Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein

Abstract

Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response.

Significance: The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.

FIGURE 1

HuB12 internalizes into FAP-expressing cells. A, Confocal microscopy images of hPrCSC-44 cells (top) or PC3 cells (bottom) after incubation with Alexa Fluor-647 (AF647)-labeled huB12 (50 nmol/L) for 1 hour. Single-channel images of huB12-AF647 localization, fluorescein-dextran labeled endosomes, and CellBrite Orange-labeled plasma membrane are shown. Composite images depicting whole-cell and enlarged regions of interest are shown as colored fluorescence overlays. B, Quantification of the organellar distribution of huB12-AF647, assessed using MCC with either fluorescein dextran (endosome marker) or CellBrite Orange (membrane marker). Values represent average ± SD of n ≥ 6 images, *, P <0.05 using two-tailed Student’s t-test. C, Representative images of CWR-R1^FAP or parental CWR-R1 cells after 16 hours incubation with the indicated antibody (40 nmol/L). A panel of phase-contrast images overlaid with an outline of individual cells (yellow) and pHrodoRed fluorescence (red) is shown. D, Time course of pHrodoRed fluorescence intensity in CWR-R1^FAP cells incubated with the indicated concentration of huB12-pHrodoRed, data shown from a representative experiment. Dose–response curves of peak pHrodoRed fluorescence values in CWR-R1 (gray) or CWR-R1^FAP (red) cells after 24 hours incubation with increasing concentrations of huB12-pHrodoRed (E) or isotyped control IgG-pHrodoRed (F). Values represent average ± SEM from n = 3 replicates.

FIGURE 2

PET/CT imaging of FAP in vivo. A, Representative PET/CT images of mice bearing subcutaneous 22Rv1/hPrCSC-44 xenografts (white arrowhead). Mice (n = 3/group) received 3.5 MBq (25–50 µg, 7.7 µg/MBq) of [⁸⁹Zr]Zr-HuB12, 3.6 MBq (25–50 µg, 7.5 µg/MBq) [⁸⁹Zr]Zr-F19 or 3.4 MBq (25–50 µg, 7.9 µg/MBq) [⁸⁹Zr]Zr-J591 via tail vein and were imaged at the indicated timepoints. B, Quantitative analysis of subcutaneous xenografts from mice (n = 3/group) revealed significantly higher [⁸⁹Zr] uptake in the tumors of [⁸⁹Zr]Zr-HuB12 administered animals compared with the [⁸⁹Zr]Zr-F19 or [⁸⁹Zr]Zr-J591 at all timepoints postinjection. Values represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. C, Radio-competition cell binding results using [⁸⁹Zr]Zr-HuB12 and either the FAP-positive hPrCSC-44 or FAP-negative 22Rv1 cell line. The IC₅₀ was only determinable for hPrCSC-44 and was measured at 11.6 nmol/L.

FIGURE 3

In vitro and in vivo therapeutic efficacy of huB12-MMAE. A, FAP-targeted ADCs show a potent and FAP-selective cytotoxic activity. Cell viability hPrCSC-44, CWR-R1FAP, PC3, and CWR-R1 cell lines. Cells were treated for 72 hours with a serial dilution of either huB12 IgG or an isotype control IgG with or without an MMAE warhead. Data are represented as mean ± SEM (n = 3 biological replicates). B, HuB12 ADC therapy demonstrates significant tumor control compared with the saline arm. Black arrows signify treatment administration (*, P ≤ 0.05; **, P ≤ 0.01, ANOVA mixed methods test). C, Kaplan–Meier survival curve for mice treated with therapeutic antibodies, huB12 ADC or Isotype Control ADC compared with saline control mice. Mice treated with huB12 ADC survived significantly longer than the controls, HR analysis used for statistical analysis.

FIGURE 4

Specificity of huB12-MMAE treatment in the Stacks. A, Workflow of treatment and analysis of CAF-tumor models using huB12-MMAE. B, Viability analysis of CAFS (hPrCSC-44/top) and tumor cells (22Rv1/bottom) in monoculture (left) and coculture (right) using indicated concentrations of huB12-MMAE for 72 hours.

FIGURE 5

Downstream effects of huB12-MMAE treatment on gene expression in CAF-tumor models in Stacks. A, Concentration of selected factors in supernatant in monoculture and coculture conditions. B, Relative mRNA expression of select genes in hPrCSC-44 cells in monoculture and coculture treated and untreated conditions. C, Relative mRNA expression of select genes in 22Rv1 cells in monoculture and coculture treated and untreated conditions. Data for B and C expressed as normalized relative quantity (NRQ). *, P < 0.05.