Anti-Human PD-1 (Spartalizumab)

Product No.: P450

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Product No.P450
Clone
PDR001
Target
PD-1
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
CD279, PD1, Anti-PD1, PDCD1
Isotype
Human IgG4κ
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 PD-1
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,
IP,
FA,
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 Spartalizumab. This product is for research use only. Spartalizumab activity is directed against human PD-1.
Background
PD-1 is a transmembrane protein in the CD28/CTLA-4 subfamily of the Ig superfamily 1, 2 . When stimulated via the T cell receptor (TCR), Tregs translocate PD-1 to the cell surface 3. Programmed cell death 1 ligand 1 (PD-L1; CD274; B7H1) and programmed cell death 1 ligand 2 (PD-L2; CD273; B7DC) have been identified as PD-1 ligands 1. PD-1 is co-expressed with PD-L1 on tumor cells and tumor-infiltrating antigen-presenting cells (APCs) 2 . Additionally, PD-1 is co-expressed with IL2RA on activated CD4 + T cells 3 .

PD-1 is an immune checkpoint receptor that suppresses cancer-specific immune responses 4. Additionally, PD-1 acts as a T cell inhibitory receptor and plays a critical role in peripheral tolerance induction and autoimmune disease prevention as well as important roles in the survival of dendritic cells, macrophage phagocytosis, and tumor cell glycolysis 2. PD-1 prevents uncontrolled T cell activity, leading to attenuation of T cell proliferation, cytokine production, and cytolytic activities. Additionally, the PD-1 pathway is a major mechanism of tumor immune evasion, and, as such, PD-1 is a target of cancer immunotherapy 2.

Spartalizumab is a humanized IgG4 anti-PD1 antibody that has been tested for the treatment of various cancers 5, 6. Spartalizumab binds PD-1 with sub-nanomolar affinity and blocks interactions with ligands PD-L1 and PD-L2, leading to T cell activation 5.
Antigen Distribution
PD-1 is expressed on activated T cells, B cells, a subset of thymocytes, macrophages, dendritic cells, and some tumor cells and is also retained in the intracellular compartments of regulatory T cells (Tregs).
Ligand/Receptor
PD-L1, CD274
NCBI Gene Bank ID
UniProt.org
Research Area
Biosimilars
.
Cancer
.
Immuno-Oncology
.
Immunology

Leinco Antibody Advisor

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Research-grade Spartalizumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to ensure accurate, precise, and comparable measurement of drug concentration across both biosimilar and reference serum samples. This approach supports robust PK bioequivalence assessments.

Key Points on Their Use:

  • Analytical Standard Role: In PK ELISA, a single analytical standard—often the biosimilar itself—is used to generate the standard curve for quantitating both the biosimilar and reference (innovator) drug concentrations in serum samples. This standardization minimizes assay variability and eliminates the need for dual calibration curves, which could introduce additional variability and complicate comparability.

  • Calibration and Validation: Research-grade biosimilar standards are calibrated against commercially sourced drugs (including the reference product) and may also be benchmarked against international standards such as those from NIBSC or WHO where available. These standards are rigorously validated for accuracy, reproducibility (precision), and sensitivity according to international regulatory guidelines (FDA, EMA, ICH).

  • Bioanalytical Comparability: Before full assay validation, both biosimilar and reference products are tested within the same ELISA to ensure they are bioanalytically equivalent (i.e., show comparability in response). Statistical methods, such as comparing 90% confidence intervals of the measured responses, are used to demonstrate equivalence within predefined limits.

  • Reference Controls: In parallel with the calibration curve, quality control (QC) samples are prepared from both the biosimilar and the reference drug at multiple concentrations and analyzed in each run to monitor assay performance (precision and accuracy) within the validated method framework.

  • Bridging Assay Utility: The approach is termed a “bridging” ELISA because the same standard curve is used to “bridge” measurement of concentrations for both biosimilar and innovator serum samples, ensuring alignment of PK data across products.

Summary Table: How Research-Grade Spartalizumab Biosimilars are Used in PK Bridging ELISA

ApplicationHow Biosimilar Standard is UsedRegulatory/Validation Considerations
Calibration (Standard Curve)Standard dilutions of biosimilar set curve for all samplesCalibrated vs. reference drug, validated vs. guidelines
Reference/Quality ControlQC samples made from both biosimilar and innovatorMonitors precision/accuracy in every run
Bioanalytical EquivalenceResponse comparability must be statistically demonstrated90% CI within preset equivalence limits
Kit/Assay ConsistencyLyophilized standards for stability and reproducibilityInter/intra assay CV requirements <10%

In summary: Research-grade Spartalizumab biosimilars, when used as calibration standards and reference controls, enable the robust quantification and direct comparison of PK profiles between biosimilar and reference drug in bridging ELISA formats for serum samples, underpinned by rigorous calibration, validation, and regulatory alignment.

The primary models used to study in vivo efficacy of research-grade anti-PD-1 antibodies, focusing on tumor growth inhibition and characterization of tumor-infiltrating lymphocytes (TILs), are syngeneic mouse models and humanized mouse models. The choice depends on whether the antibody targets mouse or human PD-1.

