Research ArticleCancer

Targeted antibody and cytokine cancer immunotherapies through collagen affinity

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Science Translational Medicine  10 Apr 2019:
Vol. 11, Issue 487, eaau3259
DOI: 10.1126/scitranslmed.aau3259
  • Fig. 1 CBD protein localizes to tumors after intravenous injection through collagen affinity.

    (A) Schematic of conjugation of a CBD, the recombinant VWF A3 domain, to CPI antibody, resulting in affinity for collagen. CBD–IL-2 was recombinantly expressed, with the CBD on the N terminus of IL-2, using a (GGGS)2 linker. (B) KD values of CBD–αPD-L1 and unmodified αPD-L1, αCTLA4, and IL-2 against type I and III collagen, recombinant mouse (rm)CTLA4, rmPD-L1, and/or rmIL-2Rα were measured by enzyme-linked immunosorbent assay (ELISA). N.D., not determined because of low signals. Graphs of concentrations versus signals are shown in fig. S2. (C) MMTV-PyMT cells (5 × 105) were inoculated in the mammary fat pad. When the tumor volume reached 500 mm3, DyLight 800–labeled CBD was injected intravenously. A pie chart represents the biodistribution of CBD protein 48 hours after injection as determined by fluorescence analysis of each organ (n = 4). (D) Intratumoral imaging was performed on MMTV-PyMT tumors when they reached 200 mm3 by injecting DyLight 594–labeled CBD–αPD-L1 or (E) DyLight 594–labeled αPD-L1 intravenously (i.v.) 30 min after injection. The tumor was then harvested, and fluorescence was analyzed by microscopy. Top: Images of whole tumors. Scale bar, 500 μm. Bottom: Images of enlarged yellow squares within top panels. Scale bar, 50 μm. Representative images of two tumors each. (F and G) Binding of (F) CBD–IL-2 or (G) unmodified IL-2 to human melanoma cryosections was imaged by fluorescence microscopy. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 100 μm. Two experimental replicates. Statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey’s test. **P < 0.01.

  • Fig. 2 CBD fusion reduces treatment-related toxicity of immunotherapeutic drugs.

    Adverse events were studied in mice bearing B16F10 melanomas. B16F10 cells (5 × 105) were inoculated on day 0. (A to F) αCTLA4 and αPD-L1 were injected intravenously on days 4 and 7. (A) On day 8, serum concentrations of TNFα in blood plasma were measured (means ± SEM). (B and C) On day 10, the numbers of leukocytic infiltration spots in histologic (B) lung and (C) liver sections were counted and divided by area (means ± SEM). (D and E) On day 10, blood serum (D) ALT and (E) AST activities were measured (means ± SEM). (F) On day 10, the liver was harvested and weighed. Water content in the liver was determined by weighing before and after lyophilization and was normalized to dry tissue weight (means ± SEM). (G and H) B16F10 cells (5 × 105) were inoculated on day 0. CBD–IL-2 or unmodified IL-2 was injected intravenously on days 7 to 9. On day 10, the (G) spleen and (H) lung were harvested and weighed. Water content in the lung was determined as described above (means ± SEM). Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (C) due to nonparametric data. Two experimental replicates. *P < 0.05, **P < 0.01. N.S., not significant; PBS, phosphate-buffered saline.

  • Fig. 3 Both CBD-CPI and CBD–IL-2 treatments reduce tumor growth in three murine tumor models.

    We used three tumor models to study the efficacy of targeted immunotherapy, namely, the B16F10 melanoma model, the CT26 colon carcinoma model, and the MMTV-PyMT breast cancer model. (A) A total of 5 × 105 B16F10 cells were inoculated on back skin, (B) 5 × 105 CT26 cells were inoculated on back skin, and (C and D) 5 × 105 MMTV-PyMT cells were inoculated on the right mammary fat pad on day 0. (A to D) CBD-αCTLA4 + CBD–αPD-L1 (CBD-CPI), αCTLA4 + αPD-L1 (CPI), or PBS was administered on (A) day 4, (B) day 5, and (C and D) day 7. (A) CBD-CPI and unmodified CPI were injected intravenously, and PlGF-2123–144–CPI was injected peritumorally (p.t.). Antibody doses per administration are indicated on the figure. Graphs depict (A to C) tumor volume until the first mouse died and (D) survival rate. (E) Thirty days after the first tumor inoculation in the right mammary gland fat pad, 5 × 105 MMTV-PyMT cells were again inoculated into the left mammary gland fat pad in CBD-CPI–treated tumor-free survivors or in naïve mice. Numbers indicate how many mice remained tumor free among total mice after 40 days of tumor rechallenge. (F) A total of 5 × 105 B16F10 cells were inoculated on the back skin, (G) 5 × 105 CT26 cells were inoculated on the back skin, and (H to K) 5 × 105 MMTV-PyMT cells were inoculated on the right mammary fat pad on day 0. (F to I) IL-2 or equimolar CBD–IL-2 was injected intravenously on (F) day 4, (G) day 5, and (H and I) day 7. Graphs depict (F to H) tumor volume until the first mouse died and (I) survival rate. (J and K) After 7, 14, and 21 days from tumor inoculation, CBD-αCTLA4 + CBD–αPD-L1 (CBD-CPI) + CBD–IL-2, αCTLA4 + αPD-L1 (CPI) + IL-2, or PBS were injected intravenously. Graphs depict (J) tumor volume until the first mouse died and (K) survival rate. Numbers indicate how many mice remained tumor free among total mice 100 days after tumor inoculation. (A) PBS, n = 9; CPI (100 μg) and CBD-CPI (25 μg), n = 8; CBD-CPI (100 μg) and PlGF-2123–144–CPI (100 μg), n = 7. (B) PBS and CPI (25 μg), n = 11; CPI (100 μg) and CBD-CPI (25 μg), n = 10; CBD-CPI (100 μg), n = 9. (C and D) CBD-CPI, n = 12; other groups, n = 11. (E to G) n = 6. (H and I) PBS, n = 10; other groups, n = 11. (J and K) PBS, n = 10; other groups, n = 13. Tumor volumes are presented as means ± SEM. Two experimental replicates. Statistical analyses were performed using ANOVA with Tukey’s test for tumor size and log-rank (Mantel-Cox) test for survival curves. *P < 0.05, **P < 0.01.

