Anti-Human CD20 (Obinutuzumab) [Clone GA101] — Purified No Carrier Protein

Anti-Human CD20 (Obinutuzumab) [Clone GA101] — Purified No Carrier Protein

Product No.: LT906

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Product No.LT906
Clone
GA101
Target
CD20
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
Obinutuzumab, CD20, MS4A1
Isotype
Human IgG1κ
Applications
ELISA
,
FA
,
FC
,
IP
,
WB

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Select Product Size
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Antibody Details

Product Details

Reactive Species
Human
Host Species
Human
Expression Host
HEK-293 Cells
FC Effector Activity
Active
Immunogen
Human lymphoblastoid cell line SB.
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
Additional Applications Reported In Literature ?
ELISA,
FA,
FC,
IP,
WB
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 Obinutuzumab. This product is for research use only. Obinutuzumab (GA101) activity is directed against human CD20.
Background
CD20 is a nonglycosylated 33-37 kDa phosphoprotein member of the MS4A family which is widely expressed on normal B cell surfaces during all stages of development as well as by most B cell malignancies1,2. The biological role of CD20 remains poorly understood; however, it is thought to be involved in calcium ion influx. CD20 has no natural ligand and is not immediately internalized upon antibody binding. Thus, mAbs directed against CD20 depend on the recruitment of a host response. Anti-CD20 mAbs bind to the 44 amino acid extracellular portion.

Obinutuzumab (GA101) is a new generation, type II, anti-CD20 antibody2. Obinutuzumab was humanized by grafting the complementarity-determining sequences of murine IgG1-κ antibody B-Ly1 onto human VH and VL acceptor frameworks3. The Fc segment was glycoengineered to attach bisected, complex, nonfucosylated oligosaccharides to asparagine 297, leading to increased affinity to FcgRIII.

Obinutuzumab causes homotypic adhesion4,5,6, induces direct cell death via largely caspase-independent mechanisms4,6,7,8,9, does not localize into lipid rafts4,10,11, displays half-maximal CD20 binding at saturating conditions7, and displays minimal complement dependent cytotoxicity7.

Compared to rituximab, obinutuzumab recognizes a distinct but overlapping CD20 epitope, in a different orientation that results in increased pro-apoptotic potential12,13,14. A modified elbow-hinge residue, characterized by a leucine to valine mutation at Kabat position 11, is key to superior phosphatidylserine exposure and cell death relative to rituximab3.
Antigen Distribution
CD20 is a general B cell marker expressed by the majority of normal B cells in all stages of their development as well as by most B cell malignancies.
Ligand/Receptor
Src family tyrosine kinases, MHC class I, II, CD53, CD81, CD82
PubMed
NCBI Gene Bank ID
UniProt.org
Research Area
Biosimilars
.
Cancer
.
Immunology
.
Oncology

Leinco Antibody Advisor

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Research-grade Obinutuzumab biosimilars are used as calibration standards (or reference controls) in pharmacokinetic (PK) bridging ELISAs by preparing a standard curve from known concentrations of the biosimilar, which is then used to quantify Obinutuzumab (both originator and biosimilar) in serum samples. This supports PK measurement and comparability assessments in biosimilar development.

Essential Context and Supporting Details

  • Calibration Standard Role: In PK bridging ELISAs for biosimilar studies, a single analytical standard—often the approved research-grade biosimilar Obinutuzumab—is selected to construct the calibration (standard) curve. Serum samples and quality control (QC) samples containing either the biosimilar or reference product are quantified by comparison to this standard curve.

  • Reference Control Role: The same preparation of biosimilar can serve as a reference control in the assay, ensuring that the assay's performance (accuracy, precision, and quantitation capability) is tracked over time and across multiple assay runs.

Why Use the Biosimilar as the Standard?

