Nivolumab

Biological Activity

In vitro: Nivolumab binds PD-1 with high affinity (KD 2.6 nmol/l by Scatchard analysis to polyclonally activated human T cells) and blocks its interactions with both B7-H1 and B7-DC. It effectively inhibits the interaction between PD-1 and its ligands. In vitro assays demonstrated the ability of nivolumab to potently enhance T-cell responses and cytokine production in the mixed lymphocyte reaction and superantigen or cytomegalovirus stimulation assays. Nivolumab inhibits the interaction between PD-1 and its ligands, PD-L1 and PD-L2, with IC50 values of 2.52 and 2.59 nmol/L, respectively, as shown by surface plasmon resonance. In a study using FACS to evaluate ligand binding to PD-1 expressed on CHO cells, the IC50 values for nivolumab-mediated inhibition of PD-1 binding to PD-L1 or PD-L2 were similar (1.04 and 0.97 nmol/L, respectively). Nivolumab binds specifically to PD-1 and not to other immunoglobulin superfamily proteins, such as CD28, CTLA-4, ICOS, and BTLA. Nivolumab can, at very low concentrations (∼1.5 ng/mL), enhance T-cell reactivity in the presence of a T-cell receptor stimulus. However, nivolumab had no stimulatory effect in the absence of antigen or T-cell receptor stimulus. Specifically, there was no significant release of inflammatory cytokines, including IFNγ, TNFα, IL-2, IL-4, IL-6, or IL-10, from unstimulated whole blood after coincubation with nivolumab. Nivolumab does not cause nonspecific lymphocyte activation.

In vivo: Nivolumab was well tolerated, dose-limiting toxicities (DLTs) were not reached and the maximum tolerable dose (MTD) was not defined in patients with advanced stage solid tumors. The measured half-life of nivolumab was 12-20 days, the pharmacodynamic effects of PD-1 receptor occupancy was even more prolonged at 85 days, indicating the biological durability of this high-affinity mAb. In monkeys, serum nivolumab has a relatively slow clearance with limited extra vascular distribution, as demonstrated by a Vss value consistent with plasma volume. Mean apparent terminal elimination half-life estimates for males and females at 1 mg/kg were similar (124 and 139 hours, respectively), and the mean half-life estimate for males at 10 mg/kg was 261 hours. Although nivolumab seems to lack toxicity in monkeys, toxicities have been observed in human clinical trials. In a phase I trial, nivolumab had a favorable safety profile. Adverse events were generally similar to those observed with ipilimumab, although with lower incidence and of less severity, and comprised gastrointestinal, endocrine, and skin toxicities, and pulmonary inflammation. Interestingly, pneumonitis has been observed in PD-1-deficient mice bred onto the MRL genetic background, but not in PD-1-deficient mice with other genetic backgrounds.

Product Citations

• 

  Mol Cell Probes. 2023 Sep 28;72:101925.

  CD3ζ as a novel predictive biomarker of PD-1 inhibitor resistance in melanoma
  Nivolumab purchased from AbMole

• 

  PeerJ. 2023 Apr 19;11:e15172.

  Sema4D silencing increases the sensitivity of nivolumab to B16-F10 resistant melanoma via inhibiting the PI3K/AKT signaling pathway
  Nivolumab purchased from AbMole

Protocol (for reference only)

Cell Experiment
Cell lines	PBMCs
Preparation method	In a cytomegalovirus (CMV)-restimulation assay, 2 × 10^5 PBMCs from a CMV-positive donor (Astarte) were stimulated using lysate of CMV-infected cells (Astarte), with serial dilutions of nivolumab added at the initiation of the assay. After 4 days, supernatants were assayed for IFNγ.
Concentrations	0-10 μM
Incubation time	4 days

Animal Experiment
Animal models	Cynomolgus monkeys (Macaca fascicularis)
Formulation	Saline
Dosages	3 mg/kg or 10 mg/kg
Administration	i.v.

Chemical Information

CAS Number	946414-94-4
Storage	Store at -20°C or -70°C. Avoid multiple freeze-thaws.

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Wai Nam Liu, et al. Cancers (Basel). Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy

[2] Jibin Zhang, et al. MAbs. HX008: a humanized PD-1 blocking antibody with potent antitumor activity and superior pharmacologic properties

[3] James L Weaver, et al. Toxicol Sci. BLT-Immune Humanized Mice as a Model for Nivolumab-Induced Immune-Mediated Adverse Events: Comparison of the NOG and NOG-EXL Strains

[4] Zheng B, et al, J Hematol Oncol. PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.

[5] Boning Liu, et al. MAbs. Acid-induced aggregation propensity of nivolumab is dependent on the Fc

[6] Borghaei H, et al, N Engl J Med. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer.

[7] Sanmamed MF, et al. Cancer Res. Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-IL2Rγnull Immunodeficient Mice.

[8] Larkin J, et al. N Engl J Med. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma.