Targeting FAK improves the tumor uptake of antibody-drug conjugates to strengthen the anti-cancer responses

Summary

Antibody-drug conjugates (ADCs), exemplified by HER2-targeted Enhertu and TROP2-targeted Trodelvy, have demonstrated significant therapeutic potential in cancers. However, a subset of patients remains refractory to ADC treatment, suggesting that the efficacy requires further optimization. Here, we demonstrate that excessive cancer-associated fibroblasts (CAFs) can form a fibrotic barrier, impeding the tissue uptake of ADCs to dampen the anti-tumor efficacy. Mechanistically, cancer cells transform normal fibroblasts into FAK-activated CAFs. The proliferation of these CAFs reduces the tumor uptake of macromolecular drugs, conferring resistance to ADCs. Targeting FAK with a small molecule inhibitor IN10018 effectively diminishes the CAF-associated tumor barrier, enhancing the tumor uptake of various ADCs irrespective of their specific targets. Combination therapy with IN10018 and ADCs targeting either HER2 or TROP2 consistently yielded superior antitumor outcomes compared to monotherapies in animal models. These findings provide compelling preclinical evidence supporting the clinical evaluation of IN10018 in combination with ADCs.

Graphical abstract

Highlights

• •CAFs can form a fibrotic barrier, hampering the tumor uptake of ADCs
• •FAK inhibitor IN10018 can break the fibrotic barrier to enhance the tumor uptake of ADCs
• •The combination of IN10018 and ADCs outperformed monotherapy in cancer treatment
• •The preclinical evidence supports further discovery in the clinical trials

Wildlife behavior; Zoo animal behavior; Evolutionary biology

Introduction

Antibody-drug conjugates (ADCs) have demonstrated efficacy in the treatment of various cancer types in clinical practice. Trastuzumab emtansine (Kadcyla), which specifically targets HER-2, represents the inaugural ADC approved for the management of HER-2-positive cancers.¹ Trastuzumab deruxtecan (Enhertu), a novel ADC based on Trastuzumab, has recently garnered significant attention for its potential in treating various cancer types, including breast cancer, gastric cancer, pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC).^2,3,4,5 Enhertu’s most significant attribute lies in its efficacy against HER2-low cancers, a population previously deemed unresponsive to HER2-targeted therapies.⁶ Besides HER2 ADCs, a series of ADCs targeting other biomarkers were also discovered and launched, including the agents targeting TROP2, Nectin4, CD30, and Folate receptor alpha.⁷ Effective profiling of ADCs is influenced by disease characteristics, target expression levels, antibody properties, linker attributes, and payload sensitivities.⁸ The molecular characterization of ADCs has been significantly enhanced through advancements in development, design, and manufacturing processes. While this approach has demonstrated promising drug responses in some patients, many others continue to experience suboptimal therapeutic outcomes with ADCs. The primary challenge in achieving robust and sustained efficacy is closely linked to the underlying disease biology of solid tumors.⁹

Cancer-associated fibroblasts (CAFs) are typically found within the microenvironment of solid tumors,¹⁰ involving multiple interactive mechanisms with cancer cells. In early-stage cancers, a small number of CAFs surround the cancer cells, resulting in low aggressiveness characterized by slow growth and weak invasion. Conversely, in late-stage and heavily treated cancers, a greater proliferation of CAFs occurs adjacent to the cancer cells, leading to a more malignant and treatment-resistant tumor environment. CAFs primarily derive from normal fibroblasts and quiescent stellate cells.¹¹ Cancer-cell-derived cytokines/chemokines can induce the transformation of normal cells into CAFs.¹² Conversely, CAFs act as a defense system to protect cancer cells from cell-toxic lymphocytes and cell-lethal therapies.¹³ Previous studies have demonstrated that CAFs can impede the interaction between drug-based therapies and cancer cells, thereby promoting drug resistance and disease progression.^14,15 Similarly, CAFs may contribute to the resistance of ADCs by interfering with the tumor uptake of macromolecular agents.¹⁶ Therapeutic strategies that overcome this physical barrier could facilitate the development of combinatorial approaches with ADCs.

Focal adhesion kinase (FAK) functions as a pivotal nonreceptor kinase, serving as a critical mediator between the tumor microenvironment and cancer cells. FAK signaling has been positively associated with the progression of various cancer types, including pancreatic cancer, ovarian cancer, NSCLC, and breast cancer.¹⁷ Previous studies, including our own, have demonstrated that the maintenance of CAFs is dependent on activated FAK signaling. Targeting FAK with small molecule inhibitors has been shown to significantly reduce the number of CAFs, thereby enhancing the efficacy of multiple therapeutic strategies, such as chemotherapy and immunotherapy.^18,19 In light of the emerging mechanisms linking FAK signaling and CAFs, we hypothesized that the combination of FAK inhibition and ADCs could enhance the tumor uptake of macromolecules by alleviating the burden of CAFs, potentially leading to superior anti-cancer treatment outcomes compared to monotherapy.

In this study, we observed that CAFs contribute to the formation of a physical barrier, and the extracellular matrix secreted by CAFs impairs the tumor uptake of ADCs, thereby disrupting their cytotoxic effects in patient-derived xenograft (PDX) and cell-line-derived xenograft (CDX) models. Cancer cells can transform normal fibroblasts into CAFs, characterized by the upregulation of fibroblast activation protein (FAP) and activated FAK signaling. Treatment with the FAK small molecule inhibitor IN10018 reduced CAF levels and enhanced ADC tumor uptake, resulting in improved anticancer efficacy compared to monotherapy. ADCs targeting either HER2 or TROP2 demonstrated synergistic benefits when combined with IN10018, suggesting that targeting FAK may be a viable strategy to overcome ADC resistance, in part due to excessive CAFs. These preclinical findings warrant further investigation and validation in clinical trials.

Results

Enhertu low-response PDX models possess more CAFs in tumors as opposed to the Enhertu high-response PDX models

We hypothesized that the CAF-mediated barrier could influence the anti-tumor efficacy of Enhertu by restricting its distribution within the tumors. To investigate whether CAFs can determine the tumor growth inhibition induced by Enhertu, we treated nine PDX models across various cancer types (NSCLC, gastric cancer, breast cancer, head and neck cancer, and esophageal cancer) with Enhertu (Figures S1A–S1I and S2A–S2I). Based on tumor growth inhibition (TGI), we categorized the PDX models into two subgroups: those with TGI less than 70% were classified as Enhertu low-response models, whereas those with TGI greater than 70% were considered Enhertu high-response models (Figure 1A).

Figure 1.

Enhertu low-response PDX models possess more CAFs in tumors compared to the Enhertu high-response PDX models

(A) TGI values for each PDX model against Enhertu. TGI = 70% was set as a threshold to determine the drug effectiveness.

(B) HER2 H score estimated from IHC staining of the PDX tumors.

(C) Positive percentages of FAP target estimated for the PDX tumors.

(D) Percentage of Sirius red positive area for the PDX tumors.

(E–F) The tumor growth curves of ST-02-0103 and ST-02-0103R upon the treatment with Enhertu.

(G) The pathological images for ST-02-0103 and ST-02-0103R. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. NS, non-significant; ∗p < 0.05. Scale bar: 50 μm.

Pathological analysis was performed for the PDX tumors (Figures S3A–S3I). We did not see statistical significance on HER2 expression between the two subgroups, although the low-response models still exhibit lower HER2 levels versus the high-response models (Figure 1B). Besides, Enhertu low-response models exhibited heavy staining of FAP and Sirius red. The high-response models exhibited a reversed trend (Figures 1C and 1D). Further, the area of FAP staining in tumors exhibited a negative correlation with TGI, suggesting that CAFs might be of importance to the anti-tumor effects of Enhertu (Figure S1J). Consistent with the findings from multiple PDX models, an acquired Enhertu-resistant gastric cancer PDX model, ST-02-0103R, displayed more CAFs within tumors as opposed to its parental model ST-02-0103, further validating our hypothesis (Figures 1F–1H).

The fibrotic barrier impedes the tumor uptake of trastuzumab, thereby diminishing the anti-tumor efficacy of Enhertu

To evaluate the anti-tumor effects of Enhertu, we utilized xenograft models derived from the human gastric cancer cell line NCI-N87, as well as a co-inoculation model of NCI-N87 and a mouse fibroblast cell line NIH/3T3 (Figures 2A and 2B). The co-inoculation model initially exhibited robust anti-tumor responses following dosing, but tumor growth resumed exponentially by the third week post-grouping. In contrast, the NCI-N87 model continued to show tumor regression throughout the study. These findings suggest that the fibrotic barrier formed by NIH/3T3 may hinder the growth inhibition of tumors by Enhertu.

Figure 2.