  • Assay Harmonization: Using the biosimilar as the calibration standard minimizes inter-assay variability, allows direct quantification of both the biosimilar and originator drug, and aligns with regulatory recommendations for demonstrating PK equivalence.
  • Bioanalytical Comparability: Before adopting the biosimilar as the standard, a method qualification study is required to show that both the biosimilar and reference Obinutuzumab are bioanalytically equivalent within the assay context (e.g., recovery, linearity, precision).
  • Standard Preparation: The biosimilar for the calibration curve is usually prepared by serial dilution in drug-naïve (blank) serum to mirror the matrix of patient samples and establish the relationship between concentration and assay signal. For example, concentrations might typically range from 50 ng/mL to 12,800 ng/mL depending on assay sensitivity and dynamic range.

PK Bridging ELISA Workflow (Summary)

  • Coating and Capture: Microplates are coated with a capture antibody or antigen specific for Obinutuzumab.
  • Addition of Standards and Samples: Serial dilutions of the research-grade biosimilar (the standard) and unknown serum samples are added.
  • Detection: Detection antibodies (sandwich or bridging format) generate a signal proportional to drug concentration.
  • Quantification: The signal from unknown samples is interpolated from the standard curve, which is based on the known concentrations of the biosimilar calibration standard.

Additional Relevant Details

  • Research-Use Only: These biosimilars and ELISA kits are strictly for non-clinical research; they are not intended for diagnostic or therapeutic use.
  • Kit Compatibility: Vendors may provide biosimilar reference standards or controls as lyophilized (freeze-dried) materials suitable for calibration in ELISAs and compatible with both serum and plasma samples.

In summary, research-grade Obinutuzumab biosimilars serve both as calibration standards and reference controls in PK bridging ELISAs, enabling reliable and comparable quantification of drug levels in serum during biosimilar development and PK equivalence assessments.

The primary in vivo models for administering a research-grade anti-CD20 antibody to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are syngeneic mouse tumor models (with either native or humanized targets) and, in certain studies, humanized or transgenic mice engineered to express human CD20.

Key model types:

  • Syngeneic murine tumor models:
    These use murine tumor cell lines implanted into immunocompetent mice of the same genetic background. A notable example is the use of mouse models such as MC38 (colon carcinoma), CT26 (colon carcinoma), RENCA (renal carcinoma), TC-1 (lung carcinoma), and B16F10 (melanoma). These models allow for the use of anti-mouse CD20 antibodies to deplete mouse B cells and permit in-depth analysis of TIL composition following treatment.

    • For example, Kim et al. demonstrated tumor growth inhibition and enhanced immunotherapy responses using a mouse anti-CD20 antibody in models such as TC-1, correlating this with changes in CD8+ T cell infiltration in tumors and spleens.
    • These models enable detailed immune profiling, as tumor microenvironments can be immunologically "hot" or "cold" (e.g., highly infiltrated RENCA vs. poorly infiltrated B16F10), thus informing on immune modulation by anti-CD20 treatment.
  • Human CD20 transgenic/syngeneic models:Another advanced model involves syngeneic murine lymphoma cells (such as A20) engineered to express human CD20, implanted into mice that are also transgenic for human CD20 and sometimes human CD3. This setup allows testing of research-grade human anti-CD20 antibodies and their bispecific variants (e.g., CD20-T cell dependent bispecifics, CD20-TDB).

    • These models are particularly useful for evaluating human therapeutic antibodies that do not cross-react with mouse CD20.
    • Tumor growth inhibition and TIL characterization (e.g., T cell activation, PD-1/PD-L1 expression) are assessed post-treatment, with combinatorial studies (e.g., anti-CD20 with anti-PD-1/PD-L1) frequently performed.
  • Humanized mouse models (less common for TIL studies):Fully humanized mice (engrafted with human immune cells and tumors) can also be used but are less common for initial mechanistic studies of TILs and anti-CD20, in part due to cost and complexity. Instead, engineered syngeneic models expressing human targets are typically preferred for preclinical research-grade antibody testing.

