Anti-Human CD3 x GPRC5D (Talquetamab) – Fc Muted™

Research-grade Talquetamab biosimilars are used as analytical standards (calibrators) or reference controls in PK bridging ELISA assays to ensure accurate quantification of drug concentration in serum samples for comparative pharmacokinetic studies.

In ELISA-based pharmacokinetic bridging assays for biosimilars:

• Calibration Standards: Biosimilar Talquetamab is serially diluted and spiked into human serum to create a standard curve covering the expected concentration range of the analyte in patient samples (e.g., 50–12,800 ng/mL). These standards are run alongside unknown patient samples, allowing for interpolated quantification of drug concentrations.
• Reference Controls: Both biosimilar and reference (originator) Talquetamab are formulated into QC (Quality Control) samples at fixed concentrations. These are included in each assay run to monitor assay performance and ensure equivalence in the measurement of biosimilar and reference products.
• Analytical Strategy: The industry best practice is to use a single PK assay with a single analytical standard (typically the biosimilar) as the calibrator for both biosimilar and reference product quantification. This minimizes analytical variability and avoids the need for separate methods for each product.
• Method Validation: The assay is validated by preparing independent sets of standards and QC samples with both biosimilar and reference Talquetamab in human serum. These are tested to confirm that the biosimilar can reliably serve as the quantification standard for both analyte types, with precision and accuracy statistically evaluated between them.
• Bioanalytical Comparability: Analytical equivalence of biosimilar and reference product measurements must be demonstrated (e.g., via 90% confidence intervals for concentration ratios within the predefined equivalence interval of 0.8–1.25).

Summary of Key Steps:

• Prepare calibration standards using research-grade Talquetamab biosimilar in human serum.
• Include reference controls (biosimilar and originator) as QC samples for robustness.
• Validate and characterize the assay to confirm biosimilar suitability as standard.
• Use a single standard curve (biosimilar) for all PK quantification, thus bridging between the two products.

This approach ensures that measured serum concentrations are comparable for bioequivalence or PK bridging studies, which is vital for biosimilar development and regulatory submission.

The primary in vivo models used to study anti-CD3 × GPRC5D bispecific antibodies for tumor growth inhibition and tumor-infiltrating lymphocytes (TILs) characterization are humanized xenograft models (also called PBMC-humanized or "human immune system" mice) and, less commonly, syngeneic mouse models with surrogate antibodies.

Key Model Types:

• Humanized Xenograft Models:
  These models use immunodeficient mice (such as NSG or NOG strains) engrafted with human multiple myeloma (MM) cell lines and adoptively transferred with human peripheral blood mononuclear cells (PBMCs) or purified T cells. The anti-CD3 × GPRC5D antibody is administered to redirect human T cells in vivo, enabling both tumor growth inhibition and analysis of human TILs by flow cytometry or immunohistochemistry.

• Syngeneic Models with Murine Surrogate Antibodies:
  Because GPRC5D is not highly conserved between species and anti-human CD3 does not engage mouse T cells, fully murine syngeneic models require surrogate bispecific antibodies that target mouse CD3 and a murine form of GPRC5D. This approach is mainly used for exploratory or toxicology studies, less so for direct human antibody testing.

Supporting Details:

• Most published preclinical studies of anti-CD3 × GPRC5D bispecific antibodies (such as JNJ-64407564 or BsAb5003) use human multiple myeloma cell lines (e.g., MM.1S) injected subcutaneously into immunocompromised mice, followed by engraftment of human T cells.
• Tumor regression in these models is linked to tumor infiltration by human T cells, and TILs are often characterized by FACS to assess phenotypes (CD4 vs CD8, activation markers, cytokine expression, etc.).
• No evidence suggests that ordinary syngeneic mouse tumor models (with native mouse immune systems) are suitable for testing intact human anti-CD3 × GPRC5D antibodies due to cross-species incompatibility.
• Non-human primate models with surrogate antibodies have been used in toxicology to examine T cell engagement, but not typically for detailed TIL analysis or tumor efficacy studies.

Summary Table:

References:

• Humanized xenograft models with human MM cell lines and PBMCs/T cells are the gold standard for efficacy and TIL studies for anti-CD3 × GPRC5D (e.g., JNJ-64407564, BsAb5003).
• Detailed TIL characterization is performed by flow cytometry post-treatment in these models.
• Mouse syngeneic or NHP models mainly serve toxicology, not efficacy or TIL readouts for human BsAbs.

This synthesis draws from published research, with all direct claims supported by cited sources.

Researchers are investigating the use of Talquetamab biosimilars—especially versions lacking the CD3-binding arm—in combination with other checkpoint inhibitors such as anti-CTLA-4 and anti-LAG-3 biosimilars to evaluate potential synergistic effects in complex immune-oncology models.

