EMP2 Antibody, Biotin conjugated

What is EMP2 and why is it a significant target in cancer research?

EMP2 (Epithelial Membrane Protein-2) is a tetraspan protein that has emerged as a promising biomarker and therapeutic target in multiple cancer types. Research indicates that EMP2 is minimally expressed in normal mammary tissue but is significantly upregulated in various cancers, particularly breast cancer. According to studies, EMP2 protein is upregulated in 63% of invasive breast cancer tumors and in 73% of triple-negative breast cancer (TNBC) tumors . This differential expression pattern makes EMP2 an attractive target for cancer therapy.

The significance of EMP2 as a research target is further supported by studies showing that it positively correlates with advanced disease stages and can identify circulating breast tumor cells . Recent gene signature analyses have identified upregulation of EMP2 mRNA in breast cancers, providing supporting evidence of its dysregulated expression in this malignancy . EMP2's location on the cell membrane makes it particularly accessible for antibody-based targeted therapies, allowing for potential therapeutic interventions with minimal effects on normal tissues.

How is EMP2 expression characterized in different breast cancer subtypes?

When examining breast cancer cell lines, EMP2 has been detected in 8 out of 9 human cell lines tested by Western blot analysis, with expression below detection in only one cell line (HS578t) . Importantly, EMP2 expression does not strictly correlate with hormone receptor status, as it is present in both triple-negative (MDA-MB-231, MDA-MB-468, and BT-20) and triple-positive (ZR-75-1) cells . Additionally, high EMP2 expression has been observed in all HER2/neu-positive cells tested (UACC812, SK-BR-3, BT-474) .

The characterization methodology typically involves:

• Tissue microarray analysis with immunohistochemistry

• Western blot analysis of cell lysates using anti-human EMP2 antisera

• Flow cytometry for cell surface expression

• RT-PCR for mRNA expression levels

What methods are used to detect EMP2 protein expression in tissue samples?

Several complementary techniques are employed to detect EMP2 protein expression in tissue samples, each with specific advantages:

• Immunohistochemistry (IHC):

  • Typically uses biotinylated anti-EMP2 antibodies for detection

  • Allows visualization of EMP2 expression patterns within intact tissue architecture

  • Can be quantified using H-score systems (combining intensity and percentage of positive cells)

  • Used in tissue microarrays for high-throughput analysis of multiple samples

• Western Blot Analysis:

  • Detects EMP2 protein in cell or tissue lysates

  • Typically uses rabbit anti-human EMP2 antisera (1:2,000 dilution) followed by HRP-conjugated secondary antibodies

  • Provides information about protein size and relative abundance

  • β-actin is commonly used as a loading control

• Flow Cytometry:

  • Measures cell surface expression of EMP2

  • Particularly useful for assessing the binding capacity and specificity of anti-EMP2 antibodies

  • Can detect differences in EMP2 expression levels between different cell populations

• ELISA:

  • Uses biotinylated 24-amino acid peptides corresponding to EMP2 extracellular loops

  • Coated onto streptavidin-coated 96-well plates

  • Detection with HRP-conjugated secondary antibodies and TMB solution

  • Provides quantitative measurement of antibody binding to EMP2 peptides

How are anti-EMP2 antibodies typically constructed and validated?

The construction and validation of anti-EMP2 antibodies involve several sophisticated processes:

Construction Process:

• Isolation of variable (V) region sequences from existing antibody fragments (e.g., diabodies KS49) by PCR

• Cloning these sequences into expression vectors (e.g., pCR-II-TOPO vector)

• Confirmation of cloning by sequencing

• Transfection of heavy and light chain expression vectors into production cells (e.g., CHO-K1 cells)

• Screening of transfected cells by ELISA using anti-human IgG and anti-human κ chain antibodies

• Selection of high-producing subclones through S-35 biosynthetic antibody labeling and immunoprecipitation

• Expansion of optimal clones in roller bottles to maximize antibody secretion

• Purification of antibodies from cell culture supernatants

Purification Method:

• Filtration of culture supernatants

• Affinity chromatography using protein A-Sepharose columns

• Elution with sequential buffer systems (citrate buffer pH 4.5, glycine-HCl pH 2.5, glycine-HCl pH 2.0)

