Recombinant HER2 Antibody
Description
Molecular Identity and Function
ARHGEF39 is located on chromosome 9p13.3 and encodes a protein belonging to the Dbl family of RhoGEFs . RhoGEFs catalyze the exchange of GDP for GTP on Rho GTPases, activating pathways that govern cytoskeletal reorganization, cell motility, and division . ARHGEF39 specifically activates RHOA, a GTPase critical for focal adhesion assembly and cell-matrix interactions .
Key Mechanistic Insights:
-
RHOA Activation: Overexpression of ARHGEF39 in HEK293FT cells increases RHOA activity, leading to cell detachment and reduced adhesion .
-
Cell Cycle Regulation: In neural progenitor cells, ARHGEF39 is co-expressed with genes involved in G2/M phase transition, suggesting a role in mitotic progression .
-
Cancer Pathways: In clear cell renal cell carcinoma (ccRCC) and hepatocellular carcinoma (HCC), ARHGEF39 promotes cell viability, migration, and invasion via AKT/ERK and Rac1-P38-ATF2 signaling .
Role in Neurodevelopment
ARHGEF39 is enriched in prenatal brain tissues and marks proliferating neural progenitor cells (NPCs) . Single-cell RNA sequencing (scRNA-seq) data reveal:
| Key Neurodevelopmental Findings | Source |
|---|---|
| Marker gene for cycling NPCs in G2/M phase | |
| Co-expressed with CDC25C, a cell cycle regulator | |
| Prenatal enrichment in BrainSpan and PsychENCODE |
Gene ontology (GO) analysis associates ARHGEF39 with cell division (adjusted -value = ) and G2/M transition (adjusted -value = ) . A functional polymorphism in ARHGEF39 disrupts microRNA regulation, linking it to developmental language disorder (DLD) .
Oncogenic Role in Human Cancers
ARHGEF39 is upregulated in multiple cancers and correlates with poor prognosis:
Cancer-Specific Mechanisms:
-
Hepatocellular Carcinoma (HCC): Drives proliferation via G2/M phase dysregulation .
-
Non-Small Cell Lung Cancer (NSCLC): Enhances invasion through Rac1-P38-ATF2 signaling .
Clinical and Prognostic Significance
ARHGEF39 expression is an independent prognostic marker in ccRCC and HCC:
Product Specs
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL
QPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC.
HEAVY CHAIN
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR
QAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK
NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK.
Q&A
What is a recombinant HER2 antibody and what advantages does it offer over conventional antibodies?
Recombinant HER2 antibodies are engineered monoclonal antibodies produced using recombinant DNA technology, where genes encoding the antibody are cloned and expressed in controlled cell culture systems. Unlike conventional antibodies produced through hybridoma technology, recombinant antibodies offer superior lot-to-lot consistency, continuous supply, and animal-free manufacturing .
The key advantages include:
-
Precisely controlled antibody characteristics (specificity, affinity)
-
Reduced batch-to-batch variability
-
Opportunity for antibody engineering (humanization, conjugation)
-
Greater reproducibility in research applications
-
Customizable properties for specific research needs
Recombinant technology allows for the development of innovative formats like antibody-drug conjugates (ADCs), such as RC48, which combines an anti-HER2 antibody with a cytotoxic payload for enhanced therapeutic potential .
How do recombinant HER2 antibodies specifically recognize the HER2 receptor?
Recombinant HER2 antibodies recognize specific epitopes on the HER2 receptor through their complementarity-determining regions (CDRs). These CDRs determine the antibody's binding specificity and affinity. For example, some monoclonal antibodies feature unique CDR sequences specifically recognizing the extracellular domain (ECD) of HER2 .
The HER2/ErbB2 (29D8) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding tyrosine 1248 of human ErbB2 protein . This specificity ensures targeted binding to particular regions of the HER2 protein, which is crucial for both research applications and therapeutic efficacy.
Recognition of HER2 by these antibodies can lead to various downstream effects depending on the antibody's design:
-
Blocking HER2 dimerization and signaling
-
Inducing receptor internalization and degradation
-
Recruiting immune effector cells for antibody-dependent cellular cytotoxicity (ADCC)
What expression systems are most effective for producing recombinant HER2 antibodies?
Several expression systems can be employed for recombinant HER2 antibody production, each with distinct advantages:
Human Embryonic Kidney 293 (HEK293) Cells
The expression of recombinant anti-HER2 humanized monoclonal antibody (rhHER2-mAb) in HEK293 cells has been successfully achieved through transient gene expression (TGE) technology . This system allows for rapid production without establishing stable cell lines, making it ideal for research applications requiring quick turnaround.
