The rat anti-PD1 antibody clone RMP1-14, an immunoglobulin of type IgG2aκ, whose sequence was identified from hybridoma sequencing (Bio X Cell), was converted to a chimeric mouse IgG2aκ antibody with rat variable domains. The mouse IgG2a∗01 heavy chain (HC; IMGT: IGHG2A∗01; accession no. V00825)64 and the κ light chain (LC; IMGT: IGKC∗01; accession no. V00807) were used as scaffolds. The following mutations were included in the HC: (1) the CH2 glycosylation site was mutated to alanine (N297A) to impair FcγR binding; (2) effector functions were further ablated by introducing L234A, L235A, and P329G mutations to the CH2 domain;65,66 and (3) a cloning site was added that introduces a K115S mutation into the CH1 domain. Optimized H5 and L1 leader sequences were used for HC and LC secretion,67 respectively. Chains were expressed using a F2A68,69 sequence with an optimized furin site (RKRR)70 for expression from a single open reading frame, with the orientation HC-F2A-LC as described previously.71

The murine IL-12 gene was generated from translated GenBank cDNA sequences for IL-12B/p40 (GenBank: BC103608.1) and IL-12B/p35 (GenBank: BC146595.1) connected by F2A peptide as above, and the murine IL-2 gene was created from the translated GenBank cDNA sequence (GenBank: NM_008366.3). The cytokine genes included their native signal sequences. All payload constructs were codon-optimized for mouse expression and synthesized by GeneArt (Thermo Fisher Scientific).

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