Encapsulated Checkpoint Blocker Before Chemotherapy: The Optimal Sequence of Anti-CTLA-4 and Doxil Combination Therapy

Figures & data

Figure 1 Encapsulation of liposomal enhances its antitumor effect in B16 large established mouse model. (A) Injection schedule for all treatment groups. (B) Survival analysis mice bearing B16 tumor, liposomal increased survival significantly, Log-rank analysis shows statistically significant differences between treatment groups. (C) Comparison between tumor size of different groups, liposomal decreased tumor size in comparison to non-liposomal and PBS. (D–F) Tumor size progression graph for each treatment groups, individually. (G) %CD8⁺ in CD 45⁺ population of DLN, increased %CD8⁺ of CD 45⁺ population in both forms. (H) %CD8⁺ of in CD 45⁺ population spleen. (I) %CD8⁺ in live cells of the tumor microenvironment, only liposomal increased %CD8⁺ in TIL. (J) TCD8⁺/T reg ratio in DLN. (K) TCD8⁺/T reg ration in spleen. (L) TCD8⁺/T reg ratio in TIL, only liposomal increased TCD8⁺/T reg ration ratio (*p<0.05).

Abbreviation: OXP, oxaliplatin.

Table 1 Characteristics and Encapsulation Efficiencies of Encapsulated CTLA-4 Blockade Antibody Liposomal Formulations

Formulations- Molar Ratio	Total Lipid Concentrations (mM)	Expected Total Phospholipid Concentrations (mM)	Observed Total Phospholipid Concentrations (mM)	Z Average (nm) _SD	PDI ^a	Zeta Potentials (mV) _SD	Encapsulation Efficacy%
PEGylated liposome: HSPC/mPEG2000– DSPE/Chol 55/5/40	48	28.8	24.6±1.7	166.2±5.9	0.16±0.063	9.14±2.6	54.7±8.9

Note: ^aPoly dispersity index.

Table 2 Therapeutic Efficacy Data of Therapeutic and Control Group Mice Bearing B16 Tumor

Groups	TTE^a (Days) ±SD	TGD^b (%)
Control	16.01 ± 1.14	–
Anti-CTLA-4	19.67 ± 2.38	22.86
Liposomal Anti-CTLA-4	33.34 ± 2.63	113.3
Doxil	22.2 ± 2.18	39.04
Anti-CTLA-4 + Doxil	41.84 ±1.93	161.32
Concomitant Anti-CTLA-4 and Doxil	32.43 ± 2.13	102.56
Doxil+ Anti-CTLA-4	23.78 ± 3.63	48.51
Doxil +liposomal Anti-CTLA-4	42.86 ± 4.45	167.69

Notes: ^aTime to reach end point. ^bTumor growth delay.

Figure 2 Administration before Doxil in combination therapy shows promising results in B16 large established mouse model. (A) Survival analysis – mice bearing B16 tumor, injection prior to Doxil increased survival significantly, Log-rank analysis shows statistically significant differences between treatment groups. (B) Comparison between tumor size of different groups, combining therapy in sequence of before Doxil reduced tumor size in comparison to other groups. (C–F) Tumor size progression graph for each treatment groups, individually. (G) %CD8⁺ in CD 45+ population of DLN. (H) %CD8⁺ of in CD 45⁺ population spleen. (I) %CD8⁺ in live cells of tumor microenvironment, only of anti-CTLA-4 + Doxil (in sequence manner) increased %CD8⁺ in TIL. (J) T CD8⁺/T reg ratio in DLN. (K) TCD8⁺/T reg ration in spleen. (L) TCD8⁺/T reg ratio in TIL, only administration before Doxil increased TCD8⁺/T reg ration ratio (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 3 Liposomal delivery increases the efficiency of combination therapy of anti-CTLA-4 with Doxil. (A) survival analysis mice bearing B16 tumor, liposomal increases survival significantly in combination therapy when replaced with non-liposomal treatment, Mantel-Cox test shows statistically significant differences between treatment groups (p<0.001). (B) Comparison between tumor size of different groups, combining therapy with liposomal before Doxil reduced the tumor size in comparison to other groups. (C–E) Tumor size progression graph for each treatment groups, individually. (F) %CD8⁺ in CD 45⁺ population of DLN. (G) %CD8⁺ of in CD 45⁺ population spleen. (H) %CD8⁺ in live cells of tumor microenvironment. (I) TCD8⁺/T reg ratio in DLN. (J) TCD8⁺/T reg ration in spleen. (K) TCD8⁺/T reg ratio in TIL, liposomal administration before Doxil increased TCD8⁺/T reg ration ratio compared to non-liposomal treatment (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).