Research ArticleCancer

Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy

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Science Translational Medicine  26 Jun 2019:
Vol. 11, Issue 498, eaaw2614
DOI: 10.1126/scitranslmed.aaw2614
  • Fig. 1 Lumican binds collagen and demonstrates prolonged retention and systemic isolation after intratumoral injection.

    (A) Schematic of a sample fusion protein used to anchor cytokines to intratumoral collagen when locally injected. Black lines represent glycine-serine linkers between collagen-binding protein lumican (red), mouse serum albumin (MSA, blue), and the cytokine. (B) Lumican binding to collagen types I and IV was measured by enzyme-linked immunosorbent assay (n = 4, mean ± SD). MSA serves as a nonspecific protein control. (C and D) Representative immunofluorescence images of B16F10 tumors excised, fixed, and frozen 4 days after intratumoral injection of fluorescently labeled lumican (middle, purple). Sections were stained for either (C) collagen type I (left, green) or (D) collagen type IV (left, green). White pixels in the merged composites (right) represent areas of colocalization between lumican and the respective collagen stain. Scale bars, 500 μm. (E) Images and (F) quantification of intratumoral retention and (G) serum fluorescence as a percentage of the injected dose (ID) of labeled Lumican-MSA and MSA after injection into B16F10-TRP2KO tumors (n = 5, mean + SD). i.tu., intratumoral.

  • Fig. 2 Anchoring IL-2 to collagen potentiates tumor-targeting antibody TA99.

    Mice were inoculated with 1 × 106 B16F10 cells on day 0. (A) Survival of these mice treated with intratumoral injections of phosphate-buffered saline (PBS; n = 7), MSA-IL2 (n = 17), Lumican (n = 7), or Lumican-MSA-IL2 (n = 17) and intraperitoneal (i.p.) TA99 on days 6, 12, and 18. (B) Representative image and (C) percentage of treated mice with localized vitiligo after indicated treatment. (D) Survival of B16F10 tumor–bearing mice treated intraperitoneally with TA99 and Lumican-MSA-IL2 injected intratumorally (n = 17), peritumorally (peri.tu.) (n = 5), or 2 cm distal to the lesion subcutaneously at the tail base (s.c. tail base) (n = 5). (E) Survival of B16F10 tumor–bearing Batf3−/− or wild-type (WT) mice treated intraperitoneally with TA99 and treated intratumorally with Lumican-MSA-IL2 and with indicated depletion antibody (n = 5 per group). (F) Representative images (top) of an IFN-γ enzyme-linked immunospot (ELISPOT) assay and quantification (bottom) of B16F10-reactive spot-forming units (SFU) from splenocytes isolated 4 days after the indicated treatments (n = 3 per group, mean ± SD). ELISPOT data analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (G) Study of mice inoculated with 1 × 106 B16F10 cells on the right flank [ipsilateral (ipsi)] and with 3 × 105 B16F10 cells on the left flank (contralateral) on day 0. Ipsilateral intratumoral treatments were administered alongside TA99 intraperitoneally on days 6 and 12. Tumor area (left, mean + SD) of the contralateral (untreated) and ipsilateral (treated) lesions and survival (right) were monitored (n = 7 per group). Tumor area for each group is shown until a mouse in the group reaches the euthanasia criterion. Arrowheads indicate times of treatment. Survival was compared by log-rank Mantel-Cox test. *P < 0.03, **P < 0.002, ***P < 0.0002, ****P < 0.0001. ns, not significant.

  • Fig. 3 Collagen-anchoring IL-12 demonstrates reduced toxicity and potentiates disparate systemic immunotherapy modalities.