  • Fig. 4 Both CBD-CPI and CBD–IL-2 treatments increase B16F10 melanoma–infiltrating cytotoxic CD8+T cells.

    We used the B16F10 model to study T cell behavior in tumors with targeted immunotherapy. B16F10 cells (5 × 105) were inoculated on day 0. CBD-αCTLA4 + CBD–αPD-L1 (CBD-CPI), αCTLA4 + αPD-L1 (CPI), or PBS was administered on day 4. CPI was injected intravenously at 100 μg each. Tumors were collected on day 8, followed by flow cytometric analysis. Frequency of (A) CD8+CD3+ and (B) CD4+CD3+ tumor-infiltrating T cells within CD45+ leukocytes and (C) Treg (Foxp3+CD25+) of CD4+CD3+ tumor-infiltrating T cells. (D) The ratio of CTL (CD62LCD44+CD8+CD3+) versus Treg (Foxp3+CD25+CD4+). (E to G) T cells were extracted from the tumors and stimulated with αCD28 and αCD3 for 6 hours. Graphs depict the percentage of (E) IL-2+, (F) TNFα+, and (G) IFNγ+ of CD8+CD3+ T cells. (H to K) CBD–IL-2, IL-2, or PBS was administered on days 7 to 9. Leukocytes were extracted from the tumor on day 10, followed by flow cytometric analysis. Graphs depict the number of (H) CD8+CD3+ T cells per tumor weight (mg), the frequency of (I) CD8+CD3+ T cells within total CD45+ leukocytes, (J) CD4+CD3+ T cells within total CD45+ leukocytes, and (K) NK1.1+CD3 NK cells within total CD45+ leukocytes. Lines represent means ± SEM. Two experimental replicates. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (H) due to nonparametric data. *P < 0.05, **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/487/eaau3259/DC1

    Fig. S1. Molecular weights are increased by CBD conjugation to αPD-L1 or fusion to IL-2.

    Fig. S2. Affinities of CBD-αCTLA4, CBD–αPD-L1, and CBD–IL-2 for collagen I and III and their target proteins were determined.

    Fig. S3. Both CBD–IL-2 and IL-2 proliferate and bind target cells.

    Fig. S4. Distribution of CBD–αPD-L1 and αPD-L1 within the tumor was analyzed.

    Fig. S5. Blood concentrations of injected CBD-CPI, CBD–IL-2, and their unmodified forms were analyzed.

    Fig. S6. Leukocyte infiltration in the liver, lung, and kidney after unmodified CPI and CBD-CPI treatment was analyzed.

    Fig. S7. Populations of leukocytes infiltrated into the liver after unmodified CPI or CBD-CPI treatment were analyzed.

    Fig. S8. Conjugation of CBD to CPI is indispensable for B16F10 tumor growth suppression.

    Fig. S9. EMT6 immune-excluded tumor is not very responsive to CBD-CPI and CBD–IL-2.

    Fig. S10. CBD-CPI treatment decreases immunosuppressive MDSCs within B16F10 tumor.

    Fig. S11. Immune cells within B16F10 tumor and spleen were analyzed after CBD–IL-2 treatment.

    Fig. S12. CBD–IL-2 treatment increases the number of CD8+ T cells and NK cells within MMTV-PyMT tumor but not within EMT6 tumor.

    Table S1. Protein sequences.

    Data file S1. Original data.

  • The PDF file includes:

    • Fig. S1. Molecular weights are increased by CBD conjugation to αPD-L1 or fusion to IL-2.
    • Fig. S2. Affinities of CBD-αCTLA4, CBD–αPD-L1, and CBD–IL-2 for collagen I and III and their target proteins were determined.
    • Fig. S3. Both CBD–IL-2 and IL-2 proliferate and bind target cells.
    • Fig. S4. Distribution of CBD–αPD-L1 and αPD-L1 within the tumor was analyzed.
    • Fig. S5. Blood concentrations of injected CBD-CPI, CBD–IL-2, and their unmodified forms were analyzed.
    • Fig. S6. Leukocyte infiltration in the liver, lung, and kidney after unmodified CPI and CBD-CPI treatment was analyzed.
    • Fig. S7. Populations of leukocytes infiltrated into the liver after unmodified CPI or CBD-CPI treatment were analyzed.
    • Fig. S8. Conjugation of CBD to CPI is indispensable for B16F10 tumor growth suppression.
    • Fig. S9. EMT6 immune-excluded tumor is not very responsive to CBD-CPI and CBD–IL-2.
    • Fig. S10. CBD-CPI treatment decreases immunosuppressive MDSCs within B16F10 tumor.
    • Fig. S11. Immune cells within B16F10 tumor and spleen were analyzed after CBD–IL-2 treatment.
    • Fig. S12. CBD–IL-2 treatment increases the number of CD8+ T cells and NK cells within MMTV-PyMT tumor but not within EMT6 tumor.
    • Table S1. Protein sequences.

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    Other Supplementary Material for this manuscript includes the following:

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