Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells

Abstract

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Conflict of interest statement

Competing financial interests

All author with affiliation to Infinity Pharmaceuticals Inc were employees and share holders at Infinity pharmaceutical Inc at the time of the study. All other authors have no competing interests.

Figures

Extended Data Figure 1. Impact of suppressive myeloid TILs in response to Checkpoint blockade

a. Individual tumor growth of subcutaneous (4T1) or intradermal (B16, B16-GMCSF) implants in anti-PD-1, anti-CTLA4 or control treated mice (n=10). b.In vitro suppressive activity of tumor-infiltrating CD11b+ cells purified from spleen of 4T1, B16, B16-GMCSF tumor-bearing mice. Representative histograms of CD8+ T cell proliferation at corresponding CD11b+ to CD8+ T cell ratio (left panel) and quantification of CD8+ T cell proliferation (right panel) (n=3), mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (non-parametric Mann–Whitney t-test).

Extended Data Figure 2. Impact of PI3K-γ selective inhibition on tumor growth and Myeloid TILs

a. Binding affinities (Kd) and cellular IC50 inhibition of pAKT by IPI-549 for class I PI3K isoforms (Left table). Percent of inhibition of ARG-1 expression on bone marrow derived macrophages polarized with M-CSF and IL-4, (right panel). b. Percent of tumor growth inhibition in LLC, MC38, 4T1, CT26, B16-GMCSF tumor bearing mice treated with IPI-549 (table). c. Quantification of CD11b, CD206, NOS2 and PD-L1 expression in CD11b+ tumor infiltrating leukocytes from IPI-549 vs vehicle treated CT26 tumor bearing mice. d. RNAseq of co-stimulatory and checkpoint molecules on whole tumors from CT26 tumor-bearing mice treated for 6 or 9 days with IPI-549 compared to vehicle. e. Mean tumor volume of subcutaneous LLC-Brei implants in IPI-549 vs Vehicle treated mice without or after CD11b+ cell depletion. Data represent analysis of 5–10 mice per group, mean ± s.e.m. *P<0.05, ***P<0.001 (non-parametric Mann–Whitney t-test).

Extended Data Figure 3. Impact of PI3K-γ selective inhibition on subsets of CD11b myeloid cells

a. Representative flow cytometry analysis and quantification of Ly6C, MHC class II expression in CD11b+Ly6G− cells infiltrating 4T1 tumors. b. mRNA expression of selected M1 and M2 markers in sorted Ly6C low-MHCII Low (TAM-M2) compared to Ly6C low-MHCII High (TAM-M1) population from 4T1 tumor, data were relative to GAPDH expression and normalized versus the mean of TAM-M1 population. Mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (non-parametric Mann–Whitney t-test).

Extended Data Figure 4. Impact of PI3K-γ selective inhibition on suppressive PBMC derived human myeloid cells

a. Inhibition of CXCL-12 activation of PI3K-γ in monocytes as measured by pAKT (S473) in human whole blood. b. Representative histograms and quantification of Human CD8 T cells proliferation after 72 hours of co-culture with or without autologous MDSCs generated from the T-cell-depleted PBMCs ± IPI-549.

Extended Data Figure 5. Impact of PI3K-γ selective inhibition on function of tumor specific T cell responses

a. Quantification of KI67, and CTLA4 expression in CD8+ T cells in TILs of 4T1 or B16-GMCSF tumors at IPI-549 compared to vehicle treatment day 7 and 14. b. Mean tumor volume of subcutaneous 4T1 tumor in IPI-549 vs vehicle treated BALB/c nu/nu mice (n=10). c. Mean tumor volume of subcutaneous CT-26 tumor in IPI-549 vs vehicle treated BALB/c mice with or without CD8+ T cell depletion by anti-CD8 antibody (n=10). d. Quantification and representative pictures of CT26 tumor specific immune responses in PBMCs from CT26 tumor-bearing mice treated with IPI-549 in comparison to vehicle by ELISPOT. PBMC were collected from tumor-bearing animals after 10 days of vehicle or IPI-549 treatment and restimulated overnight with irradiated CT26 or 4T1 stimulator cells.

