Uncoupling therapeutic from immunotherapy-related adverse effects for safer and effective anti-CTLA-4 antibodies in CTLA4 humanized mice

Abstract

Anti-CTLA-4 monoclonal antibodies (mAbs) confer a cancer immunotherapeutic effect (CITE) but cause severe immunotherapy-related adverse events (irAE). Targeting CTLA-4 has shown remarkable long-term benefit and thus remains a valuable tool for cancer immunotherapy if the irAE can be brought under control. An animal model, which recapitulates clinical irAE and CITE, would be valuable for developing safer CTLA-4-targeting reagents. Here, we report such a model using mice harboring the humanized Ctla4 gene. In this model, the clinically used drug, Ipilimumab, induced severe irAE especially when combined with an anti-PD-1 antibody; whereas another mAb, L3D10, induced comparable CITE with very mild irAE under the same conditions. The irAE corresponded to systemic T cell activation and resulted in reduced ratios of regulatory to effector T cells (Treg/Teff) among autoreactive T cells. Using mice that were either homozygous or heterozygous for the human allele, we found that the irAE required bi-allelic engagement, while CITE only required monoallelic engagement. As with the immunological distinction for monoallelic vs bi-allelic engagement, we found that bi-allelic engagement of the Ctla4 gene was necessary for preventing conversion of autoreactive T cells into Treg cells. Humanization of L3D10, which led to loss of blocking activity, further increased safety without affecting the therapeutic effect. Taken together, our data demonstrate that complete CTLA-4 occupation, systemic T cell activation and preferential expansion of self-reactive T cells are dispensable for tumor rejection but correlate with irAE, while blocking B7-CTLA-4 interaction impacts neither safety nor efficacy of anti-CTLA-4 antibodies. These data provide important insights for the clinical development of safer and potentially more effective CTLA-4-targeting immunotherapy.

Fig. 1

Human CTLA4 gene knock-in mice distinguished irAE of anti-CTLA-4 mAbs Ipilimumab and L3D10 when used alone or in combination with anti-PD-1 mAb: growth retardation and pure red blood cell aplasia. a Timeline of antibody treatment and analysis. C57BL/6 Ctla4^h/h mice were treated, respectively, with control human IgG-Fc, anti-human CTLA-4 mAb Ipilimumab, human IgG1 Fc chimeric L3D10 + human IgG-Fc, anti-PD-1 (RMP1-14) + human IgG-Fc, anti-PD-1 + Ipilimumab or anti-PD-1 + L3D10 at a dose of 100 μg/mouse/injection on days 10, 13, 16 and 19. The CBC analysis was performed on day 41 after birth and necropsy was performed on day 42 after birth. To avoid cage variation, mice in the same cages were individually tagged and treated with different antibodies. Tests were performed double blind. b Major growth retardation of female mice by Ipilimumab + anti-PD-1. One female mouse from Ipilimumab plus anti-PD-1-treated group was excluded from analysis due to death on day 22 with serious grow retardation. Data shown were means and S.E.M. of % weight gain following the first injection. hIg vs Ipilimumab + anti-PD-1, P < 0.0001; L3D10 + anti-PD-1 vs Ipilimumab + anti-PD-1, P = 0.003. c Major growth retardation of male mice by Ipilimumab + anti-PD-1. As in b, except male mice were used. hIg vs Ipilimumab + anti-PD-1, P = 0.0116; L3D10 + anti-PD-1 vs Ipilimumab + anti-PD-1, P = 0.0152. The numbers of mice used were included in the parentheses following group labels. d–g Pure red cell aplasia recapitulated in the mouse model as a typical phenotype of irAE. d Ipilimumab + anti-PD-1 combination therapy reduced HCT, Hb and MCV. Data shown are a summary of 8 independent experiments with each dot representing one individual mouse (blue for male mice and red for female mice, and n = 9–22 mice per group. e Defective generation of red cells in bone marrow. Photographs depict the change of coloration in bone (upper panel) and bone marrow flush (lower panel) in mice that received indicated treatments. f Analysis of erythrocyte development by flow cytometry. Data shown are representative FACS profiles depicting distribution of Ter119, CD71 and forward scatters (FSC-A) among bone marrow cells. The gating and % of cells at stage I–V are indicated. g Summary data of % of erythroid cells at each of the developmental stages. Data shown are means and S.E.M. of data with 3–4 female mice per group, and have been repeated at least three times in both male and female mice. Statistical tests used: b and c, two-way repeat measurement ANOVA with Bonferroni multiple comparison test; d and g, one-way ANOVA with Bonferroni multiple comparison test and non-parametric one-way ANOVA (Kruskal–Wallis test) with Dunn’s multiple comparisons test

Fig. 2

Ipilimumab caused heart defects when used in combination with anti-mouse PD-1. a Gross anatomy shows heart enlargement despite reduced body size in mice treated with anti-PD-1 + Ipilimumab. Photographs in the left panels are from formalin-fixed hearts from mice that received indicated treatments, and the data on the right panel show the sizes after normalizing against body weight. b Macroscopic images depicting enlarged heart atriums and ventricles, and corresponding thinning of heart wall. c Histology of control hIg, L3D10 + anti-PD-1 or anti-PD-1 + Ipilimumab-treated hearts. The upper four panels show H&E staining at the aorta base, while the lower four panels show inflammation in myocardium of the left ventricle. d Identification of leukocytes and T cells by immunohistochemistry (top panels) and three-color immunofluorescence staining using FITC-labeled CD4 or CD8, Rhodamine-labeled anti-CD3 or anti-Foxp3 antibodies (lower panel). e The composite pathology scores of male and female mice (n = 5–12) receiving different treatments. The scores of male mice are indicated with blue circles, while those of female mice are indicated with red circles. The samples were collected from six independent experiments and have been scored double blind. Data are mean ± S.E.M. and analyzed by one-way ANOVA with Bonferroni’s multiple comparison test

Fig. 3

Ipilimumab caused multiple organ inflammation when either used as single agent or in combination with anti-PD-1. a Representative images of H&E stained paraffin sections from different organs. Representative inflammatory foci are marked with arrows. Scale bar, 200 μm. b Toxicity scores of internal organs and glands. The scores of male mice are indicated with blue circles, while that of female mice are indicated with red circles. c Composite scores of all organs and glands. Data are mean ± S.E.M., n = 5–12 mice per group. The samples were collected from six independent experiments and have been scored double blind. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparison test

Fig. 4

Comparison of systemic T cell activation in mice that received immunotherapy drugs starting at day 10. a Minor impact of CD4 (top panel) and CD8 (bottom panel) T cell frequencies by combinational immunotherapeutics. Data shown are % of CD4 and CD8 T cells in the spleen on day 32 after the start of antibody treatment. b Representative FACS profiles depicting the increase of memory and effector CD4 (top panels) or CD8 (bottom panels) T cells in mice that received monotherapy and combination treatment of anti-PD-1 plus Ipilimumab during the perinatal period. c, d Summary data on the phenotype of CD4 c and CD8 d T cells in mice that received combination treatments with anti-PD-1 plus anti-CTLA-4 mAbs. Data shown are % of cells with phenotypes of naive, central memory and effector memory phenotypes. Data shown are summarized from four experiments involving 7–11 female mice and 2–6 male mice per group. Statistical tests used: a, one-way ANOVA with Bonferroni multiple comparison test; c and d, one-way ANOVA with Bonferroni multiple comparison test

Fig. 5

In combination with anti-PD-1, Ipilimumab preferentially expanded autoreactive Teff cells. a Diagram of the breeding scheme. b Diagram of the experimental timeline. The mice were produced in two steps. The first step was an outcross between two inbred strains as indicated. The second step was an intercross of F1s to obtain mice of designed genotypes (H-2^d+Ctla4^{h/h or h/m}Mmtv⁸⁺⁹⁺) for the studies. c Representative FACS profiles depicting the distribution of Vβ11, Vβ8 and Foxp3 markers among gated CD4 T cells from mice that received antibody treatments. d Composite ratios of Treg/Teff among VSAg-reactive (Vβ5⁺, 11⁺ or 12⁺, top panel) and non-reactive (Vβ8⁺) CD4 T cells. e Lack of impact on thymocytes. As in d, except the CD3⁺CD4⁺CD8⁻ thymocytes were analyzed. Data shown are means and S.D., n = 6–7 mice per group

Fig. 6

Humanized L3D10 clones maintained safety profiles when used in combination therapy with anti-PD-1 mAb. a Comparing humanized L3D10 clones HL12 and HL32 with Ipilimumab for their combination toxicity when used during perinatal period. Except changes in antibodies used, the experimental regimen was identical to that depicted in Fig. 1a. b Ipilimumab but not humanized L3D10 clones induced anemia when used in combination with anti-PD-1 antibody. c Pathology scores of internal organs and glands after the mice were treated with either control of given combination of immunotherapeutic drugs. d Composite pathology scores. Blue circles represent scores of male mice and the red scores represent female mice used. All scorings were performed double blind. Data are mean ± S.E.M., n = 5–12 mice per group. The samples were collected from five independent experiments and have been scored double blind. Statistical methods used were: a repeated measures two-way ANOVA with Bonferroni’s multiple comparison test; b non-parametric one-way ANOVA (Kruskal–Wallis test) with Dunn’s multiple comparisons test; c and d one-way ANOVA with Bonferroni’s multiple comparison test

Fig. 7

Comparison of the immunotherapeutic effect of HL12 and HL32 with Ipilimumab. a, b MC38-bearing-Ctla4^h/m mice (n = 5) were i.p. treated with 30 μg a or 10 μg b of either control hIg, Ipilimumab, HL12 or HL32 on day 7, 10, 13 and 16. c, d CT26 bearing-Ctla4^h/m mice (n = 6–10) were i.p. treated with 150 μg c or 100 μg d of either control Ig, Ipilimumab, HL12 or HL32 on day 7, 10, 13 and 16. e, f B16-bearing Ctla4^h/h mice (n = 5–6) were i.p. treated with 250 μg control Ig, Ipilimumab, HL12 e or HL32 f Data are mean ± S.E.M. and data were analyzed by repeated measures two-way ANOVA with Bonferroni’s multiple comparison test. In all settings, HL12 and HL32 induced statistically significant tumor rejection when compared with Control hIgG, HL12 (a, P = 0.0023; b, P = 0.0105; c, P < 0.0001; d, P = 0.0272; e, P < 0.0001); HL32 (a, P = 0.004; b, P = 0.0059; c, P < 0.0001; d, P = 0.0259; f, P = 0.1003). Tumor rejections induced by Ipilimumab were also significant in all but except one setting (a, P = 0.0026; b, P = 0.0231; c, P = 0.2; d, P = 0.0003, e, P = 0.0145; f, P = 0.0234). The differences between different therapeutic antibodies are not statistically significant

Fig. 8

Distinct genetic requirement for irAE and CITE revealed in C57BL/6.Ctla4^h/m mice. a–c Evaluation of irAE. Female mice (n = 5) of given genotypes were treated with either control human IgG (hIg), or anti-PD-1 + Ipilimumab during the perinatal period and evaluated for body weight gain, inflammation and red blood cell anemia at 6 weeks of age. a Ipilimumab + anti-PD-1 combination induced growth retardation in Ctla4^h/h but not the Ctla4^h/m mice. b Except for a modest induction in some mice in the salivary gland, Ipilimumab + anti-PD-1 did not induce inflammation in internal organs in heterozygous mice. c Ipilimumab + anti-PD-1 did not induce red blood cell anemia in heterozygous mice. d Effective tumor rejection induced by Ipilimumab. Tumor-bearing Ctla4^h/h and Ctla4^h/m mice received treatment of either control hIg or Ipilimumab (100 μg/injection × 4) on days 7, 10, 13 and 16. The tumor growth was measured every 3 days. Data are mean ± S.E.M. and all Data shown have been reproduced two times. e Ipilimumab + anti-PD-1 did not cause systemic T cell activation in Ctla4^h/m mice. Representative FACS profiles depicting the distribution of CD44 and CD62L are shown on the left and summary data are shown on the right. Data in a and d were analyzed by repeated measures two-way ANOVA with Bonferroni’s multiple comparison test; whereas those in b, c and e were analyzed by unpaired two-tailed Student’s t test

Fig. 9

irAE and CITE in 6-7-week-old young adult and 10-day old tumor- bearing mice. a–c MC38-bearing young male mice (7-week-old) were inoculated with MC38 tumor cells and treated with either control hIgG, Ipilimumab, HL12 or HL32(100 μg/injection × 4) on days 7, 10, 13 and 16 after tumor cell challenges. a Tumor volumes over time. b Serum TNNI3 levels on day 25 after tumor challenge were determined by ELISA. c H&E staining show hyalinization and inflammation in myocardium. Scale bar 100 μm. d, e MC38-bearing young male mice (6-week-old) were inoculated with MC38 tumor cells and treated with either control hIgG, Ipilimumab or Ipilimumab + anti-PD-1(100 μg/injection × 4) on days 7, 10, 13 and 16 after tumor cell challenges. d Tumor volumes over time. e Serum TNNI3 levels on day 25 after tumor challenge were determined by ELISA. f 10-day-old mice were challenged with MC38 tumors, and immunotherapies were initiated on days 14, 17, 20 and 23 days of age and tumor sizes over time were presented. Data are mean ± S.E.M. and analyzed by repeated measures two-way ANOVA with Bonferroni’s multiple comparison test. hIg vs Ipilimumab or Ipi + anti-PD-1, P < 0.0001; Ipilimumab vs Ipi + α-PD-1, ns. g Combination therapy and monotherapy-induced multiple organ inflammations. Representative H&E sections from salivary gland and lung are presented. Scale bar, 100 μm. b, e Data are mean ± S.E.M. and statistical significance was analyzed by non-parametric one-way ANOVA

Fig. 10

Loss of naive T cells and increase of effector memory T cells correlate with multiple organ inflammation. Data shown are re-analyses of data presented in Figs. 2, 3, 4, 6 and Supplementary information, Figure S8. Naive T cells: CD44^LoCD62L^Hi (a and d); effector memory T cells (E.Mem): CD44^HiCD62L^Lo (b and e); central memory T cells (C.Mem): CD44^HiCD62L^Hi (c and f). Correlation coefficient and P-value of linear regression were calculated by Pearson’s method

Fig. 11

Distinct mechanisms responsible for irAE and CITE. a irAE is caused by inhibiting the conversion of autoreactive T cells into autoreactive Treg cells, which leads to a polyclonal expansion of autoreactive T cells in the peripheral lymphoid organs. b Tumor rejection is achieved by FcR-mediated depletion of Treg cells in the tumor microenvironment and is independent of naive T cell activation in the peripheral lymphoid organs. Neither irAE nor CITE depends on blockade of B7-CTLA-4 interaction