EXPB6 Antibody

Introduction to Antibodies

Antibodies, also known as immunoglobulins (Ig), are large, Y-shaped proteins utilized by the immune system to identify and neutralize foreign objects such as bacteria and viruses . These proteins, part of the immunoglobulin superfamily, are crucial for adaptive immunity .

Key Structural Features:

• Composed of two heavy chains and two light chains linked by disulfide bonds .

• Each chain contains a series of domains, approximately 110 amino acids each .

• Light chains have one variable domain (VL) and one constant domain (CL), while heavy chains have one variable domain (VH) and three to four constant domains (CH1, CH2, etc.) .

• The antigen-binding fragment (Fab) contains one VL, VH, CL, and CH1 domain .

• The crystallizable fragment (Fc) forms the trunk of the Y shape .

• A hinge region between the Fab and Fc fragments allows flexibility for binding to epitopes at various distances .

EphB6 and Antibody Development

Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the Eph subfamily of receptor tyrosine kinases . EphB6 is expressed in various tissues and regulates cellular homeostasis by interacting with membrane-bound ephrin ligands and other receptors . Although lacking kinase activity, EphB6 is involved in cancer pathology, creating a demand for sensitive monoclonal antibodies (mAbs) for its treatment, diagnosis, and further analysis .

Development of Anti-EphB6 Monoclonal Antibodies
A novel specific and sensitive anti-human EphB6 mAb clone, Eb6Mab-3 (mouse IgG1, kappa), was developed using the Cell-Based Immunization and Screening (CBIS) method . This method allowed for the creation of antibodies with high affinity and specificity for EphB6-positive cells .

Hybridoma Production:

1. Two 6-week-old female BALB/cAJcl mice were immunized intraperitoneally with LN229/EphB6 cells .

2. Alhydrogel adjuvant 2 % was added as an adjuvant in the first immunization .

3. Three additional injections of LN229/EphB6 cells were administered intraperitoneally without adjuvant every week .

4. A final booster injection was performed two days before harvesting splenocytes .

5. Cell fusion of splenocytes with P3U1 cells was conducted using polyethylene glycol 1500 (PEG1500) .

6. Hybridomas were cultured in RPMI-1640 medium with hypoxanthine, aminopterin, and thymidine (HAT), 5 % BriClone, and 5 μg/mL of Plasmocin .

7. Hybridoma supernatants were screened by flow cytometry using CHO/EphB6 and parental CHO–K1 cells .

CBIS Method
The CBIS method was employed to develop anti-EphB6 mAbs using EphB6-overexpressed cells .

1. Mice were immunized with LN229/EphB6 cells .

2. Mouse splenocytes and P3U1 cells were fused using PEG1500 .

3. Hybridomas were seeded into 96-well plates .

4. Flow cytometric screening was conducted to select CHO/EphB6-reactive and parental CHO–K1-nonreactive supernatants .

5. The highly sensitive clone Eb6Mab-3 (mouse IgG1, kappa) was established by limiting dilution and additional analysis .

Functional Evaluation of Eb6Mab-3

Eb6Mab-3 was evaluated for its reactivity and binding affinity using flow cytometry and Western blot analyses .

Flow Cytometry Analysis
Flow cytometric analysis was conducted using Eb6Mab-3 and a commercially available anti-EphB6 mAb (T49-25) against CHO–K1, CHO/EphB6, and DLD-1 cells .

• Eb6Mab-3 and T49-25 recognized CHO/EphB6 dose-dependently .

• Reactivity was almost the same between Eb6Mab-3 and T49-25 to CHO/EphB6 .

• Neither Eb6Mab-3 nor T49-25 reacted with parental CHO–K1 cells .

• Eb6Mab-3 showed slightly higher reactivity than T49-25 at 1 μg/mL of mAbs to DLD-1 .

• Reactivity was saturated at more than 10 μg/mL .

• Eb6Mab-3 can detect exogenously and endogenously expressing EphB6 in flow cytometry .

Binding Affinity
Eb6Mab-3 demonstrated a moderate binding affinity for CHO/EphB6 (KD: 2.6 ± 1.0 × 10−8 M) and a high binding affinity for DLD-1 (KD: 3.4 ± 1.3 × 10−9 M) .

Western Blot Analysis
Eb6Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in Western blot .

Clinical Trials and Therapeutic Applications

While specific information on clinical trials for "EXPB6 Antibody" is not available, other antibodies targeting similar pathways have been explored in clinical settings. For example, BG00011, an anti-αvβ6 IgG1 monoclonal antibody, was evaluated for the treatment of idiopathic pulmonary fibrosis (IPF) .

BG00011 Clinical Trial
A phase IIb randomized, double-blind, placebo-controlled trial was conducted to evaluate the efficacy and safety of BG00011 in patients with IPF .

• Patients with IPF were randomized 1:1 to receive once-weekly subcutaneous BG00011 56 mg or placebo .

• The primary endpoint was FVC change from baseline at Week 52, but endpoints were evaluated at Week 26 due to early trial termination .

• There was no significant difference in FVC change from baseline between patients who received BG00011 or placebo at Week 26 .

• Patients in the BG00011 group showed a worsening trend after Week 26 .

• IPF exacerbation/or progression was reported in 13 patients, all in the BG00011 group .

• Serious adverse events occurred more frequently in BG00011 patients, including four deaths .

• The results did not support the continued clinical development of BG00011 .

Potential Therapeutic Applications of Monoclonal Antibodies

Monoclonal antibodies have shown promise in various therapeutic applications, including the treatment of Long COVID symptoms .

Monoclonal Antibodies in Long COVID Treatment
A study reported dramatic recovery from severe Long COVID symptoms in three patients who received monoclonal antibody therapy . The proposed mechanisms include:

1. Neutralization of persistent SARS-CoV-2, improving the immune system .

2. Displacement of autoantibodies from patients’ cells .

3. Binding to molecules on immune cells to eliminate viral particles or infected cells .