Anti-Human EGFR (Cetuximab) [Clone C225] – Fc Muted™

Anti-Human EGFR (Cetuximab) [Clone C225] – Fc Muted™

Product No.: LT605

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Product No.LT605
Clone
C225
Target
EGFR
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
ErbB-1; HER1; epidermal growth factor receptor
Isotype
Human IgG1κ
Applications
CyTOF®
,
FA
,
FC

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Antibody Details

Product Details

Reactive Species
Human
Host Species
Human
Expression Host
HEK-293 Cells
FC Effector Activity
Muted
Immunogen
Human EGFR/ErbB1
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
< 1.0 EU/mg as determined by the LAL method
Purity
≥95% by SDS Page
≥95% monomer by analytical SEC
Formulation
This biosimilar antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
State of Matter
Liquid
Product Preparation
Recombinant biosimilar antibodies are manufactured in an animal free facility using only in vitro protein free cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s recombinant biosimilar antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Regulatory Status
Research Use Only (RUO). Non-Therapeutic.
Country of Origin
USA
Shipping
2-8°C Wet Ice
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for Cetuximab biosimilar antibody for staining cells in flow cytometry is ≤ 0.25 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
Additional Applications Reported In Literature ?
FA
CyTOF®
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
This non-therapeutic biosimilar antibody uses the same variable region sequence as the therapeutic antibody Cetuximab. This product is for research use only. Cetuximab activity is directed against Human EGFR.
Background
EGFR is a 170 kD transmembrane glycoprotein that is part of the ErbB family of receptors within the protein kinase superfamily. EGFR is one of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). EGFR is essential for various processes including controlling cell growth and differentiation and ductal development of the mammary glands. Ligand binding induces dimerization and autophosphorylation. It consists of a glycosylated extracellular domain which binds to EGF and an intracellular domain with tyrosine-kinase activity necessary for signal transduction. TGFα, vaccinia virus growth factor, and related growth factors can also bind to and signal through EGFR. Abnormal EGFR signaling has been implicated in inflammatory diseases such as psoriasis, eczema and atherosclerosis. Alzheimer's disease is linked with poor signaling of the EGFR and other receptor tyrosine kinases. Furthermore, over-expression of the EGFR is linked with the growth of various tumors. EGFR has been identified as an oncogene, a gene which in certain circumstances can transform a cell into a tumor cell, which has led to the therapeutic development of anticancer EGFR inhibitors. EGFR is a well-established target for both mAbs and specific tyrosine kinase inhibitors. Anti-Human EGFR (Cetuximab) utilizes the same variable regions from the therapeutic antibody Cetuximab making it ideal for research projects.
Antigen Distribution
EGFR is ubiquitously expressed and found in the plasma membrane.
PubMed
NCBI Gene Bank ID
Research Area
Biosimilars

Leinco Antibody Advisor

Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.

Research-grade Cetuximab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to ensure accurate, reproducible measurement of drug concentration in serum samples by closely mimicking the reference product (such as Erbitux) and providing an analytically equivalent comparator.

Essential Context and Supporting Details:

  • Purpose in PK Bridging ELISA: In a PK bridging ELISA, calibration standards and reference controls are prepared using known concentrations of research-grade Cetuximab biosimilar or the reference product (e.g., Erbitux). These standards generate a standard curve against which unknown serum sample concentrations are quantified.

  • Single Analytical Standard Approach: Best practice—supported by regulatory guidance and industry consensus—is to develop a single assay with a single standard (biosimilar or reference) to quantify both biosimilar and reference products. This minimizes variability, improves comparability, and supports robust PK assessment.

  • Calibration and Validation:

    • Standards are often calibrated against both Erbitux and biosimilar Cetuximab to ensure that the assay equally recognizes both molecules and that their PK measurements are interchangeable.
    • The ELISA is validated for parameters including accuracy, precision, linearity, and specificity to confirm that the biosimilar can serve as a valid calibrator across the expected concentration range in serum.
  • Assay Procedure:

    • The microplate wells are coated with a capture antibody specific for Cetuximab (or its epitope), frequently using inhibitory antibodies that target the EGFR binding site, ensuring specificity.
    • Patient or study samples, standards, and controls are added to the wells, and biosimilar Cetuximab in the standards generates the standard curve.
    • Detection is accomplished using a secondary antibody specific for Cetuximab, often conjugated to an enzyme for colorimetric readout.
    • The biosimilar standard's curve allows unknown concentrations in test samples to be interpolated.
  • Assessment of Bioanalytical Comparability: During method development, parallelism, dilution linearity, and equivalence between biosimilar and reference standards are rigorously examined. If the biosimilar demonstrates equivalent assay performance to the reference, it may be selected as the routine analytical standard.

Additional Relevant Points:

  • Total vs. Free Drug Detection: The specific combination of capture and detection antibodies in some PK bridging ELISA formats can distinguish between free, partially bound, and total drug; the suitability of the biosimilar as a standard depends on the antibody characteristics and assay design.
  • Regulatory Relevance: Using research-grade biosimilars as standards facilitates regulatory submissions by enabling direct bioanalytical comparability, a prerequisite for biosimilar approval.

Summary Table: Use of Research-Grade Cetuximab Biosimilars in PK Bridging ELISA

StepRole of Biosimilar StandardSupporting Details
Assay CalibrationProvides the standard curveStandards are prepared with biosimilar or Erbitux
Method ValidationEnsures specificity and equivalencePrecision, accuracy, and linearity are confirmed
PK ComparisonAllows unbiased quantification of both productsSingle standard minimizes cross-assay variation

In summary: Research-grade Cetuximab biosimilars are routinely used as calibration standards or reference controls in PK bridging ELISA, enabling accurate measurement of drug concentrations in serum by serving as analytically equivalent substitutes to the original reference product.

The primary models for studying research-grade anti-EGFR antibodies in vivo for tumor growth inhibition and TIL characterization fall into several key categories, though the available literature shows more development in certain areas than others.

Syngeneic Models

Melanoma ModelsA notable advancement is the development of the first mouse syngeneic melanoma model with preserved in vivo expression of human EGFR. This model addresses a critical gap in research, as most existing syngeneic models either lack melanoma origin or don't express human EGFR, which is essential for testing anticancer agents that specifically recognize human receptors.

Other Established Syngeneic LinesSeveral tumorigenic mouse cell lines expressing significant EGFR levels have been utilized for in vivo studies, including hepatocellular carcinoma CBO140C12, metastatic Lewis lung carcinoma variant 3LLD122, mouse breast carcinoma 4T1, and mouse colon carcinoma CT26. However, availability and quality control issues limit some of these models, and EGFR expression levels can vary significantly between studies.

Glioblastoma ModelsSyngeneic C57BL/6 mice have been successfully used for glioblastoma studies, where CAR T cells targeting NKG2D (rather than EGFR directly) demonstrated the ability to migrate to tumor sites, increase survival, and cure a fraction of glioma-bearing mice. This model's success led to human clinical trials, demonstrating the translational potential of well-designed syngeneic studies.

Xenograft Models

A431 Cell Line StudiesThe most detailed characterization comes from studies using A431-derived solid tumors in athymic nude mice. In these studies, anti-EGFR Nanobodies were administered at high doses (1 mg per mouse per injection, twice weekly for 4 weeks) and successfully demonstrated significant delays in tumor outgrowth. This represents the first reported successful use of unconjugated Nanobodies to inhibit solid tumor growth in vivo.

Human Colon Cancer XenograftsRAS wild-type human colon cancer xenografts serve as relevant models for EGFR inhibitor-sensitive tumors, particularly for studying triple monoclonal antibody approaches.

Key Considerations for Model Selection

Immunocompetent vs. Immunocompromised SystemsSyngeneic models offer the advantage of a fully functional immune system, which is crucial for evaluating immune checkpoint inhibitor efficacy and characterizing tumor-immune interactions. These models are particularly valuable for understanding TIL dynamics, as they preserve the natural immune microenvironment that influences T cell infiltration and activation.

Species-Specific Receptor ExpressionA significant challenge in anti-EGFR research is that many established syngeneic models lack human EGFR expression, limiting their utility for testing human-specific therapeutic antibodies. The development of transgenic approaches using genetic knock-in of human antigens into murine cells represents a promising solution.

Functional Validation RequirementsEffective models require not just EGFR expression but also functional responsiveness to EGF signaling. The most successful approaches have utilized functional selection strategies to identify antibodies that compete for EGF binding and effectively block downstream signaling pathways.

The field continues to evolve toward more sophisticated syngeneic models that combine human target expression with intact immune systems, enabling comprehensive evaluation of both direct anti-tumor effects and immune-mediated responses including TIL characterization.

Researchers use Cetuximab biosimilars in combination with immune checkpoint inhibitors (ICIs), such as anti-CTLA-4 or anti-LAG-3 biosimilars, to explore synergistic antitumor effects in complex immune-oncology models by leveraging their complementary mechanisms to recruit and activate immune cells and to relieve immunosuppressive checkpoints.

Key mechanisms of synergy and research approaches include:

  • Enhanced Immune Cell Recruitment and Activation: Cetuximab biosimilars, by binding EGFR, induce antibody-dependent cellular cytotoxicity (ADCC), promoting natural killer (NK) cell-mediated killing of tumor cells regardless of EGFR mutation status. This is accompanied by increased expression of MHC-II, facilitating the recruitment of T cells into the tumor microenvironment (TME), and promoting dendritic cell activation.

  • Checkpoint Disinhibition: While Cetuximab-induced immune activation is robust, tumors can upregulate inhibitory pathways (e.g., PD-1/PD-L1, CTLA-4, and potentially LAG-3) as adaptive resistance. Combining Cetuximab biosimilars with checkpoint inhibitors (including anti-CTLA-4 or anti-LAG-3 agents) allows researchers to study whether blocking these additional checkpoints further unleashes T and NK cell activity, overcoming the immunosuppressive signals and resulting in greater tumor regression.