The fibrotic barrier impedes the tumor uptake of Trastuzumab, thereby diminishing the anti-tumor efficacy of Enhertu

(A and B) The anti-tumor effects of Enhertu in the treatment of gastric cancer CDX model NCI-N87 and NIH/3T3/NCI-N87 co-inoculation model. Enhertu was dosed once at 3 mg/kg by tail vein injection. Tumor volumes were recorded to show the anti-tumor effects for NCI-N87 (A) and NIH/3T3/NCI-N87 co-inoculation models (B).

(C) The red fluorescence imaging for the NCI-N87 model and NIH/3T3/NCI-N87 co-inoculation model treated with Trastuzumab Cy5.5. The mice were dosed with 3 mg/kg Trastuzumab Cy5.5. Twenty-four hours later, the in vivo fluorescence imaging was performed by IVIS system.

(D–F) The IHC staining of FAP and Sirius red staining for the NCI-N87 and NIH/3T3-NCI-N87 tumors from (C). After the red fluorescence imaging, the tumors were harvested for the pathological assays to check with the FAP expression and Sirius red intensity. The percentages of FAP positive area (E) and Sirius red positive area (F) were evaluated by ImageJ. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. ∗p < 0.05. Scale bar: 100 μm.

To investigate whether the fibrotic barrier can obstruct the tumor uptake of antibodies, we administered Trastuzumab Cy5.5 to both the NCI-N87 and NIH/3T3-NCI-N87 co-inoculation models (Figure 2C). The fluorescence signals were recorded, and tumors were collected at 24 h post-dosing (Figure 2D). The results revealed that the co-inoculation model tumors displayed significantly stronger and more extensive signals of FAP (Figure 2E) and Sirius red staining (Figure 2F) compared to the NCI-N87 model, indicating the establishment of a more substantial CAFs-based physical barrier. Notably, fluorescence measurements showed that higher proportions of Trastuzumab Cy5.5 were in the NCI-N87 tumors compared to the co-inoculation tumors. These data suggest that excessive CAFs may reduce the tumor uptake of Trastuzumab, thereby attenuating the anti-tumor efficacy of Enhertu.

FAK inhibitor IN10018 effectively eradicates CAFs derived from normal fibroblasts

Normal fibroblast cell line NIH/3T3 was co-cultured with human gastric cancer cell line NCI-N87 or mouse colorectal cancer cell line CT26 in transwell plates. After 5 days of incubation, the NIH/3T3 cells in the lower wells were subjected to immunofluorescence staining. Compared to NIH/3T3 cells alone, those co-cultured with either NCI-N87 or CT26 cells exhibited significantly elevated expression of FAP and phosphorylated FAK at Y397, indicating that cancer cells can induce the transformation of normal fibroblasts into CAFs. Treatment with a small molecule FAK inhibitor IN10018 can effectively reverse the transformation of the NIH/3T3 cells, evidenced by decreased FAK activity and FAP expression (Figure 3A). These findings were corroborated by western blot analysis (Figures 3B and 3C).

Figure 3.

FAK inhibitor IN10018 effectively eradicates CAFs derived from normal fibroblasts

(A) The IF staining figures for NIH/3T3, NIH/3T3 co-cultured with CT26 cells, and NIH/3T3 co-cultured with NCI-N87 cells. The indicated cells were treated with PBS and 3 μm of IN10018 for 5 days. Phospho FAK Y397, FAP, and DAPI signals were recorded by IF staining.

(B–C) Western blot results for the IN10018-treated NIH/3T3 co-cultured with CT26 (B) or NCI-N87 (C) cells. The cells were treated with PBS and 3 μm of IN10018 for 3 or 5 days. The protein of NIH/3T3 was harvested for western blot to check the expression of the targets.

(D) Clonogenic assay for IN10018-treated NIH/3T3 co-cultured with CT26 cells or NCI-N87 cells. The cells were treated with PBS and 3 μm of IN10018 for 10 days and then the NIH/3T3 cell colonies were stained with crystal violet.

(E) The data analysis of colony assay from (D) by ImageJ. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. NS, non-significant; ∗∗p < 0.01. Scale bar: 40 μm.

Subsequently, the co-culture system was re-established, and a clonogenic assay was conducted to assess the cytotoxic effects of the FAK inhibitor IN10018 on NIH/3T3 cells (Figure 3D). The results demonstrated that IN10018 did not inhibit the growth of untransformed NIH/3T3 cells but effectively suppressed the proliferation of NIH/3T3 cells transformed by co-culture with cancer cells (Figure 3E). These data suggest that the transformation of the normal fibroblasts elicited by cancer cells renders them susceptible to IN10018 treatment.

