Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX

In vivo efficacy of AAZ-ValCit-MMAE and AAZ⁺-ValCit-MMAE in combination with L19-IL2 in BALB/c immunodeficient nu/nu mice bearing established subcutaneous SKRC-52 renal cell carcinoma. A, Therapeutic activity of the different treatments as assessed by measurement of tumor volume (mm³) during therapy experiment. Data points represent mean tumor volume ± SEM (n = 4/group). The two treatments were administered intravenously as bolus injections with a 24-hour gap, starting from the SMDCs. B, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment when the two treatments were given with a 24-hour gap, starting from the SMDCs. C, Therapeutic activity of the different treatments given simultaneously, without time gap between the two injections. In both cases, the combination of the AAZ⁺-ValCit-MMAE with L19-IL2 exhibited a superior antitumor activity when compared with vehicle and with the combination of AAZ-ValCit-MMAE with L19-IL2. D, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment when the two treatments were given simultaneously. All treatments were well tolerated and did not have an impact on the body weight of the animals. ****, P < 0.0001; ns, P > 0.05 (two-way ANOVA test, followed by Bonferroni post-test). E, Therapeutic activity of AAZ⁺-ValCit-MMAE and L19-IL2 as single agents or in combination as assessed by measurement of tumor volume (mm³) during therapy experiment. Therapy experiment started when the average tumor volume was about 200 mm³. Data points represent mean tumor volume ± SEM (n = 4/group). The two treatments were administered intravenously as bolus injections with a 24-hour gap, starting from the SMDCs. F, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment. ****, P < 0.0001; ns, P > 0.05 (two-way ANOVA test, followed by Bonferroni post-test). CR, complete response.

In vivo efficacy of AAZ-ValCit-MMAE and AAZ⁺-ValCit-MMAE in combination with anti–PD-1 in immunocompetent BALB/c mice bearing established subcutaneous CT26-3E10 colorectal cell carcinoma. A, Therapeutic activity of the different treatments as assessed by measurement of tumor volume (mm³) during therapy experiment. Data points represent mean tumor volume ± SEM (n = 3/group). The two treatments were administered intravenously as bolus injections with a 6-hour window, starting from the SMDCs. The combination of the SMDCs with anti–PD-1 exhibited a superior antitumor activity when compared with vehicle, AAZ-ValCit-MMAE, AAZ⁺-ValCit-MMAE, or anti–PD-1 only. B, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment. All treatments were well tolerated and did not have an impact on the body weight of the animals. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (two-way ANOVA test, followed by Bonferroni post-test). CR, complete response.

In vivo efficacy of AAZ⁺-ValCit-MMAE in combination with L19-IL12 in BALB/c immunodeficient nu/nu mice bearing established subcutaneous SKRC-52 renal cell carcinoma with different initial tumor burden. A, Therapeutic activity of the different treatments as assessed by measurement of tumor volume (mm³) during therapy experiment. Therapy experiment started when the average tumor volume was about 100 mm³. Data points represent mean tumor volume ± SEM (n = 5/group). The two treatments were administered intravenously as bolus injections with a 24-hour gap, starting from the SMDCs. The combination of AAZ⁺-ValCit-MMAE with L19-IL12 exhibited a superior antitumor activity when compared with vehicle, L19-IL12, or AAZ⁺-ValCit-MMAE only. B, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment. All treatments were well tolerated and did not have an impact on the body weight of the animals. ****, P < 0.0001; ns, P > 0.05 (two-way ANOVA test, followed by Bonferroni post-test). C, Therapeutic activity of AAZ⁺-ValCit-MMAE and L19-IL12 as single agents or in combination as assessed by measurement of tumor volume (mm³) during therapy experiment. Therapy experiment started when the average tumor volume was about 200 mm³. Data points represent mean tumor volume ± SEM (n = 4/group). The two treatments were administered intravenously as bolus injections with a 24-hour gap, starting from the SMDCs. D, Tolerability of the different treatments as assessed by the evaluation of changes (%) in body weight during the experiment. ****, P < 0.0001; ns, P > 0.05 (two-way ANOVA test, followed by Bonferroni post-test). CR, complete response.

Ex vivo histologic analysis and plasma analysis after therapy in BALB/c immunodeficient nu/nu mice treated with AAZ-ValCit-MMAE or AAZ⁺-ValCit-MMAE in combination with L19-IL2. A, Representative pictures of hematoxylin and eosin staining on kidney, stomach, lung, and liver samples from different treated groups of mice collected 3 months after first exposure to the drugs (black scale bar, 100 μm; B, bile ducts in the liver; G, glomeruli structures in the kidney; arrow, inflammatory infiltrate with increase of fibrous tissue). Creatinine (B) and urea (C) plasma concentration measured 3 months after treatment with SMDC products, L19-IL2, and combinations of the drugs (n = 3/group). Administration of SMDCs and immunocytokines did not interfere with normal kidney function as demonstrated by physiologic levels of creatinine and urea in plasma.