Overcoming immunotherapy resistance and inducing abscopal effects with boron neutron immunotherapy (B-NIT)

Abstract

Immune checkpoint inhibitors (ICIs) are effective against many advanced malignancies. However, many patients are nonresponders to immunotherapy, and overcoming this resistance to treatment is important. Boron neutron capture therapy (BNCT) is a local chemoradiation therapy with the combination of boron drugs that accumulate selectively in cancer and the neutron irradiation of the cancer site. Here, we report the first boron neutron immunotherapy (B-NIT), combining BNCT and ICI immunotherapy, which was performed on a radioresistant and immunotherapy-resistant advanced-stage B16F10 melanoma mouse model. The BNCT group showed localized tumor suppression, but the anti-PD-1 antibody immunotherapy group did not show tumor suppression. Only the B-NIT group showed strong tumor growth inhibition at both BNCT-treated and shielded distant sites. Intratumoral CD8+ T-cell infiltration and serum high mobility group box 1 (HMGB1) levels were higher in the B-NIT group. Analysis of CD8⁺ T cells in tumor-infiltrating lymphocytes (TILs) showed that CD62L- CD44⁺ effector memory T cells and CD69⁺ early-activated T cells were predominantly increased in the B-NIT group. Administration of CD8-depleting mAb to the B-NIT group completely suppressed the augmented therapeutic effects. This indicated that B-NIT has a potent immune-induced abscopal effect, directly destroying tumors with BNCT, inducing antigen-spreading effects, and protecting normal tissue. B-NIT, immunotherapy combined with BNCT, is the first treatment to overcome immunotherapy resistance in malignant melanoma. In the future, as its therapeutic efficacy is demonstrated not only in melanoma but also in other immunotherapy-resistant malignancies, B-NIT can become a new treatment candidate for advanced-stage cancers.

Keywords: abscopal effect; advanced melanoma; boron neutron capture therapy; boron‐neutron immunotherapy; immune combination therapy.

FIGURE 1

Pharmacokinetics of 4‐borono‐L‐phenylalanine (BPA) in an advanced‐stage melanoma model. (A) BPA is a phenylalanine‐bound boron drug and a melanoma‐targeted boron neutron capture therapy (BNCT) drug with high melanoma biosynthesis.(B) Comparison of mRNA SLC7A5 (LAT1) with TCGA. (C) Mouse model of advanced‐stage melanoma with C57BL/6J/ and B16F10 cells transplanted intramuscularly in the right thigh and subcutaneously in the left flank. (D, E) Boron concentration pharmacokinetics in a mouse model of advanced‐stage melanoma (subcutaneous [D] or intraperitoneal administration [E], BPA 500 mg/kg, n = 4 at each time point). (F, G) Tumor‐to‐normal tissue (T/N) ratio: Ratio of boron concentration in right thigh intramuscular tumor and normal tissue (muscle, skin, blood) administered subcutaneously (F) or intraperitoneally (G).

FIGURE 2

The antitumor results of boron neutron immunotherapy (B‐NIT) in the advanced‐stage melanoma model. (A) Schematic of the time course of the B‐NIT experiments. (B, C) Tumor volume of the right thigh intramuscular tumors at the neutron irradiation site (B) and the left abdominal subcutaneous tumor at the shielded site (C) in the four groups. The tumor volume ratio was determined to tumor volume at day 7 after cell transplantation. *p < 0.05. (D, E) Serum HMGB1 concentration (ng/mL) on day 15 (D) and the final stage (E) after neutron irradiation. *p < 0.05. (F) Mouse body weight changes in the four groups over time (n = 6/group). BNCT, boron neutron capture therapy.

FIGURE 3

Overview of therapeutic radiation dose of boron neutron immunotherapy (B‐NIT) for the advanced‐stage mouse melanoma model. (A) Production of high‐energy ⁷Li and ⁴He by alpha decay between boron‐10 nuclides and neutrons (top) and a schematic diagram of boron neutron capture therapy (BNCT) for cancer therapy (bottom). (B) Experimental diagram of the neutron irradiation/shielding system and neutron/gamma ray biomarker during irradiation of the mice. (C) The total biological dose for BNCT consists of the nitrogen dose, hydrogen dose, gamma ray dose, and boron dose. (D) Average thermal neutron fluence at each mouse site after 5 MW, 12 min neutron irradiation at KURRI (n/cm², each n = 6). (E) Mean total irradiation dose results for each tissue after BNCT (Gy‐Eq, each n = 6). (F) Boron dose results, nitrogen dose results, hydrogen dose results, and gamma radiation dose results (Gy‐Eq, each n = 6) at three sites (left flank subcutaneous tumor site, right lower limb proximal, and distal intramuscular tumor) by neutron irradiation. (D–F) Red bars indicate results for neutron‐irradiated areas; blue shaded bars indicate results for shielded areas.