1. Syngeneic Mouse Models

  • These are the most widely used models for mechanistic studies with anti-PD-1 antibodies that are specific to murine PD-1.
  • MC-38 (colon adenocarcinoma, C57BL/6 background) is considered a classic, anti-PD-1-sensitive model. Upon treatment with anti-PD-1 mAb, MC-38 tumors show significant tumor growth inhibition and robust alterations in TIL composition, such as increased CD4+ and CD8+ T cells, NK cells, and elevated lymphocyte cytotoxic potential, notably marked by increased perforin expression.
  • LLC1 (Lewis Lung Carcinoma, C57BL/6 background) serves as an anti-PD-1–resistant comparator, showing little to no tumor growth inhibition and minimal change in TILs upon anti-PD-1 treatment.

2. Humanized Mouse Models

  • These models are used when assessing antibodies that bind to human PD-1, allowing evaluation of clinically relevant, research-grade anti-PD-1 therapeutics.
  • Humanized target knock-in (KI) syngeneic models: These genetically engineered mice express a chimeric (humanized) PD-1 extracellular domain. Tumor cell lines compatible with mouse syngeneic settings are implanted, and the in vivo activity of human-specific anti-PD-1 antibodies is assessed.
  • NSG (NOD scid gamma) humanized mouse xenograft models: These highly immunodeficient mice are engrafted with human immune cells (such as allogeneic human T cells), followed by implantation of human tumor cells (xenograft). Such models allow for functional characterization of human anti-PD-1 antibodies, tumor growth inhibition, and TIL analysis in the context of a reconstituted human immune system.

Summary Table:

Model TypeDescriptionTypical Use CaseTumor ExamplePD-1 Antibody Type Tested
Syngeneic mouse (e.g., MC-38, LLC1)Immunocompetent mouse + murine tumorMurine PD-1 antibody evaluationMC-38, LLC1Mouse anti-PD-1 antibodies
Humanized target knock-in syngeneicMouse with humanized PD-1 domain + mouse tumorHuman PD-1 antibody, high fidelityMC-38 (hPD-1 KI)Human anti-PD-1 antibodies
Humanized NSG xenograftNSG mouse + human immune cells + human tumorClinical antibody characterizationPC-3, HCT-116Human anti-PD-1 antibodies

Key Points:

  • Murine syngeneic models (especially MC-38) are foundational for mechanistic and screening studies with murine-reactive research anti-PD-1 mAbs.
  • Humanized models (KI or NSG) enable the use of research-grade human or humanized anti-PD-1 antibodies, directly assessing clinical reagents in a mouse setting.
  • Anti-PD-1 treatment in these models enables detailed study of TIL populations, with attention to cytolytic markers (perforin, IFN-γ), DC/MDSC ratios, and phenotype/function of T and NK cells.

Both model types are prominent in preclinical immuno-oncology research for dissecting anti-PD-1 efficacy and TIL biology.

Researchers are actively investigating spartalizumab, a humanized IgG4 anti-PD-1 monoclonal antibody, in combination with other checkpoint inhibitors to explore synergistic effects in immune-oncology models. The most extensively studied combination involves spartalizumab with anti-LAG-3 inhibitors, particularly in advanced solid malignancies.

LAG-3 and PD-1 Combination Studies

The combination of spartalizumab with ieramilimab, an anti-LAG-3 antibody, represents one of the most promising approaches in dual checkpoint inhibition research. Ieramilimab is a humanized IgG4 monoclonal antibody that inhibits LAG-3 interaction with MHC class II molecules, while spartalizumab blocks PD-1 interaction with its ligands PD-L1 and PD-L2. This combination targets two critical pathways involved in T-cell exhaustion and immune suppression.

In multicenter, open-label phase 2 studies, researchers have evaluated this combination across multiple cancer types including non-small cell lung cancer (NSCLC), melanoma, triple-negative breast cancer (TNBC), renal cell carcinoma (RCC), and mesothelioma. The rationale for this combination stems from the understanding that LAG-3 and PD-1 co-regulate T-cell exhaustion and that compensatory upregulation of LAG-3 has been linked to adaptive resistance to PD-1 checkpoint blockade.

Mechanisms of Synergistic Action

The synergistic effects observed in these combination studies operate through several key mechanisms. First-in-human dose-escalation trials have demonstrated that the ieramilimab-spartalizumab combination enhances T-cell activation in the tumor microenvironment. Specifically, combination treatment increases LAG-3 gene expression and T-cell-inflamed signature genes in most patients, suggesting enhanced immune activation compared to single-agent therapy.

The therapeutic approach addresses multiple immune-mediated resistance mechanisms simultaneously, including T-cell dysfunction marked by enhanced expression of co-inhibitory receptors, decreased T-cell priming and infiltration, and suppression mediated by regulatory T cells and myeloid-derived suppressor cells.

