Anti ctla4 clone 9d9

Anti-CD137 mAb (clone 2A, rat Ig2a) has been described previously.^{43 (link)} Antibody was purified from hybridoma supernatants by protein G affinity chromatography by a commercial vendor (BioXCell). Anti-IL-2 (clone S4B6–1) and anti-CTLA-4 (clone 9D9) were purchased from BioXCell and used for IL-2 depletion and CTLA-4 blockade, respectively, with rat IgG2a (clone 2A3) and mouse IgG2b (clone MPC-11) serving as isotype controls. Mice were administered 400 mg of anti-CD137 or rat Ig2a isotype control mAb (clone 2A3, BioXCell) via i.p. route at 2 dpi. For IL-2 depletion studies, mice were administered 500 μg of anti-IL-2 mAb on 2, 4 and 6 dpi. For CTLA-4 blockade, mice were administered 500 mg of anti-CTLA-4 mAb on 2 dpi and 250 μg on 5 dpi.

GBM initiation and growth development was monitored by BLI as described above and animals that reached >1X10⁷ p/s/cm²/sr were randomly enrolled in treatment groups. All antibodies were diluted with sterile 0.9% Saline (Hospira). Mice were injected i.p. with 200 μg of anti–PD-1 (clone RMP 1–14, BioXcell) and/or 200 μg anti-CTLA4 (clone 9D9, BioXcell), or the isotype control IgG2a mAb (clone 2A3, BioXcell), every 3 days for one cycle. For the immunodepletion experiments, mice were injected with 200 μg of anti-CD8 (clone 53.6.7, BioXcell) daily for 3 days prior to initiation of immune checkpoint blockade or with 200 μg anti-Ly6G (clone 1A8, BioXcell) one day prior to initiation of immune checkpoint blockade and depletion treatment was continued every 3 days for 3 treatments total. AZD-5069 (MedKoo Biosciences, #206473) was resuspended in Ora-Plus (Paddock) and administered daily by oral gavage at a dose of 100 mg/kg body weight for 12 days.

B6 mice were inoculated subcutaneously (s.c.) with 0.5 million B16-F10 cells on day 0. The animals were divided into 6 groups (n=5). One group of mice was administered with PBS; one group of mice was administered with unsorted tumor cell lysate-DC vaccine (H-DC), and four groups of mice were administered with CSC-DC vaccine 3 days after B16-F10 cell inoculation. The vaccination was repeated on days 6 and 9. Each mouse was inoculated s.c. with 1 million DCs per vaccine. In the CSC-DC vaccination groups, 100 μg anti-CTLA-4 (clone 9D9, Bio X Cell, West Lebanon, NH) and/or anti-PD-L1 (clone10F.9G2, Bio X Cell, West Lebanon, NH) or control rat immunoglobulin (IgG) were administered intraperitoneally, on days 3, 6, and 9 following the B16-F10 cell inoculation. Tumor size were recorded three times per week by measuring the width and length diameters of the tumors.

Mice were injected with 1 mg tamoxifen (Sigma) each day for 4 consecutive days. For diphtheria toxin-mediated cell depletion, 100 μg·kg⁻¹ of diphtheria toxin (Sigma) were injected intraperitoneally as indicated in the appropriate figure legends.
To deplete CD4⁺-TLs, mice were injected intraperitoneally with 250 μg anti-CD4 (clone GK1.5) or 250 μg isotype-matched control antibody (Rat IgG2b (clone LTF-2; both from BioXcell)) on Day 3 and Day 1 before tumour inoculation.
For immune checkpoint blockade therapy, 125 μg anti-CTLA-4 (clone 9D9) and 125 μg anti-PD-1 (clone RMP1-14), or the same amount of isotype-matched control antibody (mouse IgG2b (clone MPC-11) and rat IgG2a (clone 2A3; all from BioXcell)) were delivered into mice intraperitoneally on Day 1 and Day 8 post-tumour inoculation.

An inoculum of 10⁶ tumor cells was injected s.c. on the flank of mice in 50 μL sterile PBS. Eight days following injection, treatment was initiated as indicated (Fig. 1a). The tumor-targeting antibody (A) was administered at 100 μg per dose i.p. for B16F10 and DD-Her2/Neu models. 2.5-Fc was was administered at 500 μg per dose (i.p.). MSA-IL2 (I) was administered i.p. at 30 μg (6 μg molar equivalent IL-2) per dose. Anti-PD-1 (P) (clone RMP1-14, BioXCell) was administered at 200 μg i.p. The vaccine, composed of 1.24 nmol amph-CpG and 20 μg of amph-peptide, was administered s.c. at base of the tail, half the dose given on each side. For experiments exploring other quaternary combos, the following doses were used as indicated in Supplementary Fig. 3: anti-CTLA-4 (clone 9D9, BioXCell) was administered at 200 μg per dose i.p., IFN-α was administered at 50 μg per dose i.p., IL-15 superagonist at 4 μg per dose i.p., and anti-TGF-β at 100 μg per dose i.p. Mice were randomized into treatment groups on day 8 following tumor inoculation, immediately prior to treatment. Tumor size was measured as an area (longest dimension x perpendicular dimension) three times weekly, and mice were euthanized when tumor area exceeded 100 mm². Mice that rejected tumors were rechallenged as indicated with 10⁵ tumor cells s.c. on the opposite flank.