FIGURE 4

Results in four groups of CD8⁺ immune cells in tumor and spleen. (A, B) Immunohistochemistry image of CD8⁺ (red) and nuclear staining (blue) of right thigh intramuscular tumors (A) and left flank subcutaneous tumors (B) on day 22 in the four groups (scale bars, 100 μm). (C–E) Flow cytometry analysis of CD8⁺ T cells in CD45⁺ cells in right‐thigh intramuscular tumors (C), left‐abdominal subcutaneous tumors (D), and spleen (F) on day 15 (*p < 0.05). (F) Representative flow cytometry results of CD8⁺ T cells with CD45⁺ cells in control group, anti‐PD‐1 group, BPA‐BNCT group and B‐NIT group. B‐NIT, boron neutron immunotherapy; BNCT, boron neutron capture therapy; BPA, 4‐borono‐L‐phenylalanine.

FIGURE 5

Analysis of tumor infiltrating lymphocytes of effector memory T cells (TEMs). (A–D) Flow cytometry analysis of effector memory T cells (CD44⁺ CD62L‐ CD8⁺, TEMs) in the right‐thigh intramuscular tumors (A, B) (p < 0.0001) and in the left‐flank subcutaneous tumor areas (neutron‐shielded area) (C, D) (p < 0.0001). (E, F) Flow cytometry analysis of TEMs of four groups in the right intramuscular tumor (E) and left‐flank subcutaneous tumor (F) (p < 0.0001). (G) Comparison of TEM results for both tumors between the control and B‐NIT group (p < 0.0001). B‐NIT, boron neutron immunotherapy; BNCT, boron neutron capture therapy; BPA, 4‐borono‐L‐phenylalanine.

FIGURE 6

Analysis of tumor‐infiltrating lymphocytes of early‐activating T cells. (A, B) Flow cytometry analysis results of early‐activating T cells (CD8⁺ CD69⁺) among the four groups. (A) Right‐thigh intramuscular tumors, p = 0.0523. (B) Left‐flank subcutaneous tumor, p < 0.0001 (C) Comparison of early‐activating T cell results for both tumors between the control and B‐NIT group (p < 0.0001). B‐NIT, boron neutron immunotherapy; BNCT, boron neutron capture therapy; BPA, 4‐borono‐L‐phenylalanine.

FIGURE 7

Results of treatment effects without neutron irradiation in four groups. (A, B) Tumor volume ratio results in four groups (control group, anti‐PD‐1 mAb group, BPA group, and anti‐PD‐1 mAb + BPA group) without neutron irradiation (right intramuscular tumor (A) and left lateral abdominal tumor (B), n = 4 per group). (C) Flow cytometry analysis of CD8⁺ T cells in CD45⁺ cells in spleen on day 16 (n = 3 per group). (D) Mouse body weight changes in the four groups over time (n = 4 per group). (E) Serum HMGB1 concentration (ng/mL) on day 16. BPA, 4‐borono‐L‐phenylalanine.

FIGURE 8

Therapeutic effects of boron neutron immunotherapy (B‐NIT) with anti‐CD8‐depleting mAb administration. (A) Schematic of B‐NIT experiments with anti‐CD8‐depleting mAb (n = 6 per group). (B, C) Tumor volume ratio results at the right neutron irradiation site (B) and at the left shielded site (C) in six groups of four anti‐CD8‐depleting mAb groups (control, anti‐PD‐1 mAb, BPA‐BNCT, B‐NIT) and the two no‐anti‐CD8 mAb groups (control, B‐NIT). (D) Tumor volume graph of the right tumor at the neutron‐irradiation site (**p < 0.01) (E) Tumor volume graph of the left tumor at the shielded site (**p < 0.01). (F) Flow cytometry analysis of CD8⁺ CD45⁺ cells in spleen on day 16 (n = 4 per group). (G) Serum HMGB1 concentration (ng/mL) on day 16. (H) Mouse body weight (g) changes over time (n = 6) (p = 0.125).

FIGURE 9

Graphic abstract of boron neutron immunotherapy (B‐NIT). (A) Graphic abstract of B‐NIT.(B) Image of B‐NIT's future clinical direction.