Unexpected PD-L1 Immune Evasion mechanism in TNBC, Ovarian and other Solid Tumors by DR5 Agonists (2020)
Figure 5

Figure 5. Generation and testing of chimeric DR5 for human DR5 agonist targeting in immunocompetent murine models

(A) Schematic and genetic construction of two chimeric human-mouse DR5 (Chi-DR5, Chi-G4S-DR5) with human extracellular domain and mouse transmembrane (TM) and intracellular domains (ICD).

(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C). Also see Fig EV8A.

(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).

(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).

(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry similar to described in Fig 1I, J (n=3-4). See also Appendix Fig S9.

(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).

(I) Same as H, except average tumor weight is shown (n=4).

(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.

(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size. Also see Fig EV4B.

(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6). Also see Fig EV8C-D for additional confirmation with PD-1 inhibitor.

(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.

(O) Same as H, except KMTR2 (8 doses) antibody instead of lexatumumab was used as the DR5 agonist for the experiment.

(P) Average of tumor weights of data shown in (O).

(Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.

Data information: Mean ± SD. Statistical significance in (E), (G), (I), (L), (M) and (P) was determined by unpaired two-tailed t-test and in (N) using two-tailed paired Wilcoxon Mann-Whitney test (n=4-6) (*p <0.05, **p <0.005, ***p <0.0001, ****p <0.00005).