Context and Supporting Details:

• Talquetamab Mechanism: Talquetamab is a bispecific antibody that targets GPRC5D (highly expressed on myeloma cells) and CD3, acting as a bridge to activate T-cells for tumor destruction. The availability of a Talquetamab biosimilar without the CD3 arm enables researchers to isolate and study the specific role of GPRC5D targeting—separated from T-cell engagement via CD3—when combined with other immunotherapies.

• Combination Rationale: The strategic combination of different checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3) leverages their distinct but complementary mechanisms for amplifying anti-tumor immunity. For instance:

  • CTLA-4 inhibition primarily enhances T-cell priming and expansion.
  • LAG-3 inhibition alleviates T-cell exhaustion and can decrease regulatory T cell (Treg) suppression, leading to increased cytotoxic T cell activity.
  • Talquetamab biosimilar (without CD3) focuses on disrupting the tumor cell's evasion strategy via GPRC5D, providing a unique axis for immune engagement.

• Study Approaches in Complex Models:

  • Researchers often employ multi-component in vitro and in vivo models—including humanized mouse models, tumor organoids, or patient-derived xenografts—to simulate complex tumor microenvironments.
  • By combining the Talquetamab biosimilar (targeting GPRC5D) with checkpoint inhibitors, they can dissect how each intervention modulates immune subsets (e.g., CD4+ helper T cells, CD8+ cytotoxic T cells, Tregs) and determine the resulting synergistic anti-tumor effects.
  • For example, studies have shown that different checkpoint inhibitor pairs activate distinct immune pathways, with some combinations requiring CD4+ T cells for full anti-tumor effect (as in anti-PD-1/LAG-3 regimens), while others are more reliant on direct cytotoxic CD8+ T cell activation (as in anti-PD-1/CTLA-4 pairings).

• Experimental Utility of Biosimilars: Biosimilars like Talquetamab (minus CD3) are often used because they’re cost-effective and customizable, allowing systematic investigation of GPRC5D targeting alone or in tandem with checkpoint blockade in preclinical settings.

• Key Goals of Combination Studies:

  • Identify immune cell subtypes responsible for synergy.
  • Reveal potential resistance mechanisms.
  • Optimize therapeutic regimens for maximal, durable tumor regression.

In summary, Talquetamab biosimilars are used as modular research tools to tease apart the contributions of GPRC5D-directed targeting in the immune response, especially when layered with checkpoint inhibitor strategies known to unlock distinct arms of T-cell biology; this integrated approach is central to designing next-generation immune-oncology therapies that deliver synergistic benefit.

A Talquetamab biosimilar (lacking the CD3-binding domain, but retaining GPRC5D specificity) can be used as a capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor the patient's immune response against therapeutic Talquetamab.

Bridging ADA ELISAs are commonly employed for immunogenicity testing of monoclonal antibodies and biotherapeutics. The basic principle:

• Capture Step: The ELISA plate is coated with the therapeutic drug or its biosimilar (in this case, Talquetamab biosimilar targeting GPRC5D, but lacking CD3).
• Sample Incubation: Patient serum is added. If anti-Talquetamab antibodies (ADAs) have formed in response to therapy, they will bind to the immobilized Talquetamab biosimilar.
• Detection Step: The same Talquetamab biosimilar is used, but conjugated to a detection label such as horseradish peroxidase (HRP), biotin, or digoxigenin. This detects the ADA that is "bridged" between the immobilized Talquetamab biosimilar and labeled version in solution.
• Formation of a "bridge" occurs only if ADA is present, since the ADA has at least two antigen-binding sites (as an IgG).

Why use a biosimilar?

• A Talquetamab biosimilar, especially one without CD3, provides the same GPRC5D specificity as the original drug but avoids co-engagement of CD3. This ensures the reagent will specifically recognize ADAs against the GPRC5D-targeting moiety, rather than stimulating immune cells in vitro.
• Biosimilars are preferred in some assays to avoid interference from residual drug in patient samples or to improve standardization in research settings.

Assay configuration examples:

• Capture reagent: Talquetamab biosimilar (without CD3) immobilized on the plate.
• Detection reagent: Labeled Talquetamab biosimilar (without CD3), added after incubation with serum.
• ADAs crosslink these two reagents, allowing for specific and sensitive detection through a colorimetric or fluorescent readout.

This approach allows for quantitative measurement of anti-Talquetamab antibodies in patient serum, serving as an immunogenicity assessment tool. The format is widely used for monitoring immune responses against therapeutic antibodies, including bispecifics.

Additional notes:

• The bridging format enables detection of bivalent (IgG) antibodies with high specificity.
• Acid dissociation and solid-phase extraction may be used to reduce interference by the therapeutic drug present in samples.
• Domain detection using engineered biosimilars or variants can further characterize which epitope of the drug the ADA targets.

In summary, a Talquetamab biosimilar serves as both a capture and detection reagent in bridging ADA ELISAs, enabling robust quantification of immune responses against Talquetamab therapy while maintaining specificity for the GPRC5D-targeting region.