Combination Image-Guided and Antibody-Targeted α-Therapy Before Targeted Immunotherapy for Treatment of Solid Tumors

Abstract

The rationale of this study was to evaluate the efficacy of 3 types of targeted therapy for solid tumors, comprising image-guided radiation therapy (IGRT), low-dose targeted α-therapy (TAT), and antibody-targeted interleukin-2 immunocytokine therapy, with the expectation that the combination of 2 types of targeted radiation therapy would perform better than either monotherapy with immunocytokine therapy. Methods: Carcinoembryonic antigen (CEA)-positive syngeneic breast and colon tumors in CEA transgenic animals were treated with single-dose IGRT (10 Gy) and 2 different regimens of fractionated IGRT (4 doses of 2.5 Gy or 4 doses of 3.65 Gy), with and without low-dose TAT (37 kBq of ²²⁵Ac-DOTA-anti-CEA) to optimize doses and tumor models for the combination of 2 types of IGRT plus TAT with immunocytokine therapy. Results: In preliminary PET imaging in the breast cancer model, fractionated IGRT (4 doses of 2.5 Gy) provided better antibody tumor penetration than did single-dose IGRT (10 Gy). Similarly, tumor regression and survival were superior with IGRT when combined with low-dose TAT, followed with best rechallenge responses in the groups treated with 4 doses of 2.5 Gy combined with TAT. Since comparable results were obtained in the colon cancer model, triple therapy (fractionated IGRT plus low-dose TAT followed by immunocytokine therapy) was studied in the colon cancer model, demonstrating complete cures in the majority of mice and rejection of all rechallenges. When the study was repeated for immunophenotyping 2 d after the completion of therapy, significant increases in natural killer and CD8-positive/interferon-gamma-positive cells were observed with triple therapy. Moreover, the changes in the tumor microenvironment, as reflected by the reduction of macrophages and infiltration of granulocytes and monocytes, was an important feature of these therapies. The memory phenotypes of the CD8-positive cells in tumor-draining lymph nodes and tumors showed significant increases of T cells with central memory versus naïve phenotypes in untreated controls. Interestingly, in contrast to the spleens and tumor-draining lymph nodes, there was almost a complete lack of naïve CD4-positive cells in the control tumors, a situation that was reversed by all 3 types of therapy, with the combination of IGRT, TAT, and immunocytokine therapy exhibiting the highest increase. Conclusion: Triple targeted therapy had the best therapeutic effects in a solid tumor model as evidenced by tumor cures, rejection of tumor rechallenge, and immunophenotyping.

Keywords: image-guided radiotherapy; radioimmunoimaging; radionuclide therapy; targeted immunotherapy; targeted α-therapy.

Graphical abstract

FIGURE 1.

Effects of IGRT on antibody penetration into orthotopic breast tumors. (A) ⁶⁴Cu-DOTA-anti-CEA antibody M5A (30 µg; 3.7 MBq) was administered 1 d after 0 or 10 Gy of IGRT or 4 doses of 2.5 Gy administered daily to CEA transgenic mice bearing established CEA transfected E0771 breast tumors. PET imaging was performed at 24 h. Upper row shows maximum-intensity-projection images; lower row shows stacked coronal tumor slices. (B) Mice were euthanized after PET imaging, tumors collected, and decay-corrected radioactivity was reported as percentage injected dose per gram of tissue (%ID/g) (n = 3 per group). *P < 0.05; ***P < 0.001.

FIGURE 2.

Comparisons of monotherapies with IGRT or TAT vs. combined IGRT plus TAT in immunocompetent breast cancer model. (A and B) CEA transgenic mice bearing established CEA transfected E0771 orthotopic breast tumors (n = 6) were treated with single dose of 3.7 kBq of ²²⁵Ac-DOTA-M5A (TAT) or single 10-Gy dose of IGRT or 10 Gy of IGRT followed by TAT 1 d later vs. untreated controls (n = 6 per group). Tumor growth (A) and probability of survival (B) measured over 50 d. (C and D) Similar study but with fractionated dose of IGRT (4 doses of 2.5 Gy over 4 d) or TAT only or combined IGRT plus TAT 1 d after completion of IGRT (n = 6 per group). (E and F) Similar study with 4 doses of 3.65-Gy IGRT or TAT 1 d after completion of IGRT or combined IGRT plus TAT (n = 5 per group). (G and H) Tumor rechallenge in opposite glands at end of studies shown in A and B and C and D. P values for treatment groups vs. untreated controls. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 3.

Comparisons of monotherapies with IGRT or TAT vs. combined IGRT plus TAT in colon cancer model. (A and B) CEA transgenic mice bearing established CEA transfected MC38 tumors (n = 6) were treated with single 3.7-kBq dose of ²²⁵Ac-DOTA-M5A (TAT) or single 10-Gy dose of IGRT followed by TAT vs. untreated controls (n = 6 per group). Tumor growth (C) and probability of survival (D) measured over 80 d. Similar study but with 4 doses of 2.5-Gy IGRT over 4 d or TAT only or 4 doses of 2.5-Gy IGRT plus TAT 1 d after completion of IGRT (n = 6 per group). (E and F) Similar study with 4 doses of 3.65-Gy IGRT or TAT 1 d after completion of IGRT or combined IGRT plus TAT (n = 5 per group). (G–I) Tumor rechallenge in opposite flanks at end of studies A and B, C and D, and E and F. P values for treatment groups vs. untreated controls. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 4.

Combination IGRT/TAT/immunocytokine therapy in colon cancer model. (A and B) CEA transgenic mice bearing established CEA transfected MC38 tumors (n = 5) were treated with 4 doses of 2.5-Gy IGRT over 4 d followed by 3.7 kBq of ²²⁵Ac-DOTA-M5A TAT 1 day later, then 10 d after TAT with 1 mg/kg immunocytokine therapy daily for 4 days vs. IGRT plus TAT or untreated controls (n = 5 per group). Tumor growth (A) and probability of survival (B) measured over 80 d. (C) At 28 d after tumor injection, both treatment groups were rechallenged with tumors in opposite flanks and tumor regrowth recorded over 47 d. *P < 0.05; **P < 0.01; ICK = immunocytokine therapy.

FIGURE 5.

Selected immunophenotypes in CEA transgenic mice bearing CEA+ colon cancers after treatment with IGRT, IGRT plus TAT, or IGRT/TAT/immunocytokine therapy vs. untreated controls. (A and B) Frequency of T cells and NK cells in spleen (A) and tumor (B). CD8+/CD4+ T cell ratios (C) and percentage of CD4+ Tregs (D) in tumor, TDLN, and spleen. (E and F) Percentage of IFNγ+ CD8+ T cells (E) and their ratio to CD4+ Tregs in tumor, TDLN, and spleen (F). (G) Phenotypes of tumor myeloid cells (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ICK = immunocytokine therapy.

FIGURE 6.

Origin of CD4+ and CD8+ T cells in CEA transgenic mice bearing CEA+ colon cancers after treatment with IGRT, IGRT plus TAT, or IGRT/TAT/immunocytokine therapy vs. untreated controls. (A–F) Frequency of CD4+ and CD8+ naïve (CD62L+CD44−), central (CD62L+CD44+), and effector (CD62L−CD44+) memory T cells in TDLN (A and B), spleen (C and D) and tumor (E and F) (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ICK = immunocytokine therapy.