Fig 2: Functional inhibition of LAG3/MHC-II and PD1/PD-L1 interactions.Cell-based assessment of T cell activation measured by induction of an NFAT response element-driven luminescent reporter gene. Relative light units (arbitrary scale) are reported. a Relief of LAG3/MHC-II-mediated suppression. Standard errors of the mean are indicated. Approximate half-maximal stimulation concentrations: CB213: 20 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 22 nM, relatlimab: 9.7 nM, aLAG3 17B4 (anti LAG3 mouse monoclonal): 1.6 nM. MSA-MSA-MSA is three interlinked VH recognising mouse serum albumin as a negative control. b Relief of PD1/PD-L1-mediated suppression. Standard errors of the means are indicated. Approximate half-maximal stimulation concentrations: CB213: 105 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 115 nM, nivolumab: 6 nM, DART (MGD-013): 1.4 nM. MSA-MSA-MSA is three interlinked VH recognising mouse serum albumin as a negative control.

Fig 3: CB213 in vivo syngeneic mouse immunophenotyping.End-of study in vivo pharmacology syngeneic tumours disaggregated and analysed by flow cytometry. Treatments were HEL-MSA (negative control) or CB213 dosed 10 mg/kg. Datapoints represent single tumours from individual animals. a Tumour-infiltrating lymphocytes as a percentage of leucocytes (CD45+) in disaggregated tumours. b Expression of PD1 and/or LAG3 on CD4+ or CD8+ tumour-infiltrating lymphocytes, respectively.

Fig 4: CB213 NHP pharmacokinetics and anti-drug antibodies.a CB213 PK and ADA in two female cynomolgus macaques: animals 102 and 103. b Cartoon of CB213 structure. A Schematic of CB213 is shown. Individual VH are shown as globular domains, with stylised glycine/serine linkers intervening in dark grey. The diagram is not to scale. c Schematic of the MSD-based CB213 PK bridging assay. Molecules that can simultaneously bind to LAG3 and to PD1 are detected. d Schematic of the MSD-based ADA assay. Molecules that can bind to CB213 when bound to LAG3 that contain an IgG light chain are detected.

Fig 5: Human ex vivo T cell proliferation.a Overview of the tumour cell/T cell assay system. A549-SC3 cells [15] engage the TCR on T cells via a single chain aCD3 antibody. MHC-II and PD-L1 expressed on A549-SC3 cells suppress T cell proliferation by engaging LAG3 and PD1, respectively. b Baseline T cell proliferation with negative control treatment. Percent proliferation as shown by Ki67 positivity relative to total CD4+ and CD8+ T cell populations with HEL-MSA on board in ex vivo blood samples from 22 patients. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. c Proliferation of NSCLC patient T cells after ex vivo stimulation with A549-SC3 cells in the presence of various potential checkpoint modulators: Lg-Lg: two Lag3-binding VH connected by a glycine/serine linker; Pd: one PD1-binding VH; Lg-Lg + Pd: Lg-Lg and Pd co-administered; nivo: nivolumab analogue mAb; Lg-Lg-Pd: LAG3-LAG3-PD1 triple VH construct. All values represent the patient-specific paired difference in %Ki67+ T cells with respect to the negative control VH construct HEL-MSA for each given patient. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. Asterisk indicates P > 0.05 by two-tailed Wilcoxon signed-rank test vs. negative control; asterisk above square bracket shows P > 0.05 by two-tailed unpaired Mann–Whitney test for nivolumab vs. LAG3-LAG3-PD1 in CD8+ T cells.