    Mice were inoculated with 1 × 106 B16F10 cells on day 0. (A) Weight change from baseline and (B) corresponding survival of these mice after treatment with intratumoral injection of PBS (n = 6), IL12-MSA (n = 7), or IL12-MSA-Lumican (n = 7) or intraperitoneal injection of IL12-MSA (n = 7) on days 6 and 12. Weight change for the entire group plotted until a mouse in that group reaches the euthanasia criterion. (C) Mice were inoculated with 1 × 106 B16F10 cells on day 0. Vaccinations (Vacc) were administered subcutaneously (s.c.) at the tail base. The graphs show weight change from baseline (left, mean + SD), corresponding tumor area (middle, mean + SD), and survival (right) of mice treated with intratumoral injections of PBS (n = 12), IL-12 (n = 10 for IL12-MSA; n = 10 for IL12-MSA-Lumican), vaccine (n = 7) alone, or vaccine and IL12 (n = 7 for IL12-MSA; n = 7 for IL12-MSA-Lumican) on days 5, 11, and 17. (D) Mice were inoculated with 0.5 × 106 B16F10 cells on day 0 and lymphodepleted by total body irradiation on day 4. CAR-T treatments using 10 million CD3+ CAR-T cells were injected intravenously (i.v.) through the tail vein only on day 5. The graphs show weight change from baseline (left, mean + SD), corresponding tumor area (middle, mean + SD), and survival (right) of mice treated with intratumoral injections of PBS (n = 11), IL-12 (n = 9 for IL12-MSA; n = 5 for IL12-MSA-Lumican), CAR-T (n = 11) alone, or CAR-T and IL12 (n = 9 for IL12-MSA; n = 5 for IL12-MSA-Lumican) on days 5 and 11. (E) Mice were inoculated with 0.5 × 106 4T1-Luc cells in the mammary fat pad on day 0. Neoadjuvant therapy was administered on days 7 and 13, and the primary tumors were surgically excised on day 16. After the operation, mice were monitored for metastases by in vivo imaging. The graphs show total body weight change during neoadjuvant treatment (left, mean + SD), primary tumor growth and weight (middle, mean + SD), and metastasis-free survival (right) of mice left untreated (n = 10) or treated with intratumoral injections of IL-12 (n = 10 for scIL12; n = 9 for IL12-MSA-Lumican) and intraperitoneal injection of anti–PD-1 on days 7 and 13. Arrowheads indicate times of treatment. Tumor area for each group shown until a mouse in that group reaches the euthanasia criterion or primary tumor is excised. Weight change shown until a mouse in study reaches the euthanasia criterion or primary tumor is excised. Weight changes and excised tumor weights were compared by one-way ANOVA with Tukey’s multiple comparisons test. Survival was compared by log-rank Mantel-Cox test. *P < 0.03, **P < 0.002, ***P < 0.0002, ****P < 0.0001.

  • Fig. 4 Collagen-anchored IL-12 and IL-2 safely potentiate anti–PD-1.

    Mice were inoculated with 1 × 106 B16F10 cells on day 0. (A) Weight change from baseline (left, mean + SD) and corresponding survival (right) of B16F10 tumor–bearing mice treated with intratumoral injections of PBS (n = 5), MSA-IL2 and IL12-MSA (n = 5), or Lumican-MSA-IL2 and IL12-MSA-Lumican (n = 5) on days 5 and 11. Weight change shown until a mouse in study reaches the euthanasia criterion. (B) Quantification (top, mean ± SD) of tumor-infiltrating CD8+ T cells (gated on single cell/live/CD45+/CD3+) on day 11 and their corresponding median fluorescence intensity (MFI) of surface PD-1 (bottom, mean ± SD) after the indicated treatments on day 5. (C) Weight change from baseline (left, mean + SD) and corresponding survival (right) of B16F10 tumor–bearing mice treated with intratumoral injections of PBS (n = 5), MSA-IL2 and IL12-MSA (n = 5), or Lumican-MSA-IL2 and IL12-MSA-Lumican (n = 5) alongside intraperitoneal anti–PD-1 on days 5 and 11. (D) Percentage of treated mice with localized vitiligo after the indicated treatments. Treatment groups for which vitiligo was not observed are not included in this plot. (E) Percentage of long-term survivors, treated as indicated, which rejected a rechallenge on day 100 with 105 B16F10 cells inoculated on the contralateral flank. (F and G) Tumor area (left, mean + SD) and survival (right) of mice inoculated with (F) 1 × 106 EMT6 cells (n = 7 to 14 per group) or (G) 1 × 106 MC38 cells (n = 6 to 8 per group) on day 0 and treated as indicated on days 5, 11, and 17. Arrowheads indicate times of treatment. Weights and T cell data were compared by one-way ANOVA with Tukey’s multiple comparisons test. Survival was compared by log-rank Mantel-Cox test. *P < 0.03, **P < 0.002, ***P < 0.0002, ****P < 0.0001.

