Table 1 Preclinical and clinical studies supporting ATRA antitumor activity via immunomodulation of the tumor micro-environment

Preclinical

Vaccination

1) For immunization of the MethA fibrosarcoma tumor model, dendritic cells (infected with adenovirus encoding full-length wild-type p53) were injected s.c. into mice (4x10⁵ cells/mouse) on days 5, 10, and 15 after tumor cell inoculation. ATRA pellet (21-day release, 5 mg) was implanted on day 7 after tumor cell injection. 2) In the DA3-HA mammary tumor model, ATRA (21-day release, 5 mg) was administered 4 days after tumor inoculation. HA based vaccinia (1x10⁷ PFU) was injected 15 days after tumor implantation. 3) C3 fibrosarcoma tumor-bearing mice were treated with three rounds of immunization with control or specific peptide (50mg) at day 5, 7 and 17 after tumor inoculation. At day 7, ATRA (21-day release, 5 mg) or placebo pellets were implanted.

Breast cancer, fibrosarcoma-syngeneic mouse models

ATRA increases the antitumor effect of vaccination

1) ATRA decrease the number of MDSCs and induces their differentiation; 2) ATRA reduces CD4⁺ induced tumor tolerance; 3) ATRA increase CD8⁺T cell response

Kusmartsev et al., Cancer Res, 2003 [118]

Preclinical

Vaccination

MC38-CEA colon-adenocarcinoma or B16-OVA melanoma tumors were implanted, and animals were vaccinated against CEA or OVA (200 mg)combined with 20 mg of CpG-Oligonucleotid 1668 and 0,2 mg of anti-Galactosylceramide (aGC) after 10 days. ATRA treatment was administered simultaneously or with a 3 days delay (500 ng /day /mouse). Treatment lenght was 8 and 13 days for MC38-CEA or B16-OVA models respectivelly.

Colon adenocarcinoma; melanoma-syngeneic mouse models

ATRA potentiates tumor vaccination if administered later after first vaccination

1) ATRA treatment inhibits the suppressive capacity of monocytic MDSCs; 2) ATRA induces elevated frequencies of antigen-specific and functional CD8⁺ cytotoxic T lymphocytes

Heine et al., Oncoimmunology, 2017 [127]

Preclinical

Radiation and PD-L1 blockade

MC38 colon-adenocarcinoma, B16/F1 melanoma and Renca renal derived tumors were irradiated (15Gy) and ATRA was administered by oral gavage for 10 days. In the MC38 model, anti–PD-L1 antibody was administered intraperitoneally on days 0, 3, and 7 after radiation at 200 μg per mouse.

Colon adenocarcinoma, melanoma, renal carcinoma-syngeneic mouse models

ATRA potentiates the antitumor effect of ablative radiation (IR)

1) IR and ATRA treatment lead to an abscopal response enhanced by PD-L1 blockade; 2) IR and ATRA induces inflammatory macrofages; 3) IR and ATRA increase T cell response

Rao et al., Sci Immunol, 2021 [121]

Preclinical

CTLA4 and PD-L1 blokade

AB1-HA mesotelioma bearing mice were treated with aCTLA-4 on day 7 and aPD-L1 on day 7, 9 and 11 post tumor cell inoculation. ATRA treatment was administered simultaneously or 3 days prior the ICI treatment at 10 mg/kg for 9 consecutive days. AB1-HA bearing mice were treated with anti-GITR or anti-CD40 (anti-OX-40) antibodies (day 7 post tumor cell inoculation), with or without ATRA (10 mg/kg) beginning 4 days prior ICI administration to day 13 post tumor inoculation. Mice bearing WEHI164 fibrosarcoma were treated with anti-CTLA4 or anti-PD-L1 on day 11 post tumor injection with or without ATRA (10 mg/kg, from day 8 to day 16 post tumor injection).

