Anti-Human CD33 (Gemtuzumab) – Fc Muted™

Anti-Human CD33 (Gemtuzumab) – Fc Muted™

Product No.: C1045

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Product No.C1045
Clone
hP67.6
Target
CD33
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
SIGLEC-3, SIGLEC3, p67, gp67
Isotype
Human IgG4κ
Applications
ELISA
,
FC
,
IF
,
WB

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Antibody Details

Product Details

Reactive Species
Human
Host Species
Human
Expression Host
HEK-293 Cells
FC Effector Activity
Muted
Immunogen
Humanized antibody derived from mouse clone P67.6
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
Country of Origin
USA
Shipping
2-8°C Wet Ice
Additional Applications Reported In Literature ?
ELISA,
WB,
IF,
FC
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 Gemtuzumab but is not covalently linked to Calich-DMH. This product is for research use only. Gemtuzumab antibody activity is directed against CD33.
Background
CD33 is a sialic-acid-binding immunoglobulin-like lectin (Siglec) that acts as an endocytic receptor1. CD33 is considered an attractive target for conjugated antibody chemotherapeutic development in patients with acute myeloid leukemia (AML) because ~90% of patients express CD33 surface antigen on myeloid blast cells, but not normal stem cells2, and additionally CD33 is rapidly internalized when bound3.

N-acetyl-γ-calicheamicin is a potent, natural cytotoxic agent produced by Micromonospora echinospora that induces double-strand DNA breaks and apoptosis in rapidly proliferating cells, independent of cell cycle progression, and is therefore also of interest as a chemotherapeutic agent3, 4. The semisynthetic derivative N-acetyl-γ-calicheamicin dimethyl hydrazide (Calich- DMH; calicheamicin) is used as an enediyne antitumor antibiotic in CD33-based chemotherapy3.

Gemtuzumab is an antibody-drug conjugate composed of Calich-DMH attached via acetyl butyrate linker to hP67.6, an anti-CD33 antibody humanized from its murine progenitor by CDR grafting3. The conjugate contains a lysine attachment to the antibody as well as a hydrazone linkage which allows for hydrolytic release. When Gemtuzumab binds CD33-expressing tumor cells, the Gemtuzumab-CD33 complex is rapidly internalized and the acidic intracellular environment (presumably in the endosomes/lysosomes of target cells) triggers the release of Calich-DMH. Calich-DMH then binds to the minor groove of DNA, undergoes a structural change in its enediyne moiety that generates diradicals, and induces double-strand DNA breakage, cell cycle arrest and apoptosis3, 4, 5. Gemtuzumab temporarily arrests NB4 cells, but not clinical samples, at the G2/M phase and increases the percentage of hypodiploid cells in cell lines as well as clinical samples5, 6, 7. Gemtuzumab has a drug loading capacity of 2-3 mol of Calich-DMH per mole of antibody3. However, the effects of Gemtuzumab are negatively influenced by P-glycoprotein6.

Gemtuzumab has been approved for treatment of some patients with relapsed acute myeloma who are aged 60 and over2.

Antigen Distribution
CD33 is expressed on normal multipotent myeloid precursor cells, unipotent colony-forming cells, maturing granulocytes and monocytes, macrophages, dendritic cells, and can be displayed on subsets of B cells, activated T cells and natural killer cells. CD33 is also expressed on the surface of leukemic cell blasts in more than 90% of patients with acute myeloid leukemia, but is not present on normal stem cells.
Ligand/Receptor
sialic acid residues
NCBI Gene Bank ID
UniProt.org
Research Area
Autoimmune
.
Biosimilars
.
Cancer
.
Immunology
.
Inflammatory Disease

Leinco Antibody Advisor

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Research-grade Gemtuzumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to quantitatively measure drug concentration in serum samples, provided there is demonstrated analytical equivalence between the biosimilar and reference material.

To ensure the assay accurately measures Gemtuzumab concentrations for both biosimilar and reference (originator) products, the following steps and rationale typically apply:

  • Single PK Assay Design: The prevailing and regulatory-preferred approach is to develop a single PK ELISA that uses a single analytical standard (often the biosimilar) as the calibrator for quantifying both biosimilar and reference drug concentrations in sample matrices (like serum).
  • Analytical Equivalence Demonstration: Before a biosimilar can be used as the calibrator, rigorous method qualification is performed. This involves generating precision and accuracy datasets for both biosimilar and originator and applying statistical analyses to demonstrate bioanalytical equivalence within the assay (typically the 90% confidence interval of the ratio of means for key assay responses must fall within a predefined equivalence range, e.g., 0.8–1.25).
  • Calibration Curve Preparation: Calibration (standard) curves are prepared using known concentrations of the research-grade biosimilar in the same matrix (e.g., human serum) in which unknown samples will be measured. These calibration curves are used to interpolate the concentrations of Gemtuzumab in serum samples based on the measured signal (e.g., absorbance).
  • Reference Controls: Both the biosimilar and reference standard (originator) may be included as quality control (QC) samples prepared at several concentrations to ensure ongoing assay accuracy and precision, and to validate the assay's ability to measure both products appropriately.
  • Reduced Variability: Using a single assay and calibration standard (rather than two separate assays or standard curves) reduces analytical variability, simplifies validation, and supports direct comparison between biosimilar and reference products—critical for regulatory PK bridging studies.