• Dialysis against PBS

• Concentration measurement using NanoDrop 2000

Validation Techniques:

• ELISA: Determining binding affinity to EMP2 peptides (EC50 determination)

• Flow Cytometry: Confirming binding to native EMP2 on cell surfaces

• Western Blot: Verifying specificity by detection of appropriate band sizes

• Sensitivity Testing: Comparing binding to cells with different EMP2 expression levels

• Specificity Testing: Confirming lack of binding to EMP2-negative cell lines

• Functional Assays: Assessing biological effects such as inhibition of cell viability or induction of apoptosis

What is the binding specificity of biotin-conjugated anti-EMP2 antibodies?

Biotin-conjugated anti-EMP2 antibodies demonstrate high binding specificity to both EMP2 peptides and native EMP2 protein. According to the research, biotinylated anti-EMP2 IgG1 antibodies bind specifically to a 24-amino acid peptide corresponding to the extracellular loop of human EMP2 . This binding can be quantitatively measured using ELISA, with reported EC50 values around 10.8 ng/mL for some anti-EMP2 antibodies .

The specificity of these antibodies can be validated through multiple approaches:

• Differential Binding Tests: Comparing binding between:

  • EMP2-positive vs. EMP2-negative cell lines

  • Wild-type cells vs. cells with engineered EMP2 overexpression (e.g., HEC-1A wild-type vs. HEC-1A/EMP2 cells with 2-4 fold increased EMP2 expression)

• Cross-Species Reactivity: Some anti-EMP2 antibodies demonstrate binding to both human and murine EMP2, making them valuable for translational research using mouse models . Flow cytometry analyses have confirmed the binding of anti-EMP2 IgG1 to both murine EMP2 (on 4T1 cells) and human EMP2 (on MDA-MB-231 triple-negative breast cancer cells) .

• Competitive Binding Assays: Using unlabeled anti-EMP2 antibodies to compete with biotin-conjugated versions to confirm binding to the same epitope.

The high specificity of biotin-conjugated anti-EMP2 antibodies makes them particularly valuable for immunohistochemical analysis of tissue samples and for detection of EMP2 in complex biological specimens.

How does biotinylation affect the function of anti-EMP2 antibodies?

Biotinylation of anti-EMP2 antibodies offers several methodological advantages while generally preserving the antibody's binding characteristics when properly performed:

Methodological Considerations for Optimal Biotinylation:

• Biotin-to-Antibody Ratio: Controlling the degree of biotinylation is crucial; excessive biotinylation can interfere with antigen binding sites.

• Biotinylation Site Selection: Using site-specific biotinylation methods rather than random biotinylation of lysine residues can preserve antibody function.

• Validation After Biotinylation: Always confirm that biotinylated antibodies retain binding specificity through ELISA or flow cytometry before use in critical experiments.

Research has demonstrated that biotinylated anti-EMP2 antibodies can be successfully used in IHC to confirm EMP2 expression in tissues with known EMP2 expression , indicating that proper biotinylation preserves the essential binding characteristics of the antibody.

What are the optimal protocols for using biotin-conjugated anti-EMP2 antibodies in IHC?

Based on the research data, optimal protocols for using biotin-conjugated anti-EMP2 antibodies in immunohistochemistry (IHC) involve several critical steps:

Sample Preparation:

• Fixation: Formalin fixation and paraffin embedding (FFPE) is commonly used for tissue samples

• Sectioning: 4-5 μm tissue sections mounted on positively charged slides

• Deparaffinization: Using xylene followed by rehydration through graded alcohols

Antigen Retrieval:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Pressure cooking or microwave heating for 10-20 minutes

Blocking and Antibody Application:

• Endogenous Peroxidase Blocking: 3% hydrogen peroxide for 10 minutes

• Protein Blocking: 5% normal goat serum for 30 minutes

• Primary Antibody: Biotin-conjugated anti-EMP2 antibody at optimized dilution (typically 1:100 to 1:500), incubated overnight at 4°C or for 1-2 hours at room temperature

