Pharmacodynamic (PD) Profiling

Biocytogen provides comprehensive pharmacodynamic (PD) studies as both independent service offerings and in support of in vivo efficacy studies.

  • Immune cell profiling by flow cytometry (FACS) analysis in tumor (TILs) and other tissues for lymphoid, myeloid, and other surface or intracellular markers
  • Cytokine/chemokine profiling by multiplexed technologies
  • RNA expression profiling
  • Immunohistochemistry (IHC)
  • RO (Receptor Occupancy) assay

Case 1: Tumor Infiltrating Immune Cell Profiling of MC38 Tumor in B-hCD137 (4-1BB) Mice

Tumor infiltrating immune cells are commonly subject to investigation in tumor immunology studies. They encompass a diverse collection of cell types that include tumor infiltrating lymphocytes (TILs; T cells, B cells, NK cells) and mononuclear and polymorphonuclear myeloid cells (macrophages, dendritic cells, mast cells, neutrophils, eosinophils, basophils, and so on). Some cells promote tumor growth whereas others suppress tumor growth, as shown in the diagram below1. Their composition varies based on tumor types and therapeutic intervention. Interrogation of the composition of tumor infiltrating immune cells is important in revealing the underlying cellular mechanisms by which tumor growth is modulated. Changes of specific cell types in response to therapeutic interventions can also be used as pharmacodynamics (PD) markers.

Biocytogen has established high quality, multi-color flow-cytometry-based tumor infiltrating immune cell profile services, including TIL profiling, on variety of syngeneic models, such as genetically humanized mouse models, and immune-deficient mouse models, for pharmaceutical companies and clinical research institutions worldwide.


Typical flow cytometry analysis scheme for tumor infiltrating immune cells using B-h4-1BB (CD137) mice


Flow cytometry analysis of tumor infiltrating immune cells. MC38 cells (0.5 M) were subcutaneously inoculated into B-h4-1BB mice, where the extracellular domain of human 4-1BB (CD137) replaces that of mouse 4-1BB via genomic knock-in. Grouping (n=3) and dosing started when tumor volume reached approximately 300 ± 50 mm3. Tumors were collected for TIL profiling 2-4 days after one dose of anti-human 4-1BB antibody (Ab1, Ab2) at 3 mg/kg at day 0.

Antibody Ab1 treatment led to marked decrease of Treg cells and increase of CD8/Treg ratios 2-4 days post dosing. Expression of human 4-1BB in CD4, CD8, Treg, and CD11b cells was also decreased significantly after Ab1 treatment. In contrast, treatment of antibody Ab2 resulted in no change in Treg or CD8/Treg ratios and less changes in 4-1BB expression in CD4 and Tregs. Ab2 did lead to significant decrease of 4-1BB in CD11b+ cells. The results suggest that Ab1 has a more favorable anti-tumor PD profile in MC38 tumors via reducing the Treg population in tumor.

  1. Luen SJ, Savas P, Fox SB, et al., Tumour-infiltrating lymphocytes and the emerging role of immunotherapy in breast cancer. Pathology. 2017; 49(2):141-155.

Case 2: Cytokine Profiling in B-hCD40 Mice

Cytokines are a group of low-molecular-weight soluble proteins produced by a variety of cells. They include interleukins, interferons, chemokines, tumor necrosis factors, and so on. They play important roles in modulating body’s immune responses. CD40 is a costimulatory receptor expressed mainly in antigen presenting cells (APCs). Binding of its ligand CD154 (CD40L) expressed on T cells or binding of agonistic anti-CD40 antibodies results in activation of both APCs and T-cells and production of a variety of cytokines. Biocytogen created B-hCD40 mice where the murine CD40 extracellular domain was replaced with that of human CD40. This B-hCD40 model has been validated in efficacy study using anti-human CD40 antibody selicrelumab in syngeneic MC38 tumor model (A, B). Multiplexed cytokine analysis in MC38/B-hCD40 model treated with other anti-human CD40 antibodies indicate varied cytokine profiles for these antibodies (C). Given that cytokine-mediated toxicity is one of the important clinical safety considerations in CD40-based therapeutics development, cytokine profiling helps select efficacious anti-human CD40 antibodies with reduced cytokine-mediated toxicity.



Murine colon cancer MC38 cells were subcutaneously implanted in homozygous B-hCD40 mice. Mice were grouped when the tumor sizes reached approximately 150±50 mm3 (n=5). Selicrelumab (anti-human CD40 antibody) significantly inhibited tumor growth, validating that the B-hCD40 mice are a useful model for in vivo pharmacology studies of anti-human CD40 antibodies. Tumor volume: mean ± SEM (A).  Body weight: mean ± SEM (B).

Cytokine profiles after anti-human CD40 antibody treatment of MC38-bearing B-hCD40 mice. Multiplexed cytokine detection was performed with LEGENDplexTM (Biolegend). Anti-human CD40 antibody treatments led to significant increase of IFN-γ, TNF-α, MCP-1, and IL-27. Note that the two antibodies gave different profiles. Not only could the difference in cytokine profiles provide insights on the mode of action by individual anti-human CD40 antibodies, but also they could be used as PD biomarkers to inform selection of safe anti-CD40 therapeutics.

Case 3: Characterization of B-hCD3e mice revealed percentage of CD3+ T cells in leuocytes is an informative PD markers for antibody-induced cell death (AICD)


Murine colon cancer MC38 cells were subcutaneously implanted in C57BL/6 (A) and B-hCD3e (B) mice. Mice were grouped when the tumor size reached approximately 150 ± 50mm3 (n=5). The mPD-1 antibody significantly inhibited tumor growth, whereas the specific CD3 mouse antibody led to increased tumor growth. The enhanced tumor growth by anti-human CD3e antibody was probably due to antibody-induced cell death (AICD), as anti-human CD3e treatment resulted in loss of CD3 T cells in blood and spleen (C). These results indicated B-hCD3e mice are responsive to anti-human CD3e modulation and validated the B-hCD3 mouse model as a useful tool to study anti-human CD3e antibody-based therapy.

The ratio of B cells and T cells in the blood of B-hCD3e mice was detected by flow cytometry (C). Lymphocytes were isolated from peripheral blood at the 48 hours. In the anti-human CD3e antibody treated group, the proportion of CD3 T cells was significantly decreased. This depletion was specific to anti-human CD3e because anti-mouse CD3e, anti-mouse PD-L1, or an IgG control antibody did not show depletion of T cells (C, right). Further, no change in B cells as percent of leukocytes was also not changed (C, left). This observation was consistent with activation induced cell death (AICD).



Table 1. Group and treatment information.

Strains of mice Group Treatment
B-hCD3e G1 hCD3 antibody (OKT3)
G2 CD3/CD19 Bispecific antibody
G3 Blinatuomomab
C57BL/6 G5 mCD3 antibody (145-2C11)

The ratio of T cells in spleen and blood were analyzed at 24h, 72h and 168h by flow cytometry.



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