Key technical implementation details:

  • The ELISA method must be validated for specificity, linearity, accuracy, precision, sensitivity, and lack of interference in the relevant serum matrix.
  • Typical PK ELISA calibration ranges for antibody drugs like Gemtuzumab fall between 50-12800 ng/mL, with QCs spanning this concentration range.
  • Both calibrators (biosimilar) and controls (reference/biosimilar/QC samples) are aliquoted into the assay plate before subject samples, and assay readout is compared against the calibration curve.

In summary:
Use of a research-grade Gemtuzumab biosimilar as a calibrator in a PK ELISA is best practice if and only if comparability with the reference product is analytically validated. The biosimilar-based calibration curve enables robust, reproducible, and regulatory-compliant quantitation of Gemtuzumab concentration in serum for both test and reference article measurements.

Primary in vivo models for evaluating anti-CD33 antibodies in tumor growth inhibition and TIL characterization are human xenograft models, particularly using immunodeficient mice engrafted with human AML cells, and, less commonly, humanized mouse models. Syngeneic models are standard for immunotherapy studies and TIL analysis but are not used for anti-CD33 antibodies unless the tumor expresses CD33 and the antibody is cross-reactive to murine CD33.

Model Types Utilized:

  • Human Xenograft Models:

    • Most published studies of anti-CD33 antibodies (including antibody-drug conjugates like SGN-CD33A) use xenograft models, where human AML cell lines (e.g., HL-60, TF1-α, HEL 92.1.7) or primary patient-derived AML cells are implanted subcutaneously or systemically into immunodeficient mice, such as NSG or SCID mice.
    • These models allow assessment of tumor growth inhibition following antibody administration, including dose-dependent responses and durable remissions.
    • Since these mice lack a functional mouse immune system, TIL characterization is possible only when the model is further humanized (see below).
  • Humanized Mouse Models:

    • In some studies, immunodeficient mice are engrafted with human hematopoietic stem cells or PBMCs to partially reconstitute a human immune system, enabling the study of human-specific TILs after therapy.
    • These models can be used to assess changes in human TIL populations after anti-CD33 therapy, although this approach is less commonly reported in published literature compared to xenograft models without reconstitution.
  • Syngeneic Mouse Models:

    • Standard syngeneic tumor models use mouse tumor cell lines in immunocompetent mice; these are best for studying immunotherapy, TILs, and the full mouse immune landscape.
    • However, syngeneic models are not inherently suitable for anti-CD33 antibody studies because mouse CD33 often differs from human CD33 and human antibodies may not cross-react. A syngeneic setup can only be used if the mouse tumor expresses the target antigen and the antibody is reactive against mouse CD33. There is no evidence of wide use of anti-CD33 research-grade antibodies in standard syngeneic models for TIL studies.

TIL Characterization:

  • TIL analysis is routine in syngeneic models, using flow cytometry or immunohistochemical methods to quantify multiple immune subsets (CD8 T cells, myeloid-derived suppressor cells, etc.).
  • For xenografted or humanized models, analysis focuses on human immune cells if the model supports their engraftment and maintenance.

Summary Table

Model TypeImmune systemTumor TypeAnti-CD33 UseTIL Characterization Option
XenograftImmunodeficient (NSG/SCID)Human AML cell lines / patient-derivedYesLimited (unless humanized)
Humanized xenograftPartially human (engrafted PBMC/HSC)Human AMLYesYes (human TILs)
SyngeneicFully mouseMurine tumor linesRare (req. cross-reactive antibody)Yes (murine TILs)

Key references for anti-CD33 antibodies in vivo:

  • Xenograft models: HL-60, TF1-α, HEL 92.1.7 AML cell lines; patient-derived AML samples; treated with SGN-CD33A; robust tumor growth inhibition, survival benefit.
  • Humanized and xenograft models allow TIL characterization only for human immune cells when present.

In summary, anti-CD33 research-grade antibodies are administered principally in human AML xenograft models and, less frequently, humanized mice. TIL analysis is possible and routine in syngeneic models for immunotherapy, but anti-CD33 antibody use in these models is uncommon due to antigen specificity limitations.

Researchers investigating synergistic effects in complex immune-oncology models often use Gemtuzumab biosimilars—which target CD33-positive leukemic cells—alongside other checkpoint inhibitor biosimilars (such as anti-CTLA-4 or anti-LAG-3) to assess how multiple immune pathways can be modulated simultaneously. This combination approach helps to elucidate whether targeting more than one immune mechanism can enhance antitumor activity beyond what is achievable with monotherapy.

