Numbers of HLA alleles (as of September 2020) and their proteins. See updated information at
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.
- human leukocyte antigen (HLA)
- cytotoxic T-lymphocytes
- inhibitory receptors
- NK cells
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 . 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)” . 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) . 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 . The cytoplasmic c-terminal tail of naturally-occurring HLA-I OCs is tyrosine phosphorylated and plays a role in signal transduction .
|HLA Class I|
|PROFILES OF HLA TYPING: HLA ISOFORMS AND THEIR ALLELES|
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” . 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].
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 ; 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 . 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) . 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 , 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 . 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 . 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 CANCER||COMMERCIAL mAbs||REFERENCES|
|Melanoma Cervical Cancer||3D12||Marín R et al. Immunogenetics. 54(11):767–75.2003 |
|Melanoma||MEM-E/02||Derré L et al. J Immunol. 177:3100–7. 2006. |
|Melanoma and other cancers||MEM-E/07||Allard M et al. PLoS One 6(6):e21118, 2011 |
|Lip squamousal cell carcinoma||MEM-E/02||Goncalves et al. Human Immunol. 77(9): 785–790, 2016 |
|Laryngeal carcinoma||MEM-E/02||Silva TG et al. Histol Histopathol. 26:1487–97. 2011 |
|Vulvar intraepithelial carcinoma||MEM-E/02||van Esch EM et al. Int J Cancer. 135(4): 830–42, 2014 |
|Penile Cancer||MEM-E/02||Djajadiningrat et al. J Urol. 193(4):1245–51. 2015. |
|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 |
|Glioblastomas||MEM-E/02||Kren L et al. Neuropathology. 31: 129–34. 2011 |
|Glioblastomas stem cells||3D12||Wolpert et al. J Neuroimmunol. 250(1–2):27–34 2012 |
|Glioblastomas||3D12||Wischhusen J et al. J Neuropathol Exp Neurol. 64:523–8. 2005 |
|Neuroblastoma||3H2679||Zhen et al. Oncotarget. 7(28): 44340–44349, 2016. |
|Neuroblastoma||3D12||Morandi et al. J Immunol Res. 2016:7465741, 2016. |
|Oral Osteosarcoma||MEM-E/02||Costa Arantes et al. Oral Surg Oral Med Oral Pathol Oral Radiol. 123(6):e188-e196. 2017. |
|Intraoral mucoepidermoid carcinoma||MEM-E/02||Mosconi C Arch Oral Biol. 83:55–62, 2017. |
|Rectal Cancer||MEM-E/02||Reimers et al. BMC Cancer BMC Cancer. 14:486.1–12, 2014. |
|Colorectal carcinoma||MEM-E/08||Levy et al. Int J Oncol. 32(3): 633–41. 2008 |
|Colorectal carcinoma||MEM-E/08||Levy et al. Innate Immun. 15(2):91–100. 2009. |
|Colorectal carcinoma||MEM-E/02||Benevolo M, et al. J Transl Med. 9:184. 2011. |
|Colorectal carcinoma||MEM-E/02?||Bossard C et al. Int J Cancer. 131 (4): 855–863. 2012. |
|Colorectal carcinoma||MEM-E/02?||Zhen et al., Med Oncol. 30(1):482. 2013. |
|Colorectal carcinoma||MEM-E/02||Zeestraten et al. Br J Cancer. 110(2):459–68. 2014. |
|Colorectal carcinoma||MEM-E/02||Guo et al. Cell Immunol. 293(1):10–6, 2015. |
|Colorectal carcinoma||3H2679||Ozgul Ozdemir et al. Ann Diagn Pathol. 25:60–63, 2016 |
|Colorectal carcinoma||MEM-E/02||Huang et al. Oncol Lett. 13(5):3379–3386, 2017. |