Transient vs. Stable Expression
Transient gene expression technology enables researchers to quickly obtain significant quantities of anti-HER2 antibodies compared to traditional stable cell line methods . The TGE approach is particularly valuable for preliminary characterization studies and when multiple antibody variants need to be screened.
Optimization Parameters
Key parameters for optimization include:
-
Light chain (LC) to heavy chain (HC) ratio (optimal ranges from 4:1 to 1:2)
-
DNA to transfection reagent (polyethyleneimine) ratio (ranges from 4:1 to 1:1)
How can I optimize transient gene expression systems for recombinant HER2 antibody production?
Optimization of transient gene expression systems for recombinant HER2 antibody production involves several critical steps:
Vector Design and Transfection Conditions
-
Construct mammalian cell expression vectors containing the LC and HC genes
-
Optimize the ratio of LC to HC plasmids (test range: 4:1 to 1:2)
-
Determine optimal DNA to transfection reagent ratio (test range: 4:1 to 1:1)
Verification of Antibody Assembly
Western blotting is essential for verifying correct antibody assembly. Samples should be analyzed by SDS-PAGE and transferred to a PVDF membrane before probing with anti-human IgG secondary antibodies .
Purification Strategy
Protein A affinity chromatography is highly effective for purifying recombinant HER2 antibodies, yielding high-purity product suitable for functional studies .
Expression Monitoring and Analysis
Regular sampling and analysis during the expression process helps determine the optimal harvest time and conditions for maximum yield and quality.
What methodologies are most reliable for evaluating binding specificity and affinity of recombinant HER2 antibodies?
A multi-platform approach is recommended for comprehensive characterization of recombinant HER2 antibodies:
Western Blotting
-
Confirms recognition of HER2 at the expected molecular weight (185 kDa)
-
Essential for confirming antibody specificity under denaturing conditions
Immunoprecipitation
-
Demonstrates ability to bind native HER2 in solution
-
Useful for studying HER2 interaction partners
Immunohistochemistry (IHC)
-
Assesses antibody binding to HER2 in fixed tissue samples
-
Critical for translational applications
Immunofluorescence
-
Evaluates subcellular localization of HER2
-
Useful for co-localization studies
Flow Cytometry
-
Confirms binding to HER2 on intact cell surfaces
-
Recommended dilution: 1:200 - 1:400 for fixed/permeabilized cells
-
Important for quantifying cell surface expression levels
ELISA Development
For quantitative binding studies, sandwich ELISA can be developed using recombinant HER2 antibodies. A properly optimized ELISA can achieve HER2 quantitation within 1.56-100 ng/ml with sensitivity of 0.5 ng/ml .
How can I accurately assess antibody-dependent cellular cytotoxicity (ADCC) of recombinant HER2 antibodies?
Evaluation of ADCC activity requires careful experimental design:
Experimental Approach
Lactate dehydrogenase (LDH) release assays provide a reliable measurement of ADCC activity by quantifying LDH released from lysed target cells .
Critical Variables
-
Target cells: Use cell lines with varying HER2 expression levels
-
Effector cells: Peripheral blood mononuclear cells or isolated NK cells
-
Effector-to-target ratio: Test multiple ratios to establish optimal conditions
-
Antibody concentration: Perform dose-response experiments
-
Incubation time: Optimize for maximum ADCC activity
Controls
-
Positive control: Include clinically validated anti-HER2 antibody (e.g., trastuzumab)
-
Negative controls: Use isotype-matched control antibodies and HER2-negative cell lines
In Vivo Validation
To complement in vitro findings, ADCC activity should be evaluated in appropriate animal models, such as non-obese diabetic/severe combined immunodeficiency mice .
Comparative Analysis
Benchmark the ADCC activity of your recombinant HER2 antibody against reference standards. Previous studies have demonstrated that optimized recombinant HER2 antibodies can show significantly higher affinity and better biological activity compared to commercial antibodies like Herceptin (p<0.01) .
How do antibody-drug conjugates (ADCs) like RC48 compare with traditional HER2-targeted therapies?