Extended Data figure 6. Impact of PI3K-γ selective inhibition on the differentiation of T cells in tumors

a. Representative Flow cytometry analysis and quantification of CD62L and CD44 expression in CD8 and CD4 T cell infiltrates in tumor, LN and spleen of 4T1 tumor bearing mice treated with IPI-549 compared to Vehicle. Data represent analysis of 5 mice per group, mean ± s.e.m. NS = non statistical significance (non-parametric Mann–Whitney t-test).

Extended Data Figure 7. Impact of combination of PI3K-γ selective inhibitor with checkpoint blockade on various tumors

a. Survival to 2000 mm3 tumor volume of LLC Brei tumor in IPI-549 or vehicle treated mice in combination or not with anti-CTLA4 (Vehicle and IPI-549 groups, n=14; anti-CTLA4, n=13; IPI-549 and anti-CTLA4 combination, n=10). b. Mean tumor volume of CT26 tumor in IPI-549 or vehicle treated mice in combination or not with anti-PD-L1 (n=15 for all groups except vehicle, n = 13).

Extended Data Figure 8. Impact of combination of PI3K-γ selective inhibitor with checkpoint blockade on TILs

a. Mean tumor volume of subcutaneous 4T1 tumor bearing mice treated with IPI-549, Vehicle or anti-PD-1 in combination with IPI-549 or vehicle (n=10). b. Representative flow cytometry analysis of CD206 and MHCII labeling in CD11b+ F4/80+ cell populations in the different treatment groups of 4T1 tumor bearing mice. c. Quantification of CD11b+ F4/80+, M1/M2 ratio, CD8/Treg in TILs and Granzyme B expression in CD8 T cells from 4T1 tumors in the different treatment groups. d. Quantification of CD11b+ F4/80+, M1/M2 ratio, CD8/Treg in TILs and Granzyme B expression in CD8+ T cells from B16-GMCSF tumors in the different treatment groups, mean ± s.e.m. *P<0.05, **P<0.01 (non-parametric Mann–Whitney t-test).

Extended Data Figure 9. Impact of combination of PI3K-γ selective inhibitor with checkpoint blockade on acquisition of anti-tumor memory

a. Tumor rechallenge at 100 days (from first tumor implant) following primary tumor complete response in B16-GMCSF tumor bearing mice treated with Vehicle (blue) or IPI-549 (red) in combination with both anti-PD1 and anti-CTLA4, b. CT26 tumor-bearing mice with complete responses in the anti-PD-1 treatment group and the IPI-549 + anti-PD-1 combination treatment group were rechallenged with CT26 tumor implant. Additional mice with complete responses from the IPI-549 + anti-PD-1 combination were implanted with 4T1 tumors. There was a low or no tumor take with CT26 rechallenge, while all 4T1 tumors grew, indicating specific anti-tumor memory.

Figure 1. Resistance to checkpoint blockade is associated with suppressive Myeloid cells infiltration in tumor microenvironment

a. Mean tumor volume of subcutaneous (4T1) or intradermal (B16, B16-GMCSF) implants in anti-PD-1, anti-CTLA4 or control treated mice (n=10), upper panel. Survival of 4T1, B16 or B16-GMCSF tumor bearing mice treated with anti-PD1 or anti-CTLA4 compared to control (vehicle treated only) (n=10), lower panel. b. Representative flow cytometric analysis and quantification of CD11b+, CD8+, CD4+ and Tregs (CD4+ Foxp3+) cell populations in 4T1, B16, B16-GMCSF tumors at 14 days post implants (n=5). c. Representative flow cytometric analysis and quantification of Granzyme B, PD-1 expression on CD8+ T cell populations in 4T1, B16, B16-GMCSF tumors at 7 days post implants (n=5). d.In vitro suppressive activity of tumor-infiltrating CD11b+ cells purified from 4T1, B16, B16-GMCSF tumor-bearing mice. Representative histograms of CD8+ T cell proliferation at corresponding CD11b+ to CD8+ T cell ratio (left panel) and quantification of CD8+T cell proliferation using CFSE dilution (right panel) (n=3). Data represent analysis of n (shown above for each experiment) mice per group, mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (non-parametric Mann–Whitney t-test, Log-rank (Mantel-Cox) test survival comparison).

Figure 2. Selective targeting of PI3K-γ reduces tumor growth and metastasis in various checkpoint blockade-resistant tumor models associated with high myeloid cell infiltrates

a. Therapy regimen (upper panel) and mean tumor volume of subcutaneous (4T1) or intradermal (B16-GMCSF) implants in vehicle vs IPI-549 (15 mg/kg administered orally, daily) treated mice (n=10), lower panel. b. Representative pictures and hematoxylin and eosin-stained sections of lung from B16-GMCSF tumor bearing mice at day 7 on treatment with IPI-549 or vehicle (left panels). Quantification of lung metastasis at day 14 and 21 from tumor challenge (right panel). c. Flow cytometric analysis and quantification of CD11b+F4/80+ (TAM) cell populations in 4T1 tumors at day 7 post implants (n=5), expression of CD206 (M2) and MHCII (M1) in CD11b+F4/80+ cell populations. The histogram bars show the percent change in each cell population (CD11b+, TAM, M2 and M1) in IPI-549 treated group in comparison with vehicle-treated control d. mRNA expression of selected M1 and M2 markers in 4T1 and B16-GMCSF tumors as determined by RT-PCR (n=3), data were relative to GAPDH expression and normalized versus the mean of vehicle treated tumors. e.In vitro suppressive activity of tumor-infiltrating CD11b+ cells purified from 4T1, tumor-bearing mice at day 7 post treatment with IPI-549 or Vehicle. Representative histograms of CD8+ T cell proliferation in CD11b+ to CD8+ T cell ratio 1:1 (left panel) and percent CD8+ T cell proliferation (right panel) (n=3). Data represent analysis of 5–10 mice per group, mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001 (non-parametric Mann–Whitney t-test).

Figure 3. Reduction of myeloid suppressive phenotype correlates with higher anti-tumor T cell activity

a. Quantification by Flow cytometry of ratio of M1/M2 in CD11b+F4/80+ cell population, CD8+ T cells in CD45+ TILs and ratio of CD8+ T cell/Treg (CD4+FoxP3+) in CD45+ TILs in 4T1 and B16-GMCSF tumors at day 7 and 14 on treatment (n=5), (left panels), Quantification of Granzyme B and PD1 expression in CD8+ TILs in 4T1 and B16-GMCSF tumors at day 7 and 14 on treatment (right panels). b. Mean tumor volume of intradermal B16-GMCSF tumors in IPI-549 vs vehicle treated wild type C57Bl6 (left) or Rag−/− mice (right) (n=10). c. Flow cytometric analysis and quantification of gp-100 antigen specific T cells transferred into IPI-549 or vehicle-treated B16-GMCSF tumor bearing mice (n=5). Data represent analysis of 5–10 mice per group, mean ± s.e.m. *P<0.05, **P<0.01 (non-parametric Mann–Whitney t-test).

Figure 4. Resistance to checkpoint blockade therapy is overcome when combined with selective PI3K-γ inhibition

a. Therapy regimen; b. Above panels: mean tumor volume of subcutaneous 4T1 tumor in control, anti-CTLA4 or anti-PD-1 treated mice in combination with IPI-549 or vehicle (n=10). Individual tumor volumes of subcutaneous 4T1 implants in mice treated with anti-CTLA4 and anti-PD1 in combination with IPI-549 or vehicle (n=10), dotted red line corresponds to end of treatment day post tumor implant (day21). Survival at 150 days of 4T1 tumor bearing mice treated with anti-CTLA4 and anti-PD1 in combination with IPI-549 or vehicle compared to control (vehicle treated only) (n=10); c. Mean tumor volume of intradermal B16-GMCSF implants in anti-PD-1, anti-CTLA4 or control treated mice in combination with IPI-549 or vehicle (n=10). Individual tumor volumes of intradermal B16-GMCSF implants in mice treated with anti-CTLA4 and anti-PD1 in combination with IPI-549 or vehicle (n=10). Survival at 150 days of B16-GMSCF tumor-bearing mice treated with anti-CTLA4 and anti-PD1 in combination with IPI-549 or vehicle compared to control (vehicle treated only) (n=10). Data represent analysis of 5–10 mice per group, mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (non-parametric Mann–Whitney t-test or Log-Rank test).