Anti-Human CD3 x GPRC5D (Talquetamab) [Clone JNJ-64407564]

Research-grade Talquetamab biosimilars can be used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to enable the quantitative measurement of drug concentrations—such as Talquetamab—in serum samples when performing method bridging or bioequivalence (particularly between biosimilar and reference products).

Context and Supporting Details:

• Calibration Standards:
  In PK bridging ELISAs for biosimilars, a single analytical standard (often the biosimilar) is used to build a standard curve for quantifying both the biosimilar and the reference (originator) products in serum samples. This approach is preferred as it reduces assay-to-assay variability and eliminates the need for separate standard curves for each product.

• Reference Controls:
  Alongside the calibration curve, quality control (QC) samples or reference controls prepared from both biosimilar and reference products are used to assess the assay's performance, accuracy, and comparability. These QC samples ensure the method remains precise and bioanalytically equivalent across both products within the established equivalence range (typically evaluated with a confidence interval, such as [0.8, 1.25]).

• Implementation in PK Bridging ELISA:

  • Prepare a series of calibration standards (using the biosimilar) at known concentrations in human serum matrix.
  • Analyze patient serum samples containing Talquetamab (either originator or biosimilar) and interpolate their concentrations using the standard curve established above.
  • Include reference controls made from both the biosimilar and reference Talquetamab at low, medium, and high concentrations within each assay run.
  • Validate the assay to ensure comparable recovery, linearity, and accuracy for both biosimilar and reference when measured against the biosimilar-based standard curve.

• Rationale:This strategy is rooted in regulatory and industry guidance, which stresses the necessity for a bioanalytically robust, accurate, and comparable assay to support PK bridging and biosimilar development. The entire process—beginning with method qualification followed by full validation—ensures that the biosimilar can serve as a reliable calibrator across clinical PK studies for both biosimilar and originator products.

Additional Information:

• Such bioanalytical strategy is not specific to Talquetamab but is recognized as best practice across the development of PK assays for various biologic and biosimilar therapeutics.
• The use of a single-method PK assay with biosimilar as the standard is part of industry consensus for minimizing variability and maximizing confidence in PK comparability assessments.

If additional details or specifics about Talquetamab ELISA procedures are needed, these can usually be found in the method validation section of clinical PK assay protocols or in regulatory filings for Talquetamab biosimilars.

The primary in vivo models used to evaluate research-grade anti-CD3 x GPRC5D bispecific antibodies for tumor growth inhibition and characterization of tumor-infiltrating lymphocytes (TILs) are humanized xenograft models, specifically:

• Human MM (multiple myeloma) xenografts in immunodeficient mice (e.g., NSG mice) reconstituted with human PBMCs or T cells.

Supporting details:

• These models involve implanting human GPRC5D+ myeloma cells (such as MM.1S) into immunodeficient mice, followed by adoptive transfer of human peripheral blood mononuclear cells (PBMCs) or purified T cells, and administration of the anti-CD3 x GPRC5D bispecific antibody.
• Tumor growth inhibition is assessed, and TILs are analyzed by flow cytometry or immunohistochemistry after treatment to characterize immune cell infiltration and activation.

Key references:

• JNJ-64407564, a humanized GPRC5D x CD3 antibody, was tested in a human MM.1S xenograft model in NSG mice with adoptively transferred human T cells, showing robust tumor regression correlated with T cell infiltration in tumors.
• BsAb5003, another anti-CD3 x GPRC5D bispecific, was evaluated in humanized MM xenograft models where tumor growth was suppressed by human T cells recruited and activated in vivo after antibody administration; TIL populations were assessed post-therapy.
• Similar approaches were used for other IgG-based anti-GPRC5D/CD3 bispecific antibodies, with efficacy and TILs characterized in xenograft models with human MM cells and human T cell engraftment.

Notes on model types:

• Syngeneic models (using mouse MM cell lines in fully immunocompetent mice) are not used here, as murine T cells do not directly engage with human CD3-targeted bispecifics and mouse GPRC5D expression would differ, limiting translational relevance.
• Humanized mouse models are essential due to the need for both human target antigen (GPRC5D) and human T cells expressing CD3 for bispecific engagement and resultant immune activity.

Characterization of TILs:

• TILs are typically analyzed after antibody therapy to assess recruitment, expansion, and activation state (markers such as CD3, CD4/CD8, PD-1, granzyme B, cytokine production).

In summary, humanized xenograft MM models with reconstituted human immune effector cells are the gold standard for preclinical in vivo evaluation of anti-CD3 x GPRC5D bispecific antibodies, including both tumor inhibition and TIL analysis.

Researchers use the Talquetamab biosimilar, specifically versions that lack the CD3 arm, in combination studies with other checkpoint inhibitors—such as anti-CTLA-4 or anti-LAG-3 biosimilars—to investigate synergistic immune responses in cancer models, most frequently within complex immune-oncology preclinical systems.

