**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, 35–39, 96–98]. The results are comparable to assays such as ELISA but with greater specificity, sensitivity and resolution. The technology employs superparamagnetic 6.5-micron microspheres with a magnetic core and polystyrene surface. The beads are

#### *Monoclonal Antibodies*

[96–98]. **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,

*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.*

*Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-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 Lglutamine, 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**

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

TFL-006) (See **Table 7** in [99]).

**Figure 5.**

**(SAB) assay**

**51**

**5.2 Development of mAbs against HLA-E**

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

#### **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.*

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 antigencoated beads allows it to be evaluated for the mono- or polyreactivity of mAb

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

#### **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.*

[96–98]. **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]).

#### **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 Lglutamine, 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

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 antigencoated beads allows it to be evaluated for the mono- or polyreactivity of mAb

*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*

**Figure 4.**

*Monoclonal Antibodies*

**50**

*conclude on the expression of HLA-E on human cancers.*

(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 [35–37, 96–100]. **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

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) [35–37, 96–100]. 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**).


#### **Table 4.**

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

**Nature of mAbs**

**53**

**A** **HLA-E**  **(Category 1)**

**Monospecific**

 **mAbs**

**HLA-E**

16

 TFL-145, TFL-

33, TFL, 34, TFL-73, TFL-74

3

 TFL-001 TFL-016

TFL-013

**Antigen** 

5

 TFL-185 TFL-184

TFL-186

TFL-226

TFL-254

**Antigen** 

**immunized:**

**β2-microglobulin-free**

 **heavy chain of HLA-ER107**

> **B**

**HLA-IB**  **HLA-IA and**  **HLA-E mAbs (Category 2)**

**polyreactive**

 **and**

**HLA-Ib**

1

 TFL-050

 HLA-ER

IgG2b

4 K

 3 K

 2 K

 0

 0

 0

**specific**

**mAbs**

**Antigen** 

3

 TFL-208, TFL-

209, TFL-223,

4

 TFL-164 TFL-165

TFL-162

TFL-161

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

1

 TFL-228

 HLA-EG

1

 TFL-191

 HLA-EG

NK IgG1

19 K

 1 K

 0

 0

 0

 0

1 K

 0

 1 K

 0

 0

 0

 HLA-EG

IgG2b

14 K–15 K 8 K–

24 K–

000

9 K

25 K

**immunized:**

**β2-microglobulin-free**

 **heavy chain of HLA-EG107**

> HLA-EG HLA-ER

IgG1

21 K

 8 K

 20 K

 0

 0

 0

**non-reactive**

 HLA-EG

IgG1

19 K

 0

 0

 0

 0

 0

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

**immunized:**

**β2-microglobulin-free**

 **heavy chain of HLA-EG107**

 HLA-ER

IgG2a

0.9 K - 4 K

 0

 0

 0

 0

 0

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

**mAb**

**number**

**Examples of**

**Antigen (heavy**

**Subclass**

**HLA-E**

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

**Reactivity in MFI**

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

**specificity**

**of mAbs**

**Antigen** 

**immunized:**

**β2-microglobulin-free**

 **heavy chain of HLA-ER107**

> HLA-ER

IgG1

4 K–22 K

 0

 0

 0

 0

 0

**TFL mAbs**


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

(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 [35–37, 96–100]. **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

**5.4 The diversity anti-HLA-E mAbs**

*Monoclonal Antibodies*

**Table 4.**

**52**

*heavy chain of HLA-ER107 & HLA-EG107.*

nizing both HLA-Ib and HLA-Ia molecules (**Table 5**, **D**).

**HLA Class Ia HLA Class Ib HLA-A HLA-B HLA-C HLA-E HLA-F HLA-G**

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) [35–37, 96–100]. 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.

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 recog-

**mAbs formed after immunizing HLA-E**

Group 1 () () () **(+)** () () 24 TFL-monospecific anti-HLA-E

Group 6 **(+) (+) (+) (+) (+)** () Reactivity of the mAb MEM-E/06

Group 7 **(+) (+) (+) (+)** () **(+)** Reactivity of the mAb MEM-E/08

Group 8 **(+) (+) (+) (+) (+) (+)** Reactivity of the mAb TFL-006,

*The diverse HLA-E monospecific and polyreactive mAbs generated after immunizing mice with a recombinant*