|Colon carcinoma and leukemia (K562)||MEM-E/06||Stangl S et al. Cell Stress Chaperones. 13(2):221–30. 2008. |
|Colon carcinoma||MEM-E/02||Zeestraten EC et al. Br J Cancer. 110(2): 459–68.2014. |
|Hepatocellular carcinoma||MEM-E/02||Chen et al. Neoplasma. 58(5):371–376, 2011. |
|Non-small cell Lung Carcinoma||MEM-E/02||Talebian-Yazdi et al. Oncotarget. 7(3):3477–3488, 2016. |
|Breast cancer||MEM-E/02||de Kruijf EM et al. J Immunol. 185:7452, 2010 |
|Breast cancer||MEM-E/02||da Silva et al. Int J Breast Cancer. 2013:250435. 2013. |
|Ovarian cancer/ Cervical cancer||MEM-E/02||Gooden M et al.PNAS USA 108:10656, 2011. |
|Cervical cancer||MEM-E/02||Gonçalves MA et al. Eur J Obstet Gynecol Reprod Biol. 141:70–4. 2008. |
|Cervical cancer||MEM-E/02||Spaans VM et al., J Transl Med. 10:184. 2012. |
|Cervical squamous and adenocarcinoma||MEM-E/02||Ferns et al. J Immunother Cancer. 4:78, 2016. |
|Serous Ovarian Adenocarcinoma||MEM-E/02||Andersson et al. Oncoimmunology, 25;5(1):e1052213, 2015. |
|Serous Ovarian Adenocarcinoma||MEM-E/02||Zheng et al. Cancer Sci. 106(5): 522–528, 2015. |
|Renal Cell Carcinoma||MEM-E/02||Hanak L et al. Med Sci Monit. 15(12):CR638–43.2009. |
|Renal Cell Carcinoma||MEM-E/02||Kren L et al., Diagnostic Pathology, 7:58, 2012 |
|Thyroid cancer||MEM-E/02||Zanetti et al. Int J Immunopathol Pharmacol. 26(4):889–96, 2013. |
|Hodgkin Lymphoma||MEM-E/02||Kren L, et al., Pathology, Research and Practice 208: 45–49, 2012. |
|Chronic Lymphocytic Leukemia||3D12||McWilliams et al., Oncoimmunology. 5(10):e1226720, 2016. |
|Chronic Lymphocytic Leukemia||3D12||Wagner et al. Cancer, 23(5):814–823, 2017. |
|Many Cancers||3D12||Sensi M, et al. Int Immunol. 21(3):257–268. 2009. |
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 ).
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 Ia||HLA Class Ib|
|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|
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 mAbs||mAb specificity||number of mAbs||Examples of TFL mAbs||Antigen (heavy chain only) tested on beads||Subclass||HLA-E||HLA-F||HLA-G||HLA-A||HLA-B||HLA-C|
|Reactivity in MFI|
|A||Antigen immunized: β2-microglobulin-free heavy chain of HLA-ER107|
|HLA-E Monospecific mAbs (Category 1)||HLA-E||16||TFL-145, TFL-33, TFL, 34, TFL-73, TFL-74||HLA-ER||IgG1||4 K–22 K||0||0||0||0||0|
|3||TFL-001||HLA-ER||IgG2a||0.9 K - 4 K||0||0||0||0||0|
|Antigen immunized: β2-microglobulin-free heavy chain of HLA-EG107|
|B||Antigen 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 mAbs||1||TFL-050||HLA-ER||IgG2b||4 K||3 K||2 K||0||0||0|
|Antigen immunized: β2-microglobulin-free heavy chain of HLA-EG107|
|3||TFL-208, TFL-209, TFL-223,||HLA-EG HLA-ER||IgG1||21 K||8 K||20 K||0||0||0|
|4||TFL-164||HLA-EG||IgG2b||14 K–15 K||8 K–9 K||24 K–25 K||0||0||0|
|E + G+||1||TFL-191||HLA-EG||NK||1 K||0||1 K||0||0||0|
|E + F+||1||TFL-228||HLA-EG||IgG1||19 K||1 K||0||0||0||0|
|C||Antigen immunized: β2-microglobulin-free heavy chain of HLA-ER107|
|HLA-IA Polyreactive HLA-E mAbs (Categroy 3)||E + B + C+||31||TFL-059||HLA-EG HLA-ER||IgG1 (n = 12) IgG2A (n = 9) IgG2b (n = 9) IgG3 (n = 1)||8 K–20 K||0||0||0||1 K–17 K||1 K–7 K|
|E + A + B + C+||68||TFL-119||HLA-EG HLA-ER||IgG1 (n = 27) IgG2A (n = 23) IgG2b (n = 17) IgG3 (n = 1)||11 k–22 k||0||0||1 K–4 K||1 K–24 K||1 K–13 K|
|EG + B+||3||TFL-173||HLA-EG||IgG1||12 K||0||0||0||1 K||0|
