Research ArticleCancer

Matrix-binding checkpoint immunotherapies enhance antitumor efficacy and reduce adverse events

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Science Translational Medicine  08 Nov 2017:
Vol. 9, Issue 415, eaan0401
DOI: 10.1126/scitranslmed.aan0401
  • Fig. 1. PlGF-2123–144 peptide–conjugated IgG (PlGF-2123–144–IgG) binds to ECM proteins with high affinity and is released by plasmin.

    (A) Schematic of conjugation of the PlGF-2123–144 peptide to IgG Ab, resulting in binding to ECM proteins. (B) PlGF-2123–144–rat IgG2a and unmodified rat IgG2a were analyzed by SDS-PAGE under reducing conditions with Coomassie blue staining. (C) PlGF-2123–144–αCTLA4 (clone UC10-4F10-11: 4F10) and (D) PlGF-2123–144–αPD-L1 binding to ECM proteins, measured by enzyme-linked immunosorbent assay (ELISA). A450 nm represents absorbance at 450 nm. Bovine serum albumin (BSA) served as a negative control (n = 5, mean ± SD). (E) Affinities (Kd values are shown) of PlGF-2123–144–αCTLA4 and unmodified αCTLA4 (two clones: 4F10 and 9H10) and αPD-L1 against fibronectin, collagen type I, recombinant mouse CTLA4 (rmCTLA4), and/or rmPD-L1 were measured by ELISA. N.D., not determined because of low signal. Graphs of concentrations versus signal are shown in figs. S4 and S5. (F) PlGF-2123–144–rat IgG2a lost fibronectin and collagen I binding capacity after plasmin (0.1 U/ml) cleavage, measured by ELISA (n = 4, mean ± SD; two experimental repeats). Statistical analyses were done using analysis of variance (ANOVA) with Tukey’s test, and a two-tailed Student’s t test was used in (F). **P < 0.01.

  • Fig. 2. PlGF-2123–144 conjugation reduces systemic exposure to checkpoint blockade Abs and potential treatment-related toxicity.

    Mice were inoculated with 5 × 105 B16F10 cells on day 0. (A and B) PlGF-2123–144–αCTLA4 and PlGF-2123–144–αPD-L1 (100 μg each), αCTLA4 and αPD-L1 (100 μg each), or PBS was administered on day 4. PlGF-2123–144–Abs and PBS were injected peritumorally (pt), and unmodified Abs were injected either intraperitoneally (ip) or peritumorally. Blood plasma was collected on days 5, 7, 9, and 11. Concentrations of (A) αCTLA4 and (B) PD-L1 in blood plasma were determined by ELISA [PBS, n = 6; Abs (intraperitoneally), n = 5; others, n = 8; mean ± SEM]. (C to F) PlGF-2123–144–αCTLA4 or unmodified αCTLA4 and αPD-L1 (500 μg each per injection) were injected peritumorally on days 4 and 7. On day 9, blood serum was collected, and concentrations of (C) TNFα and (D) IFN-γ and (E) ALT activity in serum were measured (mean ± SEM). (F) Histologic liver sections were obtained on day 8. The number of lymphocytic infiltration spots was counted and divided by area (mean ± SEM). (G) Sixteen-week-old male NOD mice were given 100 μg of PlGF-2123–144–αPD-L1 or unmodified αPD-L1 on days 0 and 2 and evaluated for the development of diabetes. All Ab injections were intradermal at the back skin. Clinical diabetes was defined as blood glucose concentrations of 250 mg/dl for three consecutive days. Statistical analyses were done using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (C) due to nonparametric data. Log-rank (Mantel-Cox) test was performed for diabetes-free survival curves. Two experimental repeats. *P < 0.05, **P < 0.01. N.S., not significant.

  • Fig. 3. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 treatment reduces B16F10 melanoma growth rate.

    (A) B16F10-OVA cells (1 × 106) or (B to E) B16F10 cells (5 × 105) were inoculated on day 0. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 (PlGF-2123–144–Abs), αCTLA4 + αPD-L1 (Abs), Abs + non–cross-linked PlGF-2123–144 peptide, or PBS was administered (A and B) 25 μg on days 4, 7, and 10; (C) 100 μg on days 4, 7, and 10; (D) 100 μg on day 7; or (E) 300 μg on days 4 and 6, and 100 μg on days 9 and 12. Abs were injected either intraperitoneally or peritumorally. Ab doses per administration are indicated in the figure. αCTLA4 clones 4F10 (A and B) and 9H10 (C to E) were used. Graphs depict tumor volume until the first mouse died. Tumor volumes are presented as means ± SEM. (A) PBS (peritumorally), n = 10; Abs (peritumorally), n = 8; Abs (intraperitoneally), n = 7; PlGF-2123–144–Abs (peritumorally), n = 12; Abs + PlGF-2123–144 (peritumorally), n = 8. (B) Abs (peritumorally), n = 7; other treatment groups, n = 9. (C) n = 10. (D) PlGF-2123–144–Abs (peritumorally), n = 8; other treatment groups, n = 7. (E) Abs (intraperitoneally), n = 6; PlGF-2123–144–Abs (peritumorally), n = 8; other treatment groups, n = 7. Three experimental repeats. Statistical analyses were done using ANOVA with Tukey’s test. *P < 0.05, **P < 0.01.