Summary Table: Models Used for Anti-CD20 Efficacy and TIL Analysis

Model TypeAntibody UsedRelevance to Human TherapyTIL Analysis Possible
Syngeneic (mouse tumor in mouse)Anti-mouse CD20Directly models immune responseYes
Syngeneic (murine tumor, human CD20+)Human(ized) anti-CD20Tests human-targeting antibodiesYes
Humanized mouse with human immune systemHuman(ized) anti-CD20Closest mimic to human immune systemChallenging but possible

Conclusion:
Syngeneic mouse tumor models (sometimes expressing human CD20 in the tumor and/or host) are the dominant platform for in vivo studies with research-grade anti-CD20 antibodies aimed at understanding tumor growth inhibition and TIL profiles. Studies frequently focus on murine solid tumors or engineered lymphoma cell lines, with direct analysis of TILs post-treatment. Humanized models, while relevant for translational studies, are less commonly the first choice for mechanistic TIL characterization due to their inherent challenges.

Researchers investigating the synergistic effects of combining Obinutuzumab biosimilars with other checkpoint inhibitors like anti-CTLA-4 or anti-LAG-3 biosimilars in immune-oncology models typically follow a structured approach:

Approach to Combining Obinutuzumab Biosimilars with Checkpoint Inhibitors

  1. Target Selection:

    • Obinutuzumab Biosimilar: This targets CD20, a key marker on B cells, which is crucial in treating various lymphomas and autoimmune diseases.
    • Anti-CTLA-4 Inhibitors: These block CTLA-4, enhancing T-cell activation and proliferation, particularly affecting early T-cell responses.
    • Anti-LAG-3 Inhibitors: LAG-3 is a checkpoint that inhibits T-cell expansion and function. Blocking it can enhance antitumor immunity.
  2. Preclinical Modeling:

    • In Vitro Studies: Researchers use cell cultures to assess how these combinations affect immune cell interactions, focusing on synergistic effects like enhanced T-cell activation and B-cell depletion.
    • In Vivo Models: Animal models (e.g., xenografts or syngeneic models) are used to evaluate the efficacy and safety of these combinations in a more complex biological environment.
  3. Assessment of Synergistic Effects:

    • Cytokine Profiling: Analyze changes in cytokine levels to understand immune modulation.
    • Flow Cytometry and Immunohistochemistry: Use these techniques to assess changes in immune cell populations within tumors.
    • Tumor Growth and Survival Studies: Evaluate how these combinations impact tumor progression and animal survival.
  4. Clinical Translation:

    • Clinical Trials: Early-phase clinical trials are conducted to assess safety and efficacy in humans. These trials typically involve small cohorts and focus on dosing regimens and potential toxicities.

Rationale for Combination Therapy

  • Mechanism Diversity: Combining different immunotherapies with non-overlapping mechanisms can enhance antitumor responses by targeting multiple aspects of the immune system.
  • Overcoming Resistance: Some tumors develop resistance to single-agent therapies. Combining treatments can help overcome this resistance by targeting multiple pathways.

Challenges and Future Directions

  • Toxicity Management: Combined therapies can increase toxicities, necessitating careful monitoring and management strategies.
  • Biomarker Development: Identifying biomarkers to predict response to combination therapies is crucial for personalized medicine approaches.

By studying these combinations in preclinical models and translating findings into clinical trials, researchers can better understand how to harness the synergistic effects of Obinutuzumab biosimilars with checkpoint inhibitors like anti-CTLA-4 and anti-LAG-3 to improve cancer treatment outcomes.

A Obinutuzumab biosimilar is used in a bridging anti-drug antibody (ADA) ELISA to specifically detect antibodies generated by a patient's immune system against the therapeutic Obinutuzumab, employing the biosimilar as either the capture or detection reagent. In this assay, the Obinutuzumab biosimilar serves as both an antigen to “capture” ADA from patient serum and as a labeled version for detection, capitalizing on the bivalent binding nature of human antibodies.

Workflow and Principle:

  • Capture Step: The Obinutuzumab biosimilar (unlabeled) is immobilized on the microplate to serve as the capture reagent, or alternatively, patient serum is first added to plates pre-coated with streptavidin if a biotinylated version is used.
  • Sample Incubation: Patient serum, potentially containing anti-obinutuzumab ADA, is incubated in the well. Any ADA present will bind to the immobilized Obinutuzumab (biosimilar).
  • Detection Step: The same Obinutuzumab biosimilar, but now conjugated to a detection label (e.g., HRP or biotin), is added. If ADA is present (with two antigen-binding sites), it forms a “bridge” by simultaneously binding to both the immobilized and the labeled Obinutuzumab biosimilar.
  • Signal Generation: A substrate is added, and the presence of the signal (e.g., color change by HRP/TMB) is proportional to the amount of ADA bound, reflecting the patient’s immune response to the therapeutic.