IN10018 reduces CAFs and enhances trastuzumab uptake in tumors, thereby improving the anticancer efficacy of Enhertu

To investigate the relationship between CAFs depletion and enhanced antibody tumor uptake in vivo, we administered a combination of IN10018 and Trastuzumab Cy5.5 to the NIH/3T3-NCI-N87 co-inoculated mouse model (Figure 4A). Mice were pretreated with IN10018 for 10 days, then three mice per group were euthanized for tumor harvest. Immunohistochemical staining for FAP revealed that IN10018 treatment significantly reduced CAF levels compared to the vehicle group (Figures 4B and 4C). Concurrently, Trastuzumab Cy5.5 was intravenously injected into the remaining mice. Six hours later, IVIS imaging captured higher Trastuzumab Cy5.5 uptake in tumor spheres of IN10018-pretreated mice compared to non-pretreated mice (Figures 4D and 4E). The difference in fluorescence signals became more pronounced at the 48-h time point, indicating that FAK inhibition enhances macromolecular delivery to tumors by eliminating CAFs (Figures 4F and 4G).

Figure 4.

IN10018 reduces CAFs and enhances Trastuzumab uptake in tumors, thereby improving the anticancer efficacy of Enhertu

(A) The schematic figure for the animal experiment with NIH/3T3/NCI-N87 co-inoculation model.

(B) The representative images of FAP staining for the tumors of the NIH/3T3-NCI-N87 co-inoculation model.

(C) The percentage of FAP-positive area of the tumors from NIH/3T3-NCI-N87 co-inoculation model.

(D–G) The red fluorescence imaging for the NIH/3T3-NCI-N87 co-inoculation model treated with IN10018 and Trastuzumab Cy5.5. The mice were treated with 25 mg/kg IN10018 for 10 days and then dosed with one injection of Trastuzumab Cy5.5 (3 mg/kg). Six hours (D and E) and 48 h (F and G) later, the red fluorescence imaging was performed for the mice to check with the infiltrated amount of the antibody within tumors.

(H–I) The tumor growth curves and body weight changes of the efficacy study with NIH/3T3-NCI-N87 co-inoculation model. The animal model was treated with 25 mg/kg IN10018 once daily per oral and 3 mg/kg Enhertu twice on day 9 and day 28. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. NS, non-significant; ∗p < 0.05, ∗∗p < 0.01. Scale bar: 200 μm.

We subsequently assessed the antitumor efficacy of the IN10018 and Enhertu combination in the NIH/3T3-NCI-N87 co-inoculated model. The dual-drug regimen demonstrated superior tumor growth inhibition compared to either monotherapy, suggesting that IN10018-increased macromolecular tumor uptake translates to enhanced therapeutic benefits (Figures 4H and 4I).

IN10018 enhances the antitumor efficacy of Enhertu in human PDX models

A HER2-expressing NSCLC PDX model, LU-01-1626, was established to evaluate the therapeutic efficacy of the combination regimen of IN10018 and Enhertu. Previous data indicated de novo resistance to Enhertu treatment in this model (Figure 1A). The dual regimen demonstrated superior anticancer effects compared to monotherapy (Figure 5A). The mice tolerated the treatments well, with no observed body weight loss (Figure 5B).

Figure 5.

IN10018 enhances the antitumor efficacy of Enhertu in human PDX models

(A–B) The tumor growth curves and body weight changes for the efficacy study with NSCLC PDX model LU-01-1626. IN10018 was dosed to the model once daily by oral gavage. For day 0 to day 3, the dose was 25 mg/kg. Since day 4, the dose of IN10018 was adjusted to 12.5 mg/kg via oral gavage. Enhertu was dosed once weekly by tail vein injection. The dose was set as 10 mg/kg in week 1, 3 mg/kg in week 2, and adjusted to 6 mg/kg from week 3 till the end of the study. Scale bar: 100 μm.

(C) The representative images of the IHC staining with anti-FAP, anti-human IgG, anti-DXD, anti-Ki67, and TUNEL assay for the tumors from the LU-01-1626 model.

(D–F) The FAP-positive cells, anti-human immunoglobulin G (IgG)-positive cells, and anti-DXD-positive cells per unit area of the tumors from LU-01-1626 model.

(G–H) The Ki67 and TUNEL staining analysis of the tumors from LU-01-1626 model. The data from pathological assays were analyzed by ImageJ. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. ∗p < 0.05, ∗∗