**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

**Peptide [# 1] specific for HLA-E**

**59**

**HLA Class Ib**

**α1** **65**

*R*

*R*

**E\*01010101** **G\*01010101**

**F\*01010101**

**A\*110101**

**B \*1401**

**B** **B** **B**

**B \*5801** **CW\*050101** **CW\*080101**

**CW\*1802** **Qa-1(murine**

**Table 7.** *Identifying*

 *HLA-E specific epitope or amino acid sequences: Comparing*

 **eq:HLA-E)**

W

 K

**A**

 **R**

D

 M

 *the two HLA-E restricted sequences with other HLA-I amino acid sequences at the same position.*

**Qa-1(murine**

 **eq:HLA-E)**

 **S**

K

 H

**K**

 **S**

E

 A

 VD

**\*530101**

**\*40060101**

**\*350101**

**R** Q

Q

Q

Q

 I

RNMK

QKYKRQ

QKYKRQ

QKYKRQ

**A**

S

**B \*5801** **CW\*050101** **CW\*080101**

**CW\*1802**

FKTN

**B**

**\*530101**

 I

SKTN

**B**

**\*40060101**

 I

FKTN

**B**

**\*350101**

 I

CKTN

**B \*1401**

NVK

**A**

Q

**A\*110101**

 **66**

 **67**

 **68** *SARDT*

NTKAH

GYAKAN

**E\*01010101** **G\*01010101** **F\*01010101**

**S**

K

TQR

TKR

TQR

TQR

TQR

TQR

TQR

TQR

TQR

TQR

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

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

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

R

**K**

WE

 A

**A**

H

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

 R

**K**

CEA

FYEAEE

**A**

N

*S*

*EQK*

 **69**

 **70**

**α1**

**α1**

**α1**

**α1**

**α1**

 **HLA Class Ib**

**Peptide [# 2] specific for HLA-E**

> **α2**

**143**

 **144**

 **145**

 **146**

 **147**

 **148**

 **149** *SNDAS*

 **150**

 **151**

**α2**

**α2**

**α2**

**α2**

**α2**

**α2**

**α2**

**α2**


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

> **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.*

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

*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.*

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

> **Classical HLA-Ia**

**A B Cw F G**

**Nonclassical HLA-Ib**

**Specificity**

3306 restricted

Multispecific

8201-restricted

**Number of amino acids**

47PRAPWMEQE55 9 10000A\*

59EYWDRETR65 8 5 0 0 0 0 A-restricted **65RSARDTA71** 6 **0 0 0 0 0 E-monospecific** 90AGSHTLQW97 8 1 10 48 0 0 Multispecific 108RFLRGYE123 7 24 0 0 0 0 A-restricted 115QFAYDGKDY123 9 1 104 75 0 0 Multispecific 117AYDGKDY123 7 491 831 271 21 30 Highly

126LNEDLRSWTA135 10 239 219 261 21 30 Multispecific 137DTAAQI142 6 0 824 248 0 30 Multispecific 137DTAAQIS143 7 0 52 4 0 30 Multispecific **143SEQKSNDASE152** 10 **0 0 0 0 0 E-monospecific**

163TCVEWL168 6 282 206 200 0 30 Multispecific 182EPPKTHVT190 8 0 0 19 0 0 C-restricted

157RAYLED162 6 01000 B\*

**HLA-E peptide sequences**

*Monoclonal Antibodies*

**Table 6.**

**58**

epitopes. The level of inhibition differed between the two epitopes.

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

**5.6 Diagnostic potential of HLA-E monospecific mAbs**

#### **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 allelespecific amino acid sequences (private epitopes) in α1 & α2 helical groove and (C) shared peptide amino acid sequences (public epitopes).*

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

*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.*

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

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

monospecific mAbs are reliable for diagnosis and therapeutic purposes.

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

**versus polyreactive mAbs**

**Figure 8.**

**61**

**5.7 Differences in the immunoregulatory potentials of HLA-E monospecific**

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

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**.

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

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

#### **Figure 8.**

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

*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 allelespecific amino acid sequences (private epitopes) in α1 & α2 helical groove and (C) shared peptide amino acid*

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 adeno-

carcinomas, 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

immunizing HLA-EG, is presented in **Figure 9D**.

**Figure 7.**

**60**

*sequences (public epitopes).*

*Monoclonal Antibodies*

*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.*

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


#### **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.*

both HLA-Ia and HLA-Ib reactivities [16, 17]. IVIg preparations were reported to suppress CD4+ T cells [102–113], CD20+ B cells [108–113] and expand CD4 + CD25+ T-regs [114, 115]. The polyreactive anti-HLA-E mAbs performed the major immunoregulatory functions better than IVIg [101, 116–118]. 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**).