• Detection System: Streptavidin-HRP conjugate (1:100 to 1:500) for 30 minutes

Visualization and Counterstaining:

• Chromogen: DAB (3,3'-diaminobenzidine) development for 5-10 minutes

• Counterstain: Hematoxylin for 1-2 minutes

• Mounting: Permanent mounting medium

Scoring System:
For quantitative assessment of EMP2 expression in IHC samples, a hybrid (H) score system is commonly employed:

• Calculate H score = Σ (i × Pi) where i = intensity (0-3) and Pi = percentage of positive cells (0-100%)

• Intensity scale: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

• H scores typically range from 0-300

• Samples with H scores ≥1 are generally considered positive for EMP2 expression

Controls:

• Positive Control: Include known EMP2-positive tissue (e.g., certain breast cancer tissues)

• Negative Control: Include EMP2-negative tissue or omit primary antibody

• Internal Control: Non-neoplastic elements within the section can serve as internal controls

How do different formats of anti-EMP2 antibodies (IgG1 vs. diabodies) compare in experimental settings?

Anti-EMP2 antibodies have been developed in different formats, primarily as diabodies (Dbs) and full IgG1 antibodies, each with distinct characteristics that influence their experimental applications:

Structural Differences:

• Diabodies: Bivalent antibody fragments composed of two single-chain variable fragments (scFvs) joined together; smaller size (~55 kDa)

• IgG1 Antibodies: Full-length antibodies with complete Fc regions; larger size (~150 kDa)

Comparative Properties:

Experimental Performance Comparison:

• In Vitro Efficacy:

  • Both formats induce significant cell death in cancer cell lines expressing EMP2

  • Anti-EMP2 diabodies induce cell death and caspase 3 cleavage in endometrial cancer cells

  • Anti-EMP2 IgG1 shows dose-dependent reduction in cell viability with IC50 values ranging from 2 μg/mL to 140 μg/mL depending on the cell line

• In Vivo Efficacy:

  • Anti-EMP2 diabodies reduce tumor growth in endometrial and ovarian cancer xenograft models

  • Anti-EMP2 IgG1 retards tumor growth in both human xenograft and syngeneic metastatic tumor monotherapy models without detectable systemic toxicity

  • The longer half-life of IgG1 format may provide advantages for in vivo applications

• Mechanism of Action:

  • Both formats inhibit EMP2-mediated signaling

  • IgG1 format provides additional antitumor effects through antibody-dependent cell-mediated cytotoxicity (ADCC)

These differences highlight the importance of selecting the appropriate antibody format based on the specific research application and experimental design.

What are the molecular mechanisms by which anti-EMP2 antibodies inhibit tumor growth?

Anti-EMP2 antibodies inhibit tumor growth through multiple molecular mechanisms that affect cancer cell signaling, survival, and interactions with the tumor microenvironment:

Disruption of EMP2-Mediated Signaling Pathways:

• Anti-EMP2 antibodies (both IgG1 and diabodies) directly interfere with EMP2's role in cellular signaling

• This disruption affects downstream pathways that regulate cell proliferation, survival, and invasion

Inhibition of FAK/Src Signaling:

• Treatment with anti-EMP2 IgG1 (100 μg/ml) blocks FAK/Src signaling in breast cancer cells

• Specifically inhibits phosphorylation of FAK at residues 576/577 and Src at residue 416

• FAK/Src pathway inhibition leads to decreased cell adhesion, migration, and survival signals

Induction of Apoptosis:

• Anti-EMP2 antibodies trigger programmed cell death in EMP2-expressing cancer cells

• Anti-EMP2 diabodies induce significant cell death and caspase 3 cleavage in endometrial cancer cells

• Anti-EMP2 IgG1 promotes apoptosis in breast cancer cell lines in a dose-dependent manner

• The apoptotic response correlates with cellular EMP2 expression levels

Inhibition of Invasion and Metastasis:

• Anti-EMP2 IgG1 inhibits invasion of breast cancer cells

• This may be mediated through effects on cell adhesion molecules and extracellular matrix interactions

Immune-Mediated Tumor Cell Killing:

• For full IgG1 antibodies, a significant portion of the antitumor effect is attributable to antibody-dependent cell-mediated cytotoxicity (ADCC)

• The Fc region of IgG1 engages immune effector cells to target and eliminate antibody-bound tumor cells

Effects on Tumor Vasculature:

• Some evidence suggests that targeting EMP2 may affect tumor angiogenesis, though this mechanism requires further investigation

The multi-faceted mechanisms of anti-EMP2 antibodies make them promising therapeutic agents, particularly for aggressive cancers with limited treatment options, such as triple-negative breast cancer.