Key approach:

• The Talquetamab biosimilar targets the GPRC5D antigen on tumor cells, facilitating highly selective engagement and killing of malignancies typical of multiple myeloma, while checkpoint inhibitors modulate immune brakes to further unleash T-cell activity.
• By removing the CD3 engagement arm on Talquetamab biosimilars, researchers can isolate and study GPRC5D-specific targeting without introducing direct T-cell activation, thus minimizing confounding variables when dissecting synergistic or additive effects with checkpoint blockade.

Combination studies typically involve:

• Pairing Talquetamab biosimilar (GPRC5D-targeted only) with checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3) to assess how de-repression of T-cell activity (checkpoint blockade) interacts with tumor antigen specificity.
• Using immune-oncology models (murine xenografts, patient-derived xenografts, organoids) to track changes in:
  • Tumor cell killing—How the combined antibody therapies drive cytotoxic CD8+ T cell responses.
  • Immune cell activation and phenotype—Checkpoint inhibition can increase helper T cell (CD4+) and cytotoxic T cell (CD8+) populations, while Talquetamab directs this activity to GPRC5D+ tumor cells.
  • Regulatory cell suppression—Combinations (especially anti-LAG-3) may decrease suppressive Tregs, enhancing overall tumor rejection.

Synergistic effects observed:

• Mechanistic synergy: Combining Talquetamab biosimilar and checkpoint inhibitors may result in stronger T-cell infiltration, broader immune activation, and deeper tumor killing than either agent alone, as different immune pathways are targeted.
• Studies suggest anti-CTLA-4 promotes cytotoxic CD8+ T cell infiltration, while anti-LAG-3 regimens decrease Treg activity and boost CD4+ helper T cell functions, which in turn drive CD8+ cell-mediated tumor destruction.
• Talquetamab’s focused GPRC5D targeting can further direct the unleashed immune response specifically to tumor cells, minimizing off-target toxicity.

Research applications:

• Modeling combination efficacy: Early-phase research uses these biosimilars to optimize dosages, timing, and patient selection for future clinical trials.
• Cost-effective mechanistic studies: Biosimilars enable detailed exploration of drug interactions before committing to expensive, full-scale clinical-grade therapies.

Limitations and ongoing challenges:

• Most published work focuses on preclinical models, with limited direct clinical trial evidence in the public domain for Talquetamab biosimilar combinations specifically.
• The exact molecular interplay—especially how checkpoint inhibitor-induced T-cell activation interacts with GPRC5D-targeted effects—is the subject of ongoing mechanistic research and will benefit from further immune profiling and multi-omics approaches.

In summary, researchers employ Talquetamab biosimilars alongside checkpoint inhibitors in complex models to dissect and leverage their synergistic immune effects, aiming to enhance targeted cancer immunotherapy while reducing toxicity and overcoming resistance.

In immunogenicity testing for Talquetamab, a biosimilar version (that binds only GPRC5D and not CD3) can be used as either a capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor patient immune responses against the therapeutic drug.

A bridging ADA ELISA works by detecting antibodies generated by the patient's immune system against the therapeutic drug (i.e., Talquetamab) through the following approach:

• Capture Reagent: The biosimilar Talquetamab (lacking CD3 binding, retaining GPRC5D specificity) is coated on the ELISA plate. Patient serum is added, and if anti-Talquetamab antibodies (ADAs) are present, they bind the immobilized biosimilar.
• Detection Reagent: A second, labeled form of the biosimilar Talquetamab is added (for example, biotinylated or enzyme-conjugated). This molecule binds to a different epitope on any ADA that is already captured by the plate-bound biosimilar, forming a “bridge.”
• This bridging complex generates a measurable signal (such as color change), which is proportional to the amount of ADA in the serum.

The Talquetamab biosimilar without CD3 is ideal for this application because:

• It preserves the GPRC5D-binding domain, ensuring that it closely mimics the therapeutic epitope composition recognized by the immune system.
• Lacking CD3 reduces unnecessary T cell interaction or off-target binding, providing assay specificity and minimizing background signals.

Key Benefits and Technical Context:

• Using a biosimilar as both capture and detection reagent ensures the assay detects antibodies specific to the therapeutic regions of Talquetamab most likely to be immunogenic.
• Bridging ELISAs are the industry standard for ADA detection against monoclonal and bispecific antibodies, offering high sensitivity and specificity.
• This approach allows monitoring of patient immunogenicity risk, which is especially important for novel biotherapeutics like Talquetamab, where immune responses could impact efficacy and safety.
• Variations of the biosimilar, such as those lacking one domain (CD3), allow fine mapping of ADA specificity.

In summary, a Talquetamab biosimilar without CD3 is used in a bridging ELISA as both capture and detection reagent to sensitively and specifically detect patient-derived anti-Talquetamab antibodies, providing a critical tool for monitoring immunogenicity in clinical and research settings.