**Figure 9.**

*10.1002/ijc.28484.*

**63**

*Immunolocalization of HLA-E in cancer tissues with culture supernatants (s) or ascites (A) of TFL*

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

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

*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:*

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

#### **Figure 9.**

both HLA-Ia and HLA-Ib reactivities [16, 17]. IVIg preparations were reported to suppress CD4+ T cells [102–113], CD20+ B cells [108–113] and expand CD4 + CD25+ T-regs [114, 115]. The polyreactive anti-HLA-E mAbs performed the major immunoregulatory functions better than IVIg [101, 116–118]. 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

*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*

*(1:50)* 17898 *(1:100)* 16246 *(1:200)* 14004 *(1:400)* 12520

**Sample Dilution TFL-033 TFL-034 TFL-073 TFL-074** Culture Supernatant *Neat* 11273 11601 7781 8493 Protein-G purified Culture supernatant *(1:10)* 4424 2730 1974 2507

> *(1:10)* 11953 10364 7708 8467 *(1:20)* 9423 8146 6861 7500 *(1:40)* 8167 6347 5324 5883 *(1:80)* 6203 4622 3792 4176 *(1:160)* 4139 1379 2683 2438 *(1:320)* 2862 626 1454 943 *(1:640)* 1434 198 590 474 *(1:1280)* 694 98 275 220

Protein-G purified Culture supernatant

Protein-G purified Ascites Concentrate

Concentrate

*Monoclonal Antibodies*

(Eluate # 2)

*effects on T-lymphoblasts.*

**Table 8.**

117AYDGKDYLT125, 126LNEDLRSWTAV136, and 137DTAAQI142) (**Figure 7C**). Possibly such binding affinity of polyreactive but not monospecific mAbs contributes to

In contrast to polyreactive anti-HLA-E mAb, monospecific mAbs (TFL-033)

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

and TFL-007 bind to shared amino acid sequences of HLA-I molecules

the unique immunoregulatory functions mimicking IVIg [101, 118].

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*

*5.7.2 Therapeutic potential of anti-HLA-E monospecific mAbs*

(

**62**

*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.*


**Presence or absence of**

**65**

**CD3+ NAÏVE T-CELLS**

**No PHA**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

*CD4-/*

**CD4+/**

**CD4-/**

**CD8-**

3,088

65

*NS*

 *0.018*

 *0.013*

 *NS*

 *NS*

 *NS*

 *0.019*

 *0.006*

 *NS*

 *NS*

 *of CD4+ T* 

*lymphoblasts*

 *occurs only after*

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

*…*

 *NS*

 *NS*

 16

 55

 48

 23

*7*

17

 4

 26

*18*

8

 21

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

 667

 1,075

 491

 230

*107*

193

 80

 892

*443*

122

 339

Mean

SD

2-tail p [<]

**Table 9.** *TFL-033 promotes* 

*PHA activation.*

*T-lymphoblast*

 *proliferation*

 *of CD8+ naïve T cells and* 

*T-Lymphoblasts*

 *in the absence or the presence of PHA. The proliferation*

**CD8+**

**CD8-**

**CD8+**

**CD8-**

**CD8+**

**CD8+**

**CD8-**

**CD8-**

*CD8+*

**CD8+**

**CD8-**

**With PHA**

**No PHA**

**CD3+** 

**LYMPHOBLASTS**

**With PHA**

**CD4/CD8**

*Monoclonal Antibodies*


**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 proliferationPHAactivation.*

 *of CD4+ T* 

*lymphoblasts*

 *occurs only after*

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

**Presence or absence of**

**64**

**CD3+ NAÏVE T-CELLS**

**No PHA**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

**CD4-/**

**CD4+/**

*CD4-/*

**CD4+/**

**CD4-/**

*Monoclonal Antibodies*

**CD8-**

No mAb *[n = 5]*

Mean

SD 2-tail p [<]

**mAb TFL-033** (IgG1) [n = 3]

[1/30]

Mean

SD 2-tail p [<]

[1/150]

Mean

SD 2-tail p [<]

**mAb TFL-007**

[1/10]

Mean

SD 2-tail p [<]

[1/50]

2876

136

*NS*

 *NS*

 *NS*

 *NS*

 *NS*

 *NS*

 *NS*

 *NS*

 *0.027*

 *NS*

 *NS*

 *NS*

 72

 19

 26

 33

*2*

3

 17

 79

*25*

4

 29

 451

 1183

 444

 164

*63*

145

 52

 676

*317*

100

 222

(Polyreactivec

 anti-HLA-E,

 IgG2a) [n = 3]

3238

14

*NS*

 *NS*

 *NS*

*NS*

*0.047*

*0.001*

*0.020*

 *NS*

*0.001*

 *0.003*

*NS*

 *0.001*

 64

 21

 22

 30

*17*

13

 9

 80

*31*

14

 16

 681

 1149

 508

 252

*120*

205

 68

 1266

*572*

157

 412

3185

180

*NS*

 *NS*

 *NS*

*0.009*

*NS*

*0.015*

*NS*

 *0.005*

*0.010*

 *0.016*

*NS*

 *0.014*

 146

 58

 12

 40

*27*

80

 13

 86

*23*

16

 20

 755

 1170

 536

 223

*163*

153

 99

 1129

*505*

152

 412

3063

149

 86

 99

> *<0.0001*

 37

 33

*14*

35

 15

 115 *0.001*

*0*

 *NS*

 *0*

*126*

43

 84

 547

 1249

 475

 197

*65*

141

 52

 867

*325*

128

 289

**CD8+**

**CD8-**

**CD8+**

**CD8-**

**CD8+**

**CD8+**

**CD8-**

**CD8-**

*CD8+*

**CD8+**

**CD8-**

**With PHA**

**No PHA**

**CD3+** 

**LYMPHOBLASTS**

**With PHA**

**CD4/CD8**

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.

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

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

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

activity of CD8+ cytotoxic T lymphocytes (CTLs) and NKT cells.

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

anti-HLA-E may be potentially considered for anti-cancer NK therapy.

tool, than those binding to specific but cryptic epitopes.

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

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

**6. Conclusion**

**67**