Key elements of these investigations:

  • Gemtuzumab biosimilar mechanism: Gemtuzumab is an antibody-drug conjugate targeting CD33 on AML cells, delivering a cytotoxic agent to induce cell death with minimal damage to healthy tissues.
  • Checkpoint inhibitors: Agents like anti-CTLA-4 and anti-LAG-3 biosimilars block immune checkpoints—regulatory pathways that suppress immune responses in cancer. CTLA-4 blockade mainly acts in lymph nodes to restore T cell activation, while anti-LAG-3 works to enhance T cell function and overcome immune exhaustion.
  • Rationale for combination: By targeting both CD33-positive tumor cells (direct cytotoxic effect) and checkpoint pathways (immune system activation), researchers can evaluate whether simultaneous direct killing and immune activation produce additive or synergistic effects against tumors, as suggested by preclinical and early clinical studies.
  • Experimental design: In immune-oncology model systems (e.g., mouse models of AML or patient-derived xenografts), researchers co-administer Gemtuzumab biosimilars with checkpoint inhibitors and measure outcomes such as tumor regression, increased cytotoxic T cell infiltration, and survival. Immune profiling is performed to assess changes in T cell phenotypes, macrophage polarization, and immune activation markers.

Synergy evaluation:

  • Researchers compare outcomes from monotherapy arms (e.g., Gemtuzumab alone, anti-CTLA-4 alone) versus the combination arm, frequently observing enhanced antitumor activity and increased immune activation with combinations.
  • Studies often address mechanistic questions, for example, whether cytotoxic payload delivery by Gemtuzumab leads to increased antigen release and presentation, thereby enhancing the effects of checkpoint inhibition.

Challenges in biosimilar research:

  • The use of biosimilars rather than originator biologics allows for expanded preclinical and translational studies due to lower cost and easier accessibility, but requires careful immunogenicity profiling to avoid confounding synergistic effects with non-specific immune responses.

Summary table: Gemtuzumab Biosimilar + Checkpoint Inhibitor Combinations

AgentTargetMechanismSynergy Rationale
Gemtuzumab biosimilarCD33Direct cytotoxicity to AML cellsIncreased antigen release, possible immune priming
Anti-CTLA-4 biosimilarCTLA-4Blocks T cell inhibition in lymph nodesRestores T cell activation, complements cytotoxicity
Anti-LAG-3 biosimilarLAG-3Enhances T cell activation/exhaustionOvercomes immune escape, boosts combination effect

This multifaceted approach is increasingly central in studies aiming to overcome resistance and improve outcomes in complex cancers like AML by engaging both direct cytotoxicity and immune-system reactivation.

A Gemtuzumab biosimilar can be used as both the capture and detection reagent in a bridging ADA ELISA to detect anti-drug antibodies (ADAs) that develop in response to gemtuzumab therapy in patients with AML.

Bridging ADA ELISA works as follows:

  • Capture Step: The gemtuzumab biosimilar is immobilized on the ELISA plate. Patient serum is added, and any ADAs present will bind to the drug.
  • Detection Step: A labeled form of the same gemtuzumab biosimilar (commonly conjugated to biotin or HRP) is added. If ADAs are present, they “bridge” between the capture and detection gemtuzumab, forming a drug-ADA-drug complex. The label enables quantification.

Key details for using a biosimilar in this assay:

  • The biosimilar must have the same antigenic structure as the therapeutic gemtuzumab, ensuring it can bind ADAs targeting the original drug.
  • Using the biosimilar is cost-effective and ensures batch-to-batch consistency, which is important for reliable immunogenicity results.
  • Detection reagents can be directly conjugated or biotinylated, with subsequent addition of streptavidin-HRP and chromogenic substrate for signal generation.
  • The biosimilar should be pure, endotoxin-free, and validated for ELISA applications.

Applications:

  • This approach monitors a patient’s immune response by quantifying the presence and level of ADAs specific to gemtuzumab, which can impact therapeutic efficacy and safety.

Additional notes:

  • Bridging ELISA is widely used for ADA detection due to its specificity, sensitivity, and ability to detect diverse isotypes of ADAs (e.g., IgG, IgM). It is critical to validate the assay for selectivity, interference, and sensitivity for clinical immunogenicity monitoring.

In summary, Gemtuzumab biosimilar is both plate-bound (capture) and labeled (detection) in bridging ADA ELISA, allowing sensitive detection of patient antibodies directed against the therapeutic gemtuzumab.

References & Citations

1 Clark MC, Stein A. Best Pract Res Clin Haematol. 33(4):101224. 2020.
2 McGavin JK, Spencer CM. Drugs. 61(9):1317-1322; discussion 1323-4. 2001.
3 Hamann PR, Hinman LM, Hollander I, et al. Bioconjug Chem. 13(1):47-58. 2002.
4 Thota S, Advani A. Eur J Haematol. 98(5):425-434. 2017.
5 Naito K, Takeshita A, Shigeno K, et al. Leukemia. 14(8):1436-1443. 2000.
6 Matsui H, Takeshita A, Naito K, et al. Leukemia. 16(5):813-819. 2002.
7 Takeshita A, Shinjo K, Naito K, et al. Leukemia. 19(8):1306-1311. 2005.
8 Larson RA, Sievers EL, Stadtmauer EA, et al. Cancer. 104(7):1442-1452. 2005.
Indirect Elisa Protocol
Flow Cytometry
IF
General Western Blot Protocol

Certificate of Analysis

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