The MW assignment by MALS indicated that the monomeric component had a MW of 144,300 Da. Size-exclusion chromatography (SEC; gel filtration) coupled with multi-angle light scattering (MALS) was performed to determine the monomer content and MW distribution of the major impurities of ICOS.33 IgG1f S267E. N-linked glycans present on ICOS.33 IgG1f S267E comprised a mixture of asialo-biantennary sugars without fucose that varied with respect to the level of galactose incorporation. TABLE-US-00029 TABLE 26 Pharmacokinetic Parameters of ICOS.33 IgG1f S267E after Intravenous (IV) Administration to Cynomolgus Monkeys Area Under Volume at the Curve T1/2 Clearance steady state Monkey Dose (AUC)(0-INF) (half-life) (CLT) (Vss) Study number (mg/kg) (.mu.M .times. Anti-drug antibodies (ADA) were detected at seven days post dose in three out of four cynomolgus monkeys in the 1 mg/kg dose group and continued to increase up to 42 days post dose. The increase in the ADA signal corresponded with the rapid clearance of the antibody in these monkeys, and this portion of the data affected by ADA were not included in the PK data analysis. Cells were confirmed to be virus-free via mouse antibody production testing. T cells (whole blood and spleen) with across all doses of groups treated with anti-ICOS.4 mIgG1.



Tumor growth inhibition was .gtoreq.74% for each of the four doses (1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg) anti-ICOS treatment groups in combination with anti-PD-1, with at least 8/15 mice tumor free in each of these groups. These results showed comparable levels of antitumor activity of the anti-ICOS mIgG1 in combination with anti-PD-1 mIgG1 treatments across all doses. Results suggested mg2a promoted the best antitumor activity. Results from this study showed that choice of isotype is an important determinant of anti-ICOS antibody treatment. Antibody fluorescence was detected by flow cytometry on a Fortessa cytometer (BD Biosciences, San Jose, Calif.), and the results were analyzed using FlowJo software (FlowJo, LLC, Ashland, Oreg.). The right flank of each mouse was subcutaneously implanted with 1.times.10.sup.6 CT26 cells in 0.2 mL PBS using a 1-cm syringe and a 26-gauge half-inch needle. CT26 cells were cultured in RPMI-1640 medium (HyClone/GE Healthcare, Logan Utah, Catalog 10-040-CM, Lot 16915003) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Catalog 26140-079, Lot 1704315). Cells were split 1:10 every three to four days. 10% heat-inactivated fetal bovine serum (FBS), for less than 10 passages.



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