Antibody-drug conjugates represent an evolution in HER2-targeted therapies with distinct mechanisms and efficacy profiles:
Mechanism of Action Comparison
| Feature | Traditional Anti-HER2 Antibodies | HER2 Antibody-Drug Conjugates (e.g., RC48) |
|---|---|---|
| Primary mechanism | Receptor signaling blockade | Targeted cytotoxic payload delivery |
| Immune engagement | ADCC and ADCP | Reduced dependence on immune effectors |
| HER2 expression requirement | Typically requires high expression | Effective against low/heterogeneous HER2 expression |
| Bystander effect | Limited | Significant (affects nearby cells regardless of HER2 status) |
Clinical Efficacy (Neoadjuvant Setting)
RC48 treatment has demonstrated promising results in clinical studies:
Response by Receptor Status
Safety Profile
No relevant serious (grade 3/4) toxicities were reported with RC48 treatment in the neoadjuvant setting , suggesting a favorable safety profile for this class of therapeutics.
How can I develop a validated assay for quantifying soluble HER2 in biological samples?
Developing a validated assay for soluble HER2 quantification requires careful consideration of several factors:
Assay Design Principles
-
Sandwich ELISA using monoclonal antibodies specifically recognizing HER2 ECD
-
Antibodies should target different, non-overlapping epitopes
-
Optimize capture and detection antibody pairs for maximum sensitivity and specificity
Performance Characteristics
A well-designed assay should achieve:
-
Linear quantitation range: 1.56-100 ng/ml
-
Detection sensitivity: approximately 0.5 ng/ml
-
High specificity for HER2 ECD
Sample Considerations
The assay should be validated for multiple biological matrices:
Validation Parameters
Comprehensive validation should include:
-
Linearity assessment
-
Recovery experiments
-
Precision determination (intra- and inter-assay)
-
Specificity testing
Cross-Platform Applications
The same antibodies can be used across multiple platforms for comprehensive HER2 analysis:
How do I troubleshoot inconsistent results when using recombinant HER2 antibodies across different experimental platforms?
When facing inconsistent results with recombinant HER2 antibodies, consider these systematic troubleshooting approaches:
Antibody-Related Factors
Epitope Accessibility Issues:
-
Different experimental conditions affect epitope accessibility
-
The HER2/ErbB2 (29D8) antibody targets residues around tyrosine 1248 , which may be variably accessible
-
Solution: Try antibodies targeting different HER2 epitopes
Concentration Optimization:
-
Recommended dilutions vary significantly by application:
-
Solution: Perform titration experiments for each application
Sample Preparation Factors
Protein Conformation:
-
Native vs. denatured HER2 may affect antibody recognition
-
Solution: Ensure antibody validation for your specific application
Fixation and Permeabilization:
-
Different fixation methods alter epitope recognition
-
Solution: Optimize and standardize fixation protocols
Detection System Considerations
Signal-to-Noise Ratio:
-
Background signal varies across platforms
-
Solution: Include appropriate blocking steps and controls
Cross-Reactivity:
-
Potential reactivity with related proteins (other ErbB family members)
-
Solution: Include specificity controls (HER2-negative samples)
A systematic approach to troubleshooting with proper controls and standardized protocols will help resolve inconsistencies across experimental platforms.
What are the critical quality attributes to monitor for recombinant HER2 antibodies used in research applications?
Maintaining consistent quality of recombinant HER2 antibodies is essential for reliable research outcomes. Key quality attributes to monitor include:
Physical Characteristics
-
Molecular weight confirmation (145-150 kDa for full IgG)
-
Purity assessment (>95% by SDS-PAGE)
-
Aggregation level (<5% by size exclusion chromatography)
Functional Properties
-
Binding specificity (target recognition with minimal cross-reactivity)
-
Binding affinity (consistent KD values between lots)
-
Biological activity (consistent ADCC potency, if applicable)
Stability Indicators
-
Thermal stability
-
Freeze-thaw resistance
-
Long-term storage stability at recommended conditions
Application-Specific Performance
-
IHC: Reproducible staining patterns in control tissues
-
Flow cytometry: Consistent mean fluorescence intensity on reference cell lines
Documentation Requirements
-
Certificate of analysis for each lot
-
Validation data across relevant applications
-
Species reactivity information
-
Recommended storage and handling conditions
Regular monitoring of these attributes will ensure consistent performance in research applications and enhance experimental reproducibility.
How are recombinant HER2 antibodies evolving for applications beyond breast cancer?
While HER2 has been primarily associated with breast cancer, recombinant HER2 antibodies are finding expanded applications:
Expanded Cancer Indications
Research is demonstrating efficacy of anti-HER2 therapies in other cancers:
-
Ovarian cancer: Mouse models have shown effective inhibition of HER2 high-expressing ovarian cancer cell lines both in vitro and in vivo
-
Gastric cancer
-
Lung cancer
-
Colorectal cancer with HER2 amplification
Novel Detection Methodologies
Beyond conventional applications, researchers are developing:
-
Multiplexed detection systems for HER2 alongside other biomarkers
-
Integrated assays combining HER2 protein detection with genetic analysis
Diagnostic Innovations
-
Blood-based HER2 assays present an attractive alternative to tissue testing
-
Validated assays for circulating HER2 concentration may correlate with disease clinical manifestations
-
Combined assessment of membrane-bound and soluble HER2 could provide complementary diagnostic information
What experimental considerations are important when developing novel recombinant HER2 antibody-based therapeutics?
Development of novel HER2-targeting therapeutics requires careful experimental design:
Target Selection and Validation
-
Define specific HER2 domain/epitope targeting strategy
-
Consider bispecific approaches (HER2 plus another target)
-
Validate target accessibility in relevant tumor models
Antibody Engineering Approaches
-
Affinity optimization (may require balancing between too high and too low affinity)
-
Fc engineering for modified immune engagement
-
Linker chemistry optimization for ADCs
Combination Strategies
RC48 study demonstrates a combination approach:
-
RC48 alone as neoadjuvant therapy
-
Conventional chemotherapy (EC) postoperatively
-
HER2-targeted therapy (trastuzumab with pertuzumab) in adjuvant setting
Predictive Biomarkers
-
Evaluate response based on HER2 expression levels
-
Consider impact of hormone receptor status:
-
Develop companion diagnostics for patient selection
Resistance Mechanisms
-
Design experiments to identify and overcome potential resistance pathways
-
Consider HER2 mutation status and downstream signaling alterations
Product Science Overview
Recombinant human anti-HER2 antibodies are a class of therapeutic proteins designed to target the human epidermal growth factor receptor 2 (HER2). HER2 is a member of the ErbB family of receptor tyrosine kinases, which play a crucial role in the regulation of cell growth and differentiation. Overexpression of HER2 is observed in approximately 20-30% of breast cancers and is associated with aggressive tumor growth and poor prognosis .
HER2, also known as ErbB2, is a ligand-less receptor that forms heterodimers with other members of the ErbB family, such as HER1 (EGFR), HER3, and HER4. This dimerization leads to the activation of downstream signaling pathways that promote cell proliferation and survival . In normal cells, HER2 expression is tightly regulated, but in cancer cells, gene amplification or protein overexpression leads to uncontrolled cell growth and tumor development .
The development of recombinant human anti-HER2 antibodies was driven by the need for targeted therapies that could specifically inhibit the activity of HER2 in cancer cells. Trastuzumab (Herceptin) was the first humanized monoclonal antibody approved for the treatment of HER2-positive breast cancer . Trastuzumab binds to the extracellular domain of HER2, preventing receptor dimerization and subsequent activation of downstream signaling pathways . Additionally, trastuzumab induces antibody-dependent cellular cytotoxicity (ADCC), leading to the destruction of cancer cells .
Recombinant human anti-HER2 antibodies, such as trastuzumab, work through multiple mechanisms to inhibit tumor growth:
- Inhibition of HER2 Signaling: By binding to the extracellular domain of HER2, these antibodies prevent the receptor from dimerizing with other ErbB family members, thereby blocking the activation of downstream signaling pathways that promote cell proliferation and survival .
- Induction of ADCC: The Fc region of the antibody interacts with immune cells, such as natural killer (NK) cells, leading to the targeted killing of HER2-overexpressing cancer cells .
- Inhibition of HER2 Shedding: Trastuzumab has been shown to inhibit the proteolytic cleavage of HER2, preventing the release of the extracellular domain and the formation of a truncated, active receptor fragment known as p95HER2 .
Recombinant human anti-HER2 antibodies have shown significant clinical benefits in the treatment of HER2-positive breast cancer. Trastuzumab, in combination with chemotherapy, has been demonstrated to improve overall survival and reduce the risk of disease recurrence in patients with early-stage and metastatic HER2-positive breast cancer . Other anti-HER2 antibodies, such as pertuzumab and ado-trastuzumab emtansine (T-DM1), have also been developed and approved for clinical use .
Ongoing research aims to develop novel anti-HER2 therapies with improved efficacy and reduced resistance. Bispecific antibodies, which can simultaneously target HER2 and another receptor, are being investigated for their potential to enhance anti-tumor activity . Additionally, antibody-drug conjugates (ADCs) that deliver cytotoxic agents directly to HER2-overexpressing cancer cells are being explored as a means to improve therapeutic outcomes .
