Research ArticleCancer

Selective inhibition of TGFβ1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape

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Science Translational Medicine  25 Mar 2020:
Vol. 12, Issue 536, eaay8456
DOI: 10.1126/scitranslmed.aay8456
  • Fig. 1 TGFβ1 isoform expression is prevalent in most human tumors and correlates with transcriptional signatures of TGFβ pathway activation.

    (A) Heatmap showing percentage of patient tumor samples from TCGA database scoring positive (fragments per kilobase million, FPKM ≥30) for each TGFβ isoform. Tumor types are stratified on the basis of TCGA annotation. (B) Correlation (R, Pearson’s coefficient) analysis of TGFβ isoform expression (FPKM ≥10 cutoff) and a TGFβ pathway activation gene signature from Plasari et al. (33) across selected TCGA-defined tumor types, for which CBT therapies are currently used for therapeutic intervention; significance of association determined by two-sided t test. (C) Similar analysis as described in (B) but using the IPRES transcriptional signature (5) across the same set of tumor types. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; ESCA, esophageal carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma.

  • Fig. 2 SRK-181 is an isoform-specific, anti-latent TGFβ1 antibody that inhibits TGFβ1 activation.

    (A) SRK-181 was captured on anti-human Fc biosensors, and binding to human LTBP1 LLCs with TGFβ1, TGFβ2, or TGFβ3 was measured by biolayer interferometry. Association phase was measured over 10 min by immersing the sensor into 100 nM antigen solution, and subsequent dissociation was recorded in buffer over 10 min. (B) Affinities of SRK-181 for TGFβ1 LLCs from different species were determined by equilibrium titration. Best-fit KD values were calculated in Prism by nonlinear regression analysis of binding data and are indicated as means ± SE from one to two experiments, each performed in duplicate; n.d., not determined. (C) LN229 cells were transfected with a plasmid encoding human proTGFβ1 or human proTGFβ3 (LLC formation with endogenous LTBP) or cotransfected with plasmids encoding human proTGFβ1 plus either GARP or LRRC33 for LLC formation on the cell surface. TGFβ activity was measured with CAGA12 reporter cells. Data are averages ± SD from two independent experiments, each performed in triplicate. Curves are best fit to dose-response inhibition model. (D) CAGA12 reporter cells were cultured for about 18 hours with TGFβ1 SLC in the absence or presence of human plasma kallikrein (KLK). Data are averages ± SD of one experiment run in quadruplicate and representative of at least three independent experiments. (E) Human CD4+ T effector cells (Teff) and autologous regulatory T (Treg) cells were isolated from freshly isolated human PBMCs. Teff cells were labeled with CellTrace Violet, activated, and cultured in absence or presence of Treg cells and the indicated antibodies (1 or 10 μg/ml) under T cell–activating conditions for 5 days. Teff division was determined by measuring CellTrace Violet dilution using flow cytometry. Shown are individual data points plus mean from one experiment run in duplicate, which is representative of two independent experiments run with cells from two separate donors. **P ≤ 0.01; ***P ≤ 0.001; Student’s two-tailed t test.

  • Fig. 3 SRK-181 binding protects three regions on latent TGFβ1 from hydrogen/deuterium exchange.

    (A) Region 1 (red), region 2 (orange), and region 3 (yellow) on latent TGFβ1, which are protected from hydrogen/deuterium exchange (H/DX) upon SRK-181 Fab binding, are mapped onto a model structure of the human TGFβ1 SLC homodimer in surface representation. The TGFβ1 prodomain is shown in blue, the TGFβ1 growth factor is shown in green, and the RGD integrin binding site in the prodomain is highlighted in magenta for orientation. The location of H/DX-protected regions is consistent with SRK-181 binding to the latency lasso region of latent TGFβ1. (B) Amino acid sequences of the three H/DX-protected regions. Region 1 is in the TGFβ1 prodomain, and regions 2 and 3 are on the TGFβ1 growth factor. Highlighted in region 1 are the proteolytic sites for both plasmin and kallikrein in the prodomain. TGFβ2 and TGFβ3 sequences corresponding to the three H/DX-protected regions on TGFβ1 are shown as well. The dots (•) represent amino acid sequence diversity across the three TGFβ isoforms.

  • Fig. 4 Antitumor effects and survival benefit of SRK-181-mIgG1 combination with anti–PD-1 in checkpoint blockade–resistant syngeneic mouse tumors.

    (A) Experimental design for the MBT-2 study. MBT-2 cells were subcutaneously implanted, and dosing was initiated 14 days later, when group mean tumor volumes reached 56 to 57 mm3. Study ended on day 32. (B) Tumor growth (mm3) over time during treatment of established subcutaneous MBT-2 tumors. SRK-181-mIgG1 or its isotype control was dosed once weekly at 10 mg/kg except where indicated. Anti–PD-1 or its control antibody was dosed twice weekly at 10 mg/kg. Tumor ulceration is a common feature of this model, and animals with pronounced ulcerations were euthanized per veterinarian discretion and removed from subsequent survival analyses, yet represented here as dotted lines and further described in table S2. Red dashed line is at 1200 mm3, the IACUC-defined study endpoint. Blue dashed line is at 300 mm3 or 25% of endpoint volume, defining responders, which are indicated as a fraction of evaluable animals in each group. Data are representative of two independent studies. (C) Kaplan-Meier survival plots from (B). ***P < 0.001 log-rank test versus anti–PD-1. (D) Experimental design for the Cloudman S91 study. Cloudman S91 tumor cells were implanted, and dosing was initiated 16 days later, when group mean tumor volumes reached 126 to 132 mm3. Dosing continued for 72 days. (E) Tumor growth (mm3) over time during treatment of established subcutaneous Cloudman S91 tumors. SRK-181-mIgG1 or its isotype control were dosed once weekly at 30 mg/kg, except where indicated. Anti–PD-1 or its control antibody was dosed twice weekly at 10 mg/kg. Red dashed line is at 2000 mm3, IACUC-defined study endpoint. Blue dashed line is at 25% endpoint tumor volume (500 mm3), defining responders, indicated as a fraction of evaluable animals in each group. Limb fractures are common in this model and are considered non–treatment related. Animals with broken limbs were euthanized per veterinarian discretion and were not included in subsequent survival analysis but plotted here as dotted lines and further detailed in table S2. Data are representative of two independent studies. (F) Kaplan-Meier survival plots from (E). **P < 0.01; ***P < 0.001 log-rank test versus anti–PD-1. (G) Experimental design for the EMT-6 study. EMT-6 cells were implanted, and dosing was initiated 3 days later, when group mean tumor volumes reached 39 to 41 mm3 and continued throughout the length of the study, which ended on day 56. (H) Tumor growth (mm3) during treatment of established subcutaneous EMT-6 tumors. SRK-181-mIgG1 or its isotype control was dosed once weekly at 10 mg/kg. Anti–PD-1 or its control antibody was dosed twice weekly at 10 mg/kg. The pan-TGFβ antibody 1D11 was dosed twice a week at 5 mg/kg. Red dashed line is at 2000 mm3, IACUC-defined study endpoint. Blue dashed line is at 25% of endpoint tumor volume (500 mm3), defining responders, which are indicated as a fraction of evaluable animals in each group. Data are representative of two independent studies. (I) Kaplan-Meier survival plots from (H). **P < 0.01; log-rank test versus anti–PD-1.

  • Fig. 5 Blockade of latent TGFβ1 in combination with anti–PD-1 overcomes immune exclusion.

    (A) Flow cytometric analysis of immune cell populations in MBT-2 tumors at day 10 after treatment initiation, five to six animals per group. Mϕ, macrophages; MDSC, myeloid-derived suppressor cells; *P < 0.05; **P < 0.01, ***P < 0.001 Student’s two-tailed t test against anti–PD-1. (B) Representative immunohistochemical staining for CD8 in MBT-2 tumors at day 10 after treatment initiation. Scale bars, 200 μm. (C) Quantification of data exemplified in (B). Area of CD8+ signal per whole-tumor slide scan, n = 5 to 6 animals per group. *P < 0.05, Student’s two-tailed t test against anti–PD-1 group. (D) Representative 40× magnification of immunofluorescence staining for CD8 (cyan) and CD31 (green) in MBT-2 tumors 10 days after treatment initiation. Scale bars, 30 μm. (E) Distribution of CD8+ cells from CD31+ objects from data exemplified in (D) using nearest-neighbor analysis binned by average CD8+ object diameter of 12.4 μm. A distance of “0” is touching a CD31+ object. Data show percentage of CD8+ signal of total tumor CD8+ signal for five to six whole-slide scanned tumors per group.

  • Fig. 6 Lack of cardiovascular findings in rats treated with SRK-181.

    (A) Hematoxylin and eosin histologic staining of rat heart tissue sections. Left panel: Control rat with normal heart valves. Middle panel: Heart valve displaying hemorrhage and inflammatory cells in rat treated with ALK5 inhibitor LY2109761 (300 mg/kg administered daily). Right panel: Heart valves displaying hemorrhage and endothelial hyperplasia in rat treated with a single intravenous dose of pan-TGFβ antibody (30 mg/kg). (B) Summary of incidence and severity of cardiac lesions observed across different treatment groups (n = 5 per group). Animals were administered four weekly intravenous doses of SRK-181 on days 1, 8, 15, and 22 and euthanized on day 29 for histopathology evaluation. Animals receiving pan-TGFβ inhibitors were dosed daily (LY2109761) or once on day 1 (pan-TGFβ antibody) and examined after 1 week of exposure. The control group received four weekly doses of buffer; po, oral gavage; iv, intravenous; qwk, weekly dosing; qd, daily dosing; Ab, antibody.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/536/eaay8456/DC1

    Materials and Methods

    Fig. S1. TGFβ isoform mRNA expression and pathway activation in individual tumors.

    Fig. S2. Characterization of recombinant latent TGFβ1 complexes.

    Fig. S3. SRK-181 does not bind active TGFβ growth factors.

    Fig. S4. Characterization of LLC-presenting molecule expression in LN229 cells.

    Fig. S5. SRK-181 inhibits activation of mouse latent TGFβ1 LLC.

    Fig. S6. Activation of peripheral human Treg cells induces GARP and TGFβ1 LAP expression on their cell surface.

    Fig. S7. SRK-181 Fab binding protects three regions on TGFβ1 SLC from hydrogen/deuterium exchange.

    Fig. S8. SRK-181 and integrin αVβ6 can bind to TGFβ1 SLC simultaneously, suggesting an allosteric mechanism of inhibition.

    Fig. S9. Selection of syngeneic mouse tumor models that recapitulate profiles from CBT-resistant human tumors.

    Fig. S10. Induction of prolonged tumor control with SRK-181-mIgG1/anti–PD-1 combination treatment in multiple tumor models.

    Fig. S11. Further characterization of the treatment effect of SRK-181/anti–PD-1 in MBT-2 tumors.

    Table S1. Percent amino acid sequence identity across human TGFβ isoforms.

    Table S2. Animals euthanized early due to health reasons or found dead.

    Table S3. qPCR reagent list.

    Data file S1. Nonclinical toxicology study summary.

    Data file S2. Original data.

    References (6270)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. TGFβ isoform mRNA expression and pathway activation in individual tumors.
    • Fig. S2. Characterization of recombinant latent TGFβ1 complexes.
    • Fig. S3. SRK-181 does not bind active TGFβ growth factors.
    • Fig. S4. Characterization of LLC-presenting molecule expression in LN229 cells.
    • Fig. S5. SRK-181 inhibits activation of mouse latent TGFβ1 LLC.
    • Fig. S6. Activation of peripheral human Treg cells induces GARP and TGFβ1 LAP expression on their cell surface.
    • Fig. S7. SRK-181 Fab binding protects three regions on TGFβ1 SLC from hydrogen/deuterium exchange.
    • Fig. S8. SRK-181 and integrin αVβ6 can bind to TGFβ1 SLC simultaneously, suggesting an allosteric mechanism of inhibition.
    • Fig. S9. Selection of syngeneic mouse tumor models that recapitulate profiles from CBT-resistant human tumors.
    • Fig. S10. Induction of prolonged tumor control with SRK-181-mIgG1/anti–PD-1 combination treatment in multiple tumor models.
    • Fig. S11. Further characterization of the treatment effect of SRK-181/anti–PD-1 in MBT-2 tumors.
    • Table S1. Percent amino acid sequence identity across human TGFβ isoforms.
    • Table S2. Animals euthanized early due to health reasons or found dead.
    • Table S3. qPCR reagent list.
    • References (6270)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.pdf format). Nonclinical toxicology study summary.
    • Data file S2 (Microsoft Excel format). Original data.

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