How does FAK/Src signaling inhibition by anti-EMP2 antibodies contribute to cancer cell apoptosis?

The inhibition of FAK/Src signaling by anti-EMP2 antibodies creates a cascade of molecular events that ultimately leads to cancer cell apoptosis:

Disruption of FAK/Src Activation:

• Under normal conditions, EMP2 promotes the activation (phosphorylation) of FAK at residues 576/577 and Src at residue 416

• Anti-EMP2 antibodies (100 μg/ml) block this activation, as demonstrated in MDA-MB-468 breast cancer cells

• This inhibition occurs within 12 hours after antibody treatment and cell plating

Anoikis Sensitization Mechanism:

• FAK activation typically provides survival signals that protect cells from anoikis (detachment-induced apoptosis)

• When FAK signaling is blocked by anti-EMP2 antibodies, cancer cells become vulnerable to anoikis

• This is particularly significant for invasive cancer cells that would otherwise survive during metastatic spread

Cell Adhesion and Cytoskeletal Alterations:

• FAK/Src signaling regulates focal adhesions and cytoskeletal organization

• Inhibition leads to disruption of these structures, compromising cell attachment and migration

• Changes in cell morphology and attachment precede apoptotic events

Downstream Signaling Effects:

• Blocked FAK/Src signaling affects multiple downstream pathways:

  • Reduced activation of PI3K/Akt pathway (cell survival)

  • Decreased MAPK/ERK signaling (proliferation)

  • Altered integrin-mediated signaling (adhesion and migration)

Mitochondrial Pathway of Apoptosis:

• FAK inhibition ultimately leads to activation of the intrinsic (mitochondrial) apoptotic pathway

• This involves:

  • Decreased expression of anti-apoptotic proteins (Bcl-2, Bcl-xL)

  • Increased expression of pro-apoptotic proteins (Bax, Bad)

  • Mitochondrial outer membrane permeabilization

  • Release of cytochrome c

  • Activation of caspase cascade including caspase 3 cleavage

Cell Type-Specific Sensitivity:

• The efficiency of this pathway depends on cellular EMP2 expression levels

• Higher EMP2 expression generally correlates with greater sensitivity to anti-EMP2 antibody treatment

• Different cell lines show varying IC50 values for anti-EMP2 IgG1, ranging from 2 μg/mL to 140 μg/mL

This detailed understanding of the molecular mechanisms helps explain why anti-EMP2 antibodies show promise as targeted therapeutics and provides rationale for potential combination therapies that could enhance their efficacy.

What factors influence the sensitivity of cancer cells to anti-EMP2 antibody treatment?

Multiple factors determine the sensitivity of cancer cells to anti-EMP2 antibody treatment, creating a complex landscape of treatment response:

EMP2 Expression Levels:

• The most significant determinant of sensitivity is cellular EMP2 expression level

• Higher EMP2 expression generally correlates with greater sensitivity to anti-EMP2 antibodies

• EMP2-negative cell lines (e.g., HS578t breast cancer cells) show no response even at high antibody concentrations

• Cell lines with variable EMP2 expression show corresponding differences in sensitivity

Antibody Format and Characteristics:

• Different antibody formats (IgG1, diabodies) have varying efficacy profiles

• The EC50 of anti-EMP2 IgG1 binding to EMP2 peptide is approximately 10.8 ng/mL

• IC50 values for cell viability vary widely among cell lines:

  • SK-BR-3: approximately 2 μg/mL

  • MDA-MB-468: approximately 140 μg/mL

  • 4T1 (murine): approximately 108 μg/mL

Cell Type and Genetic Background:

• Intrinsic cellular characteristics beyond EMP2 expression affect sensitivity

• Hormone receptor status does not directly correlate with EMP2 expression or antibody sensitivity

• EMP2 is expressed in both triple-negative (MDA-MB-231, MDA-MB-468, BT-20) and hormone-positive cell lines

Hormonal Factors:

• Progesterone treatment augments the response to anti-EMP2 diabodies

• This is likely due to progesterone's ability to induce EMP2 expression

• Suggests potential for combination therapies in hormone-responsive tumors

FAK/Src Signaling Dependency:

• Cells with greater dependency on FAK/Src signaling show enhanced sensitivity

• The degree to which cancer cells rely on these pathways varies and affects treatment response

Apoptotic Machinery Integrity:

• Functional apoptotic pathways are necessary for full response

• Defects in caspase activation or other apoptotic machinery may confer resistance

• Anti-EMP2 antibodies induce caspase 3 cleavage as part of their mechanism

Tumor Microenvironment Factors:

• For full IgG1 antibodies, ADCC contributes significantly to the antitumor effect

• The presence and function of immune effector cells in the tumor microenvironment influence therapeutic efficacy

• This factor is particularly relevant for in vivo applications rather than cell culture studies

Understanding these factors can help in selecting appropriate patient populations for potential clinical applications, designing rational combination therapies, and predicting and overcoming resistance mechanisms.

How can biotin-conjugated anti-EMP2 antibodies be optimized for targeted drug delivery systems?

Biotin-conjugated anti-EMP2 antibodies offer significant potential for targeted drug delivery systems. Based on the research findings, several optimization strategies can be employed:

Conjugation Chemistry Optimization:

• Site-specific biotinylation at non-binding regions to preserve antibody function

• Controlled biotin-to-antibody ratio to maintain binding affinity while providing sufficient biotin for streptavidin interaction

• Use of cleavable linkers that can release the payload in response to tumor-specific conditions (pH, proteases)

Payload Selection and Attachment:

• Conjugation of biotin-streptavidin complexes with:

  • Cytotoxic drugs (doxorubicin, paclitaxel)

  • Small molecule inhibitors targeting complementary pathways

  • Radionuclides for theranostic applications

  • siRNA or microRNA for gene silencing

• Optimization of drug-to-antibody ratio for maximal efficacy while maintaining antibody properties

Formulation Design:

• Development of nanoparticle-based delivery systems incorporating:

  • Biotin-conjugated anti-EMP2 antibodies on the surface

  • Therapeutic agents encapsulated within

  • Potential for co-delivery of multiple agents

• Optimization of nanoparticle size, charge, and surface properties for enhanced tumor penetration

Target Population Selection:

• Focusing on tumors with high EMP2 expression

• Particularly promising for triple-negative breast cancers, where 73% of tumors express elevated EMP2

• Potential for combination with therapies that upregulate EMP2 expression (e.g., progesterone )

Format Selection and Engineering:

• Comparison of different antibody formats:

  • Full IgG1 (longer circulation time, ADCC capability)

  • Diabodies (better tissue penetration, shorter half-life)

  • Fab fragments (smaller size, reduced immunogenicity)

• Engineering antibodies with enhanced tumor penetration properties while maintaining EMP2 specificity

Validation and Testing Methodology:

• In Vitro Validation:

  • Confirmation of specific binding to EMP2-expressing cells

  • Verification of internalization kinetics

  • Assessment of drug release and intracellular trafficking

• In Vivo Evaluation:

  • Biodistribution studies to confirm tumor targeting

  • Pharmacokinetic analysis of antibody-drug conjugates

  • Efficacy testing in relevant tumor models expressing EMP2

  • Toxicity assessment

Combination Strategy Development:

• Combining anti-EMP2 antibody-drug conjugates with:

  • FAK/Src inhibitors to enhance the disruption of these pathways

  • Immune checkpoint inhibitors to promote anti-tumor immune responses

  • Conventional chemotherapy for synergistic effects

These optimization strategies should be guided by the understanding that anti-EMP2 antibodies can induce direct effects on cancer cells through FAK/Src signaling inhibition and apoptosis induction, which may complement the effects of delivered payloads.