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-

mAbs

E/02 & MEM-E/07 & TFL series

& TFL-series

& TFL series

TFL-007 & other TFL mAbs


**Nature of mAbs**

**55**

**mAb**

**number**

**Examples of**

**Antigen (heavy**

**Subclass**

**HLA-E**

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

**Reactivity in MFI**

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

TFL-168

TFL-205

**E+A+B+**

35

 TFL-243 TFL-246

TFL-244

TFL-245

TFL-172

TFL-171

**Nature of mAbs**

**Immunogen**

**mAb**

**number of**

**Examples of**

**Subclass**

**HLA-E**

**ER107**

**EG107**

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

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

**Reactivity in MFI**

**mAbs**

**TFL mAbs**

**used**

**specificity**

**D. Category 4. HLA- IA and IB** 

> **HLA = IA**

**mAbs (Category 4)**

**Polyreactive**

 **HLA-IB**

**HLA-EG**

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

4

TFL-232 TFL-177

TFL-176

TFL-198

> **E+/G+**

> 16

TFL-236

TFL-238

TFL-256 TFL-229

> **E+/F+**

10

TFL-210

 IgG1 (n = 10)

 18–21

 17–

19

IgG2b

30

 22

18

*…*

IgG3

22

 27

1

 IgG1 (n = 14)

 18–22

0

 0

 18–22

> (n = 11)

> (n = 13)

 IgG1 (n = 3)

IgG3

13–22

0

0

**polyreactive**

 **anti-HLA-E**

 **mAbs. (n = 36)**

 HLA-EG/R

 IgG1 (n = 22) IgG2A

13 K–26 K

 0

 0

 1 K–

1 K–

1 K–

> 9 K

> 24 K

20 K

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

(n = 6) IgG2b (n = 6)

IgG3 (n = 1?)

**C+**

**specificity**

**of mAbs**

**TFL mAbs**

#### *Monoclonal Antibodies*


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

**Nature of mAbs**

**54**

**C** **HLA-IA**  **mAbs (Categroy 3)**

**Polyreactive**

 **HLA-E**

**E + B + C+**

31

 TFL-059 TFL-143

TFL-158

TFL-076

TFL-159

**E+A+B+**

68

 TFL-119 TFL-142

TFL-153

TFL-118

TFL-133

TFL-141

TFL-095

**EG + B+**

3

 TFL-173 TFL-174

TFL-175

**E + B+**

> **EG**

**+A+B+**

6

 TFL-167 TFL-170

TFL-169

TFL-166

 HLA-EG

**C+**

1

 TFL-219

 HLA-EG/R

IgG1 IgG1

15 K-25

 0

 0

 1 K–

1 K

1 K–

> 9 K

> 20 K

> 20 K

21

 0

 0

 0

 2 K

 0

 HLA-EG

IgG1

12 K

 0

 0

 0

 1 K

 0

 HLA-EG

IgG1 (n = 27) IgG2A

11 k–22 k

 0

 0

 1 K–

1 K–

1 K–

> 4 K

> 24 K

13 K

HLA-ER

(n = 23) IgG2b (n = 17) IgG3 (n = 1)

**C+**

 HLA-EG

IgG1 (n = 12) IgG2A

8 K–20 K

 0

 0

 0

 1 K–

1 K–7 K

*Monoclonal Antibodies*

17 K

HLA-ER

(n = 9) IgG2b (n = 9)

IgG3 (n = 1)

**mAb**

**number**

**Examples of**

**Antigen (heavy**

**Subclass**

**HLA-E**

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

**Reactivity in MFI**

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

**specificity**

**of mAbs**

**Antigen** 

**immunized:**

**β2-microglobulin-free**

 **heavy chain of HLA-ER107**

**TFL mAbs**


#### **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.*

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

shows HLA-E restricted amino acid sequences found in

from the initial concentration of 100

**Figure 6.**

**57**

The mAbs were further diluted with 14

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

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**

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

used for peptide inhibition assays. **Figure 7A** illustrates locations of private and public epitopes. **Figure 7B** shows allele-specific amino acid sequences in

Peptide inhibition analyses were performed to confirm the monospecificity of HLA-E mAbs. Various concentrations of HLA-E-restricted peptides (serially diluted

μL to 100

*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.*

helical groove and **Figure 7C** shows shared peptide amino acid sequences.

α1 and

μL) were added to the mAbs (7

μL PBS-BSA (pH 7.0; final dilution 1/1200),

α2 helices, which were

α1 & α 2

μL).

*…*