  • Fig. 4. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 treatment promotes T cell activation and increases B16F10 melanoma-infiltrating CD8+ T cells.

    B16F10 cells (5 × 105) were inoculated on day 0. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 (PlGF-2123–144–Abs), αCTLA4 + αPD-L1 (Abs), or PBS was administered on days 4 and 7. Abs were injected at 100 μg of each per injection either intraperitoneally or peritumorally. Clone 9H10 was used as αCTLA4. Tumor and tdLN were collected on day 8 and analyzed by flow cytometry. (A) Number and (B) frequency of CD8+CD3+ tumor-infiltrating T cells. (C) Frequencies of CD62LCD44+ effector cells and (D) PD-1+ cells among CD8+CD3+ tumor-infiltrating T cells. (E) Number and (F) frequency of CD4+CD3+ tumor-infiltrating T cells. (G) Frequencies of CD62LCD44+ effector cells and (H) CD25+Foxp3+ Tregs among CD4+CD3+ tumor-infiltrating T cells. (I to L) T cells were extracted from tumors and stimulated with αCD28 and αCD3 for 6 hours. Graphs depict the % of (I) Gzmb+, (J) IL-2+, (K) TNFα+, and (L) IFN-γ+ cells among CD8+CD3+ T cells. (M to P) Graphs depict the % of (M) CD62LCD44+ effector cells of CD8+CD3+ T cells, (N) CD25+Foxp3+ Tregs of CD4+CD3+ T cells, (O) CD62L+CD44+ memory, and (P) PD-1+ cells among CD8+CD3+ T cells in tdLN. Two experimental repeats. Statistical analyses were done using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (E), (F), and (P) due to nonparametric data. *P < 0.05, **P < 0.01.

  • Fig. 5. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 treatment induces systemic antitumor immunity.

    (A) Schedule of tumor inoculation and Ab administration throughout the experiment. B16F10 cells (5 × 105) were inoculated intradermally on day 0 in the left side of mouse back skin and then repeated on day 2 in the right side. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 (PlGF-2123–144–Abs), αCTLA4 + αPD-L1 (Abs), or PBS was administered on days 4, 7, and 10. Abs were injected at 100 μg of each per injection either intraperitoneally or peritumorally. Peritumoral injections were performed only beside the left tumor, but not the right tumor. (B) Tumor volumes of the tumor on the left back and (C) the tumor on the right back were measured (n = 9, mean ± SEM). Two experimental repeats. Statistical analyses were done using ANOVA with Tukey’s test. *P < 0.05, **P < 0.01.

  • Fig. 6. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 treatment exhibits antitumor activity in clinically relevant cancer models.

    (A) Tyr:Cre-ER+/LSL-BrafV600E/Ptenfl/fl mice received 50 μg of 4-OH-tamoxifen on their back skin to induce melanoma development. Day 0 is defined as the time point when tumors first become visible. PlGF-2123–144–αCTLA4 + PlGF-2123–144–αPD-L1 (PlGF-2123–144–Abs), αCTLA4 + αPD-L1 (Abs), or PBS was injected peritumorally on days 0, 3, and 6. Abs were injected at 100 μg of each per injection. (B and C) MMTV-PyMT cells were obtained from spontaneously developed breast cancer in FVB-Tg(MMTV-PyVT) transgenic mice and cultured in vitro. MMTV-PyMT cells (8 × 105) were inoculated into the right mammary gland fat pad. After 7, 10, and 13 days, Abs were injected at 100 μg of each per injection peritumorally. (D) Thirty days after the first tumor inoculation, MMTV-PyMT cells (8 × 105) were again inoculated into the left mammary gland fat pad in PlGF-2123–144–Ab–treated tumor-free survivors or in naïve mice. Numbers indicate how many mice remain tumor-free among total mice at the end of the experiment. Graphs depict (A, B, and D) tumor volume until the first mouse died and (C) survival rates. αCTLA4 clone 9H10 was used. Tumor volumes are presented as means ± SEM. (A) PBS (peritumorally), n = 9; Abs (peritumorally), n = 11; PlGF-2123–144–Abs (peritumorally), n = 12. (B and C) PBS (peritumorally), n = 13; Abs (peritumorally), n = 15; PlGF-2123–144–Abs (peritumorally), n = 16. (D) Naïve, n = 7; survivors, n = 9. Three experimental repeats. Statistical analyses were done using ANOVA with Tukey’s test. Kruskal-Wallis test followed by Dunn’s multiple comparison was used in (B) due to nonparametric data. For single comparisons, a two-tailed Student’s t test was used. Log-rank (Mantel-Cox) test for survival curves. *P < 0.05, **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/415/eaan0401/DC1

    Fig. S1. MALDI-TOF MS analysis revealed that molecular weights are increased by PlGF-2123–144 conjugation to αCTLA4 (4F10) and αPD-L1.

    Fig. S2. PlGF-2123–144 conjugation to αCTLA4 (9H10) and αPD-L1 does not yield aggregates.

    Fig. S3. Unmodified αCTLA4 (4F10) and αPD-L1 do not bind to ECM proteins.

    Fig. S4. Affinities of PlGF-2123–144–αCTLA4 and PlGF-2123–144–αPD-L1 for fibronectin and collagen I were measured.

    Fig. S5. Affinities of PlGF-2123–144–αCTLA4 or unmodified αCTLA4 and αPD-L1 for their antigens were measured.

    Fig. S6. Heparin inhibits fibronectin binding of PlGF-2123–144–Abs.

    Fig. S7. Plasmin cleaves PlGF-2123–144.

    Fig. S8. PlGF-2123–144–Abs are retained in collagen in vitro.

    Fig. S9. PlGF-2123–144–Abs are retained in the melanoma tissue.

    Fig. S10. PlGF-2123–144 conjugation does not affect binding of αPD-L1 and FcRn receptor.

    Fig. S11. Liver histologies after unmodified and PlGF-2123–144–checkpoint blockade Ab treatment are shown.

    Fig. S12. PlGF-2123–144–Ab treatment extends survival of B16F10 tumor-bearing mice.

    Fig. S13. Single-agent treatment with PlGF-2123–144–αCTLA4 or PlGF-2123–144–αPD-L1 does not affect tumor growth.

    Fig. S14. PlGF-2123–144 peptide does not affect B16F10 cells’ proliferation.

    Fig. S15. PlGF-2123–144–Ab treatment activates T cells in the spleen.

    Fig. S16. PlGF-2123–144–Ab treatment activates tumor antigen–specific T cells in tdLN, tested in the B16F10-OVA model.

    Fig. S17. Individual tumor growth curves for Fig. 5 are shown.

    Table S1. Original data (provided as an Excel file).

  • Supplementary Material for:

    Matrix-binding checkpoint immunotherapies enhance antitumor efficacy and reduce adverse events

    Jun Ishihara, Kazuto Fukunaga, Ako Ishihara, Hans M. Larsson, Lambert Potin, Peyman Hosseinchi, Gabriele Galliverti, Melody A. Swartz, Jeffrey A. Hubbell*

    *Corresponding author. Email: jhubbell{at}uchicago.edu

    Published 8 November 2017, Sci. Transl. Med. 9, eaan0401 (2017)
    DOI: 10.1126/scitranslmed.aan0401

    This PDF file includes:

    • Fig. S1. MALDI-TOF MS analysis revealed that molecular weights are increased by PlGF-2123–144 conjugation to αCTLA4 (4F10) and αPD-L1.
    • Fig. S2. PlGF-2123–144 conjugation to αCTLA4 (9H10) and αPD-L1 does not yield aggregates.
    • Fig. S3. Unmodified αCTLA4 (4F10) and αPD-L1 do not bind to ECM proteins.
    • Fig. S4. Affinities of PlGF-2123–144–αCTLA4 and PlGF-2123–144–αPD-L1 for fibronectin and collagen I were measured.
    • Fig. S5. Affinities of PlGF-2123–144–αCTLA4 or unmodified αCTLA4 and αPD-L1 for their antigens were measured.
    • Fig. S6. Heparin inhibits fibronectin binding of PlGF-2123–144–Abs.
    • Fig. S7. Plasmin cleaves PlGF-2123–144.
    • Fig. S8. PlGF-2123–144–Abs are retained in collagen in vitro.
    • Fig. S9. PlGF-2123–144–Abs are retained in the melanoma tissue.
    • Fig. S10. PlGF-2123–144 conjugation does not affect binding of αPD-L1 and FcRn receptor.
    • Fig. S11. Liver histologies after unmodified and PlGF-2123–144–checkpoint blockade Ab treatment are shown.
    • Fig. S12. PlGF-2123–144–Ab treatment extends survival of B16F10 tumor-bearing mice.
    • Fig. S13. Single-agent treatment with PlGF-2123–144–αCTLA4 or PlGF-2123–144–αPD-L1 does not affect tumor growth.
    • Fig. S14. PlGF-2123–144 peptide does not affect B16F10 cells’ proliferation.
    • Fig. S15. PlGF-2123–144–Ab treatment activates T cells in the spleen.
    • Fig. S16. PlGF-2123–144–Ab treatment activates tumor antigen–specific T cells in tdLN, tested in the B16F10-OVA model.
    • Fig. S17. Individual tumor growth curves for Fig. 5 are shown.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 Original data (provided as an Excel file).

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