|E + B+||1||TFL-219||HLA-EG/R||IgG1||21||0||0||0||2 K||0|
|EG + A + B + C+||6||TFL-167||HLA-EG||IgG1||15 K-25||0||0||1 K–9 K||1 K 20 K||1 K–20 K|
|E + A + B + C+||35||TFL-243||HLA-EG/R||IgG1 (n = 22) IgG2A (n = 6) IgG2b (n = 6) IgG3 (n = 1?)||13 K–26 K||0||0||1 K–9 K||1 K–24 K||1 K–20 K|
|Nature of mAbs||Immunogen used||mAb specificity||number of mAbs||Examples of TFL mAbs||Subclass||HLA-E||HLA-F||HLA-G||HLA-A||HLA-B||HLA-C|
|Reactivity in MFI|
|D. Category 4. HLA- IA and IB polyreactive anti-HLA-E mAbs. (n = 36)|
|HLA = IA Polyreactive HLA-IB mAbs (Category 4)||HLA-EG||E+/F+/G+||4||TFL-232||IgG3||13–22||21||2 to 10||11 to 21||1 to 13||1 to 20||1 to 20|
|TFL-177||IgG1 (n = 3)||0|
|E+/G+||16||TFL-236||IgG1 (n = 14)||18–22 (n = 13)||0||0||18–22 (n = 11)||1 to 9||1 to 25||1 to 24|
|E+/F+||10||TFL-210||IgG1 (n = 10)||18–21||17–19||5 to 11||0||1 to 15||1 to 20||1 to 22|
|HLA-ER||E+/F+/G+||3||TFL-049||IgG2b||15–22||8 To 12||2 to 7||1 to 10||1 to 10||1 to 17|
|TFL-006||IgG2a (n = 2)|
|E+/G+||2||TFL-103||IgG1 (n = 2)||17,18||0||4||1 to 6||1 to 11||1 to 11|
|E+/F+||1||TFL-063||IgG2b||22||3||0||2-Jan||1 tp 7||3 to 8|
5.5 Unique (private) and common (public) epitopes of HLA-E
The international immunogenetics project (
|Comparison of the amino acid sequences of HLA-E with other HLA-I antigens|
|HLA-E peptide sequences||Number of amino acids||Classical HLA-Ia||Non-classical HLA-Ib||Specificity|
|Peptide [# 1] specific for HLA-E||Peptide [# 2] specific for HLA-E|
|HLA Class Ib||α1||α1||α1||α1||α1||α1||HLA Class Ib||α2||α2||α2||α2||α2||α2||α2||α2||α2|
|Qa-1(murine eq:HLA-E)||W||K||A||R||D||M||Qa-1(murine eq:HLA-E)||S||K||H||K||S||E||A||V||D|
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.
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.
|Protein-G purified Culture supernatant||(1:10)||4424||2730||1974||2507|
|Protein-G purified Culture supernatant Concentrate||(1:10)||11953||10364||7708||8467|
|Protein-G purified Ascites Concentrate (Eluate # 2)||(1:50)||17898|
Detailed immunodiagnostic analyses were performed using a tissue microarray (TMA) of normal gastric mucosal and primary gastric cancer tissues . 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 . 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 . 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).
126.96.36.199 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 . 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/CD8||CD3+ NAÏVE T-CELLS||CD3+ LYMPHOBLASTS|
|No PHA||With PHA||No PHA||With PHA|
|No mAb [n = 5]|
|2-tail p [<]||<0.0001||0.001||0||NS||0|
|mAb TFL-033 (IgG1) [n = 3]|
|2-tail p [<]||NS||NS||NS||0.009||NS||0.015||NS||0.005||0.010||0.016||NS||0.014|
|2-tail p [<]||NS||NS||NS||NS||0.047||0.001||0.020||NS||0.001||0.003||NS||0.001|
|mAb TFL-007 (Polyreactivec anti-HLA-E, IgG2a) [n = 3]|
|2-tail p [<]||NS||NS||NS||NS||NS||NS||NS||NS||0.027||NS||NS||NS|
|2-tail p [<]||NS||0.018||0.013||NS||NS||NS||0.019||0.006||NS||NS||NS||NS|
188.8.131.52 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 . 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 cell
s 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.
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.
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.
- 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).