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

The international immunogenetics project (http://www.ebi.ac.uk; or http://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.

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),


#### **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.*

**Nature of mAbs**

**56**

**mAb**

**number**

**Examples of**

**Antigen (heavy**

**Subclass**

**HLA-E**

 **HLA-F HLA-G HLA-A HLA-B HLA-C**

**Reactivity in MFI**

*Monoclonal Antibodies*

**chain only)** **tested on beads**

TFL-211

TFL-212

TFL-235

**HLA-ER**

**E+/F+/G+**

3

TFL-049 **TFL-006** **TFL-007**

**E+/G+** **E+/F+**

> *mAbs in Bold are highly polyreactive,*

**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.*

1

TFL-063

IgG2b

22

 3

 0

 2-Jan

2

TFL-103

TFL-104

 IgG1 (n = 2)

17,18

 0

 4

IgG2a (n = 2)

IgG2b

15–22

**specificity**

**of mAbs**

**TFL mAbs**


**Comparison of the amino acid sequences of HLA-E with other HLA-I antigens**

#### **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.*

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.