  • Fig. 5 Collagen-anchoring IL-2 and IL-12, anti–PD-1, and TA99 control large BrafV600E/Ptenfl/fl tumors.

    Melanoma in BrafV600E/Ptenfl/fl mice was initiated by application of 4-hydroxytamoxifen on the right flank on day 0. (A) Tumor volume (mean + SD) of tumor-bearing mice treated either with an intratumoral injection of PBS (n = 4) or with intratumoral collagen-anchoring cytokines Lumican-MSA-IL2 and IL12-MSA-Lumican and intraperitoneal TA99 and anti–PD-1 (n = 5) on days 25, 31, 37, 43, 49, 55, and 61. For each group, tumor volume shown until a mouse reaches the euthanasia criterion (>1200 mm3). (B) Representative images of BrafV600E/Ptenfl/fl flank tumors at onset of indicated treatment on day 25 (top) and before the sixth treatment on day 55 (bottom). (C) Overall survival of tumor-bearing mice treated with intratumoral PBS (n = 10), with intratumoral Lumican-MSA-IL2 and IL12-MSA-Lumican and intraperitoneal TA99 and anti–PD-1 (n = 14), with intratumoral Lumican-MSA-IL2 and IL12-MSA-Lumican and intraperitoneal anti–PD-1 (n = 10), with intratumoral MSA-IL2 and IL12-MSA and intraperitoneal TA99 and anti–PD-1 (n = 9), or with intratumoral MSA-IL2 and IL12-MSA and intraperitoneal anti–PD-1 (n = 8) on days 25, 31, 37, 43, 49, 55, and 61. Arrowheads indicate times of treatment. Overall survival graph enumerates mice that succumbed to tumor burden (>1200 mm3) or to treatment-related weight loss (>20%); the latter is indicated by a blue “×” for each mouse. Survival was compared by log-rank Mantel-Cox test. *P < 0.03, ***P < 0.0002, ****P < 0.0001.

Additional Files

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Expressibility criterion hones potential collagen-binding proteins to lumican.
    • Fig. S2. Recombinant lumican is stable and monomeric.
    • Fig. S3. Collagen types I and IV are abundantly expressed in several syngeneic murine tumor models.
    • Fig. S4. Molecular weight and collagen affinity contribute to intratumoral retention.
    • Fig. S5. Recombinant lumican is amenable to C-terminal IL-2 fusion.
    • Fig. S6. Lumican-MSA-IL2 and MSA-IL2 have identical bioactivity.
    • Fig. S7. IL-2 or TA99 as monotherapies impart limited survival enhancement.
    • Fig. S8. Intranodal administration of Lumican-MSA-IL2 is insufficient for survival.
    • Fig. S9. Individual innate immune populations appear dispensable for treatment efficacy of TA99 and Lumican-MSA-IL2.
    • Fig. S10. TA99 and Lumican-MSA-IL2 induced systemic tumor-specific cellular immunity and established protective memory.
    • Fig. S11. Recombinant lumican is amenable to N-terminal IL-12 fusion.
    • Fig. S12. IL-12 demonstrates dose-dependent toxicity and efficacy.
    • Fig. S13. Vaccination yields antigen-specific CD8+ T cells.
    • Fig. S14. Combination of reduced-dose IL-12 and IL-2 yields supra-additive toxicity.
    • Fig. S15. Adaptive immunity is critical for therapeutic efficacy of Lumican-MSA-IL2 and IL12-MSA-Lumican treatment.
    • Fig. S16. IL12-MSA-Lumican and Lumican-MSA-IL2 treatment expands intratumoral and tumor-specific splenic CD8+ T cells.
    • Fig. S17. Collagen-anchoring cytokines are not immunogenic.
    • Table S1. Lumican and cytokine fusion protein sequences.

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    Other Supplementary Material for this manuscript includes the following:

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