  • Preclinical and Clinical Experimental Design:

    • In preclinical models, researchers employ syngeneic murine tumor models or humanized mouse models where Cetuximab biosimilars are administered along with checkpoint inhibitor biosimilars to evaluate changes in tumor growth, immune infiltrate composition, cytokine production, and survival. These studies use immunophenotyping, flow cytometry, and transcriptomic profiling to dissect mechanisms.
    • In clinical studies (so far, mainly with innovator Cetuximab, but methods are applicable to biosimilars), researchers assess endpoints such as overall response rate, disease control rate, and immune-related toxicities in patient cohorts with tumors known to resist checkpoint inhibition alone (e.g., MSS/pMMR colorectal cancer).
  • Examples and Observed Mechanisms:

    • Cetuximab-enriched environments foster T and NK cell infiltration and induce pro-inflammatory cytokines (IFN-γ, TNF-α), which prime the TME for checkpoint inhibitor efficacy.
    • The two-step synergy hypothesis: Cetuximab brings in and activates new immune cells, while checkpoint inhibitors "disinhibit" or rescue these cells from exhaustion, giving a compounded anti-tumor effect in immune-evasive cancer models.

Immunogenicity and Biosimilar Considerations:

  • Recent comparative studies show that biosimilar Cetuximab is noninferior to the innovator product regarding efficacy, safety, and pharmacokinetics, but may induce a higher frequency of antidrug antibodies, which researchers need to monitor in combinatorial studies for potential immune-related confounders.

Summary Table: Cetuximab + Checkpoint Inhibitors Research Focus

Cetuximab EffectCheckpoint Inhibitor ActionCombined Impact
ADCC via NK cellsRelieve T/NK inhibitionIncreased tumor cell killing
T cell recruitmentBlockade of T cell exhaustion pathways (CTLA-4, LAG-3)Robust T cell anti-tumor response
Pro-inflammatory cytokinesPrevent immunosuppressionProlonged, durable immunity

Thus, Cetuximab biosimilars in combination with anti-CTLA-4 or anti-LAG-3 biosimilars are investigated in immune-oncology models to dissect and enhance the multiple layers of anti-tumor immunity, mainly by escalating both innate and adaptive immune responses while blocking adaptive resistance mechanisms. Ongoing clinical and translational studies aim to refine these combinations and validate their safety, immune impact, and clinical benefit.

In a bridging ADA ELISA for immunogenicity testing of a Cetuximab biosimilar, the biosimilar is used both as the capture and detection reagent to specifically detect anti-drug antibodies (ADAs) generated against Cetuximab in patient serum.

Assay Principle and Workflow:

  • Capture Phase: Microtiter plate wells are coated with the Cetuximab biosimilar, which functions analogously to the reference molecule Erbitux®. This allows any ADA present in the patient’s serum sample (anti-Cetuximab antibodies) to bind the immobilized therapeutic.
  • Bridging Phase: After washing away unbound serum proteins, a labeled (often biotinylated or enzyme-conjugated) version of the same Cetuximab biosimilar is added. If ADA is present, it has two binding sites (since it is bivalent IgG), so one arm of the ADA can bind to the plate-bound Cetuximab while the other arm binds to the labeled Cetuximab—creating a “bridge”.
  • Detection: The presence of this sandwich is then detected via enzymatic (e.g., HRP and TMB substrate) or amplification systems, depending on the conjugate used. The signal produced is proportional to the amount of anti-Cetuximab ADA in the sample.
ComponentRole in ADA ELISA
Cetuximab biosimilarCoated on plate for capture; labeled for detection
Patient ADABridges capture and detection Cetuximab molecules
Enzyme/SubstrateEnables readout after bridging has occurred

Rationale:

  • This setup ensures only antibodies capable of binding two Cetuximab molecules are detected (typically bivalent IgG class)—enhancing specificity for true, high-affinity ADA against the drug.
  • The same biosimilar is used for both steps, so the assay robustly reflects patient immunogenicity to the actual clinical product or its biosimilar version.
  • Utilizing a biosimilar (instead of the originator) can reduce costs and support studies during biosimilar development or post-marketing surveillance.

Notes:

  • Use of a biosimilar as both capture and detection reagent requires careful validation to confirm equivalent antigenicity to the originator and appropriate negative/positive control performance.
  • Modifications may be required to address interference from circulating drug or preexisting anti-mouse or anti-chimeric antibodies in patient serum.

This approach is standard for ADA monitoring across various monoclonal antibody therapies, with the biosimilar substituting wherever regulatory and analytical comparability have been established.

References & Citations

1. Tortora, G. et al. (1999) Clin Cancer Res. 5(4):909-16.
2. Myers, J. et al. (2006) Clin Cancer Res. 12(2): 600–607.
CyTOF®
FA
Flow Cytometry

Certificate of Analysis

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Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.