Mesotelioma, fibrosarcoma-syngeneic mouse models

ATRA improves the anti-tumour response to anti CTL4, anti PDL1, anti-GITR and anti-OX40. ATRA /ICI combination efficacy is schedule-dependent; ATRA was most effective at increasing the αCTLA-4/αPD-L1 mediated antitumour response when given 3 days prior to ICI

1) ATRA induces an inflammatory, Interferon-driven tumour microenvironment; 2) ATRA further enhances the inflammatory tumour microenvironment induced by ICI; 3) activation of inflammation distinguishes responders from non-responders following ATRA/ICI combination therapy

Tilsed et al., Front Oncol, 2022 [122]

Preclinical

PD-1 blockade

ATRA (200 μg/mouse) was administered daily, starting on day 5 post tumor inoculation (KPL-3M cells). Anti-PD-1 antibody (200 μg) was dministered 2–3 times per week for 5 doses starting from day 7 post inoculation.

LKB1-Deficient Non–Small Cell Lung Cancer- genetically engineered syngeneic murine models

ATRA potentiates the antitumor efficacy of anti PD1

1) ATRA suppresses the proliferation and function of G-MDSCs; 2) ATRA enhanced T-cell infiltration, proliferation, and activation; 3) combination therapy with ATRA and anti–PD-1 induces systemic tumor-specific immune memory

Li et al., Cancer Res, 2021 [123]

Preclinical

PD-L1 blokade

Tumor bearing mice were treated with ATRA (7.5 mg/kg) daily from day 6 post U14 cells injection. Anti-PD-L1 antibody (10 mg/kg) was administered on day 6, 9 and 12 and mice were sacrificed on day 21 post tumor cells inoculation.

Cervical cancer-syngeneic mouse models

ATRA enhances the antitumor efficacy of anti PDL1

1) ATRA partially reverses MDSCs suppression; 2) ATRA enhances CD8⁺ T cell infiltration

Liang et al., Scientific Reports, 2022 [128]

Clinical trial

Vaccination

Patients were randomized into three arms: Arm A (standard of care control patients or observation), Arm B (patients treated with p53 vaccine only) and Arm C (patients treated with vaccine in combination with ATRA, 150 mg/mq for 3 days beginning one day prior to each vaccine). Each vaccine consisted of 2–5 × 10⁶ autologous dendritic cells expressing p53 after infection with adenovirus encoding full-length wild-type p53 gene. Cells were injected intradermally, at two-week intervals three times (three vaccine doses). Patients were restaged approximately 2 weeks after the 3rd vaccine dose. Patients without disease progression underwent a second leukapheresis and were then vaccinated 3 more times at 4-week intervals.

Small cell lung cancer, extensive stage

ATRA improves the immune response to vaccination

1) ATRA decreases MDSCs; 2) ATRA increases Granzyme B positive CD8⁺T cells

Iclozan et al., Cancer Immunol Immunother, 2013 [120]

Clinical trial

CTLA4 blokade

Ipilimumab (anti CTL4) was administered in four adjuvant infusions of 10 mg/kg for stage III patients or four infusions of 3 mg/kg for stage IV patients every three weeks in the Ipilimumab arm. Patients in the Ipilimumab plus ATRA arm were treated with ATRA at 150 mg/mq at days −1, 0, and +1 from Ipilimumab infusion.

Melanoma, stage III and IV

The addition of ATRA to standard of care Ipilimumab therapy appears safe

1) ATRA significantly decreased the frequency of circulating MDSCs

Tobin et al., Int Immunopharmacol, 2018 [125]

Clinical trial

PD-1 blockade

Pembrolizumab (anti PD-1) was administered at a fixed dose regimen of 200 mg every 3 weeks for 5/6 cycles. ATRA (VESANOID) at 150 mg/mq was used for three days surrounding each (days −1, 0, and +1) of the first four infusions of pembrolizumab.

Melanoma, stage IV

The combination was well tollerated. The overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%

1) ATRA significantly decreased the frequency of circulating MDSCs

Tobin et al., Clin Cancer Res, 2023 [126]