Dosing optimization of CCR4 immunotoxin for improved depletion of CCR4+ Treg in nonhuman primates

Abstract

Recently, we have developed a diphtheria toxin-based recombinant anti-human CCR4 immunotoxin for targeting CCR4⁺ tumors and Tregs. In this study, we further optimized the dosing schedule for improved CCR4⁺ Treg depletion. We have demonstrated that up to a 90% depletion was achieved and the depletion extended to approximately 2 weeks in the peripheral blood and more than 48 days in the lymph node at 25 μg·kg^-1 , BID for 8 consecutive days in cynomolgus monkeys. Expansion was observed including monocytes and NK cells. Antibody against the CCR4 immunotoxin was detected after approximately 2 weeks, affecting further depletion efficacy for multiple course treatment.

Keywords: CCR4; Treg; diphtheria toxin; immunotoxin.

Figure 1

Schematic diagram of the designed dosing schedule.

Figure 2

Monkey CCR4⁺ Treg depletion for animals M11016 and 11216 in the peripheral blood using the CCR4 immunotoxin. (A) Representative flow cytometry analysis of the CCR4⁺ cell depletion in the peripheral blood using the antibodies against human CD4 and CCR4. (B) CCR4⁺ cell depletion in the peripheral blood was monitored by flow cytometry using the antibodies against human CD4 and CCR4 (CD4⁺ CCR4⁺). (C) Representative flow cytometry analysis of the CCR4⁺Foxp3⁺ Treg depletion in the peripheral blood using the antibodies against human CCR4 and Foxp3 (CCR4⁺Foxp3⁺ among the gated CD4⁺ cells). (D) CCR4⁺ Treg depletion in the peripheral blood was monitored by flow cytometry using the antibodies against human CCR4 and Foxp3 (CCR4⁺Foxp3⁺ among the gated CD4⁺ cells). (E) The other CD4⁺ cells in the peripheral blood were monitored by flow cytometry using antibodies against human CD4 and CCR4 (CD4⁺ CCR4⁻). (F) The CD8⁺ T cells in the peripheral blood were monitored by flow cytometry using the antibodies against human CD3, CD4, and CD8 (CD3⁺ CD4⁻ CD8⁺). (G) The B cells in the peripheral blood were monitored by flow cytometry using antibodies against human CD20, CD3, CD16, and HLA‐DR (CD20⁺ CD3⁻ CD16⁻ HLA‐DR ⁺). (H) The NK cells in the peripheral blood were monitored using antibodies against human CD3, CD16, and NKp80 (CD3⁻ CD16⁺ NKp80⁺). (I) Monocytes in the peripheral blood were monitored by flow cytometry using antibodies against human CD14, CD16, and CD11b (CD14⁺ CD11b⁺ or CD14⁺ CD16⁺, PBMC gating). (J) The effector Tregs in the peripheral blood were monitored by flow cytometry using antibodies against CD45RA and Foxp3 (CD45RA ^‐Foxp3⁺ among the gated CD4⁺ cells).

Figure 3

ELISA analysis of the antibody formation against the CCR4 immunotoxin for all experimental animals M11016, M11216, M10916, and M11116. Data are representative of three different individual experiments. Three repeats were included for each serum sample of individual experiment. Error bars are SEM.

Figure 4

Monkey CCR4⁺ Treg depletion for animals M11016 and 11216 in the lymph node using the CCR4 immunotoxin. The lymph node biopsies were performed before and after each course of the immunotoxin treatment as well as in the end of the study. (A) Flow cytometry analysis of the lymph node biopsy samples using antibodies against human CD4 and CCR4. (B) Flow cytometry analysis of the lymph node biopsy samples using antibodies against human CCR4 and Foxp3 (CCR4⁺Foxp3⁺ among the gated CD4⁺ cells). (C) Lymph node CCR4⁺ Treg depletion was monitored by flow cytometry (CCR4⁺ cells: CD4⁺ CCR4⁺, CCR4⁺ Tregs: CCR4⁺Foxp3⁺ among the gated CD4⁺ cells, effector Tregs: CD45RA ^‐Foxp3⁺ among the gated CD4⁺ cells). Other cell populations in the lymph node were also monitored by flow cytometry (other CD4⁺ cells: CD4⁺ CCR4⁻; CD8⁺ cells: CD8⁺ CD3⁺ CD4⁻; B cells: CD20⁺ CD3⁻ CD16⁻ HLA‐DR ⁺).