Safety Profile and Tolerability

A critical advantage of the spartalizumab-ieramilimab combination is its favorable safety profile. Unlike combination checkpoint blockade with anti-CTLA-4 and anti-PD-1 agents, the immune-mediated toxicity of ieramilimab combined with spartalizumab was comparable to that seen with spartalizumab alone. Phase 1 studies showed no increase in incidence of immune-mediated serious adverse events, with the most commonly occurring treatment-related adverse events being low-grade fatigue, gastrointestinal side effects, pruritus, and fever.

Biomarker-Driven Patient Selection

Researchers utilize sophisticated biomarker analyses to identify patient populations most likely to benefit from combination therapy. Studies have shown that patients with higher expression of T-cell-inflamed gene signatures in baseline tumor samples were more likely to respond to the ieramilimab-spartalizumab combination. Interestingly, LAG-3 expression by immunohistochemistry did not predict benefit of dual LAG-3/PD-1 blockade, though LAG-3 gene expression by RNA sequencing was associated with treatment response.

Comparative Mechanism Studies

Research has also examined how different checkpoint inhibitor combinations operate through distinct mechanisms. Studies comparing anti-PD-1/CTLA-4 versus anti-PD-1/LAG-3 treatments in advanced melanoma have identified different subtypes of immune cells activated by each combination, providing insights into their unique mechanisms of action.

Clinical Trial Design and Efficacy Assessment

The clinical development of these combinations follows rigorous phase 1/2 study designs that assess safety, pharmacokinetics, and preliminary efficacy in patients with advanced or metastatic solid tumors. Researchers evaluate both treatment-naive patients and those previously treated with PD-1/PD-L1 inhibitors to understand the potential for overcoming resistance mechanisms.

The research demonstrates that while single-agent anti-LAG-3 therapy shows limited anti-tumor activity in solid tumors, the combination approach with PD-1 inhibition provides a promising strategy for enhancing immune checkpoint blockade efficacy and overcoming resistance mechanisms in complex immune-oncology models.

A Spartalizumab biosimilar can be employed as both the capture and detection reagent in a bridging anti-drug antibody (ADA) ELISA to detect and monitor anti-Spartalizumab immune responses in patients treated with the reference or biosimilar drug.

How this works:

  • In a bridging ADA ELISA, the goal is to detect antibodies (ADAs) generated by the patient’s immune system against the therapeutic antibody (here, Spartalizumab or its biosimilar).
  • The assay leverages the fact that ADAs are typically bivalent or multivalent—meaning they have at least two binding sites.

Assay setup:

  • Capture step: Plates can be coated with Spartalizumab biosimilar (or more commonly, a biotinylated version immobilized on a streptavidin-coated plate). Patient serum is then added.
  • Binding: If ADAs are present in the sample, each antibody will bind to the coated Spartalizumab biosimilar via one of its antigen-binding arms.
  • Detection step: After washing, a second preparation of Spartalizumab biosimilar, labeled with an enzyme (e.g., HRP), is added. This “bridges” to any ADA already bound on the plate by the other antigen-binding site on the ADA.

Therefore, positive signal is generated only if the patient sample contains antibodies capable of binding simultaneously to two molecules of Spartalizumab—i.e., true anti-drug antibodies.

Summary table:
| Step | Reagent Used | Purpose ||----------------|---------------------|----------------------------------------------|| Plate coating | Spartalizumab biosimilar (biotinylated) | Captures ADA from patient serum || Detection | Spartalizumab biosimilar (enzyme-labeled) | Binds to ADA and generates signal |

Key rationale for biosimilar use:

  • Regulatory and scientific guidelines accept the use of well-characterized biosimilars for immunogenicity assays if they are functionally and structurally equivalent to the reference product.
  • Using a biosimilar as both capture and detection reagent enables monitoring ADA responses against the functional epitope of the therapeutic, whether the patient received the reference or biosimilar form.
  • This approach is well-established for multiple therapeutic mAbs and their biosimilars, and bridging ELISAs are the most common assay format for ADA detection for monoclonal antibody therapies.

Caveats:

  • Bridging ELISAs are sensitive and suitable for screening, but can yield false positives due to interference from circulating drug or soluble target in the patient’s serum; careful assay optimization, good quality reagents, and blocking are critical for reliable data.

In summary, a Spartalizumab biosimilar serves as both the capture and detection reagent in a bridging ADA ELISA, exploiting the bivalency of patient ADAs to monitor immune responses against the therapeutic, with the assay design being widely accepted for immunogenicity testing of biosimilars and their reference products.

References & Citations

1 Matsumoto K, Inoue H, Nakano T, et al. J Immunol. 172(4):2530-2541. 2004.
2 Zhao Y, Harrison DL, Song Y, et al. Cell Rep. 24(2):379-390.e6. 2018.
3 Raimondi G, Shufesky WJ, Tokita D, et al. J Immunol. 176(5):2808-2816. 2006.
4 Pardoll DM. Nat Rev Cancer. 12(4):252-264. 2012.
5 Kaplon H, Reichert JM. MAbs. 11(2):219-238. 2019.
6 Dummer R, Long GV, Robert C, et al. J Clin Oncol. 40(13):1428-1438. 2022.
Indirect Elisa Protocol
FA
Flow Cytometry
Immunoprecipitation Protocol
General Western Blot Protocol

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