (Table 7) to specific amino acids on CD94 and NKG2A (Figure 10) [22, 27, 135, 136]. This specific interaction is attributed to the loss of anti-tumor activity of CD8+ CTLs as well as that of NK or NKT cells [22, 27, 135, 136]. We have used the synthetic peptides of these sequences to ascertain the specific binding affinity of anti-HLA-E mAbs (Figure 8). The ability of monospecific anti-HLA-E mAbs to bind at the site of epitopes of CD94 and NKG2A on HLA-E favor the use of the monospecific anti-HLA-E mAbs to mask binding sites of inhibitory receptors on HLA-E. Such blocking of HLA-E may help restore the antitumor efficacy of NK cells and CD8+ T cells that were lost due to the interaction of inhibitory receptors and HLA-E. Possibly humanized monospecific anti-HLA-E may be potentially considered for anti-cancer NK therapy.

Figure 10.

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 anti-tumor 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 Immunodiagn Immunother. 2015,34(3):135–53).

6. Conclusion

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.

Acknowledgments

We wish to express sincere thanks to Professor Dr. Edwin L. Cooper, Distinguished Professor, at Collaborative Centers for Integrative Medicine, University of California, Los Angeles, for critically editing this manuscript. We wish to dedicate this chapter to Professor Edwin L Cooper. We also wish to thank Professor Mark Terasaki and Terasaki Family Foundation for providing financial support to carry out the research reported in this chapter at Terasaki Foundation Laboratory (TFL), Santa Monica, CA. We wish to thank the following research scientists at TFL, Drs. Dong Zhu, Toshiyuki Sasaki, Vadim Jucaud, Mr. Tho Pham, Mr. Satoru Kawakita, Mr. Curtis Y. Maehara, and Mrs. Judy Hopfield were directly involved in the experiments reported in this chapter.

A patent was filed based on our research on monospecific anti-HLA-E mAb TFL-033 and a U. S. Patent No 10,656,158 B2 was issued on May 19, 2020, to Dr. Mepur H. Ravindranath & Late Professor Paul Ichiro Terasaki. Hybridoma of TFL-033 is deposited with ATCC Patent Depository (ID: PTA-125908) at Manassas, Virginia 20110, USA.

Conflict of interest

The authors declare no conflict of interest.

Notes

  • Formerly, Recipient of Fulbright Award at the University of Madras India and came to the University of California, Los Angeles, and appointed as Adjunct Associate Professor in the Department of Anatomy. Senior Research Scientist (2008-2018), Terasaki Foundation Laboratory, Santa Monica, CA, Director, Department of Glycoimmunomics (1990-2008), John Wayne Cancer Institute, Santa Monica CA. Adjunct Faculty, University of California, Los Angeles (1983-1990), Faculty and Fulbright Recipient (1983-1984), University of Madras, Chennai, Tamilnadu, India 60005 (1972-1983).

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Mepur H. Ravindranath and Fatiha E.L. Hilali (January 22nd 2021). Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E: Diagnostic and Therapeutic Relevance [Online First], IntechOpen, DOI: 10.5772/intechopen.95235. Available from:

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