Key Points:

  • This bridging ELISA approach exploits the bivalency of ADA: one arm binds to immobilized drug, the other to the labeled form.
  • Using a biosimilar rather than the original drug ensures detection of ADA that recognize both, but the biosimilar must be highly similar structurally to avoid missing relevant immune responses.
  • The assay can detect all classes of ADA (e.g., IgG, IgM) that bind to the therapeutic.
  • The format is sensitive and high-throughput but can be prone to matrix effects and interference from circulating drug.

Application to Immunogenicity Monitoring:

  • By tracking ADA levels, clinicians and researchers can assess potential loss of efficacy or risk of hypersensitivity in patients receiving Obinutuzumab therapy.
  • Data from nonclinical studies show that immunogenicity can result in decreased drug exposure and altered pharmacodynamics, reinforcing the importance of sensitive ADA detection.

Summary Table: Obinutuzumab Biosimilar in Bridging ADA ELISA

Component/StepRole of Obinutuzumab BiosimilarFunction
CaptureImmobilized (unlabeled or biotinylated)Binds to ADA in patient serum
DetectionLabeled (e.g., HRP-conjugated)Forms bridge via ADA, allows signal readout
Target/AnalytePatient anti-Obinutuzumab ADAIndicates patient immune response

This bridging ADA ELISA, using an Obinutuzumab biosimilar as both capture and detection reagent, is a validated, widely used method in immunogenicity programs for monoclonal antibodies.

References & Citations

1. Middleton O, Wheadon H, Michie AM. Classical Complement Pathway. In MJH Ratcliffe (Ed.), Reference Module in Biomedical Sciences Encyclopedia of Immunobiology Volume 2 (pp. 318-324). Elsevier. 2016.
2. Freeman CL, Sehn LH. Br J Haematol. 182(1):29-45. 2018.
3. Mössner E, Brünker P, Moser S, et al. Blood. 115(22):4393-4402. 2010.
4. Chan HT, Hughes D, French RR, et al. Cancer Res. 63(17):5480-5489. 2003.
5. Ivanov A, Beers SA, Walshe CA, et al. J Clin Invest. 119(8):2143-2159. 2009.
6. Alduaij W, Ivanov A, Honeychurch J, et al. Blood. 117(17):4519-4529. 2011.
7. Herter S, Herting F, Mundigl O, et al. Mol Cancer Ther. 12(10):2031-2042. 2013.
8. Honeychurch J, Alduaij W, Azizyan M, et al. Blood. 119(15):3523-3533. 2012.
9. Golay J, Zaffaroni L, Vaccari T, et al. Blood. 95(12):3900-3908. 2000.
10. Cragg MS, Morgan SM, Chan HT, et al. Blood. 101(3):1045-1052. 2003.
11. Cragg MS, Glennie MJ. Blood. 103(7):2738-2743. 2004.
12. Niederfellner G, Lammens A, Mundigl O, et al. Blood. 118(2):358-367. 2011.
13. Klein C, Lammens A, Schäfer W, et al. MAbs. 5(1):22-33. 2013.
14. Könitzer JD, Sieron A, Wacker A, Enenkel B. PLoS One. 10(12):e0145633. 2015.
15. Terszowski G, Klein C, Stern M. J Immunol. 192(12):5618-5624. 2014.
16. Bologna L, Gotti E, Manganini M, et al. J Immunol. 186(6):3762-3769. 2011.
17. Ysebaert L, Laprévotte E, Klein C, Quillet-Mary A. Blood Cancer J. 5(11):e367. 2015.
18. Cartron G, Hourcade-Potelleret F, Morschhauser F, et al. Haematologica. 101(2):226-234. 2016.
Indirect Elisa Protocol
FA
Flow Cytometry
Immunoprecipitation Protocol
General Western Blot Protocol

Certificate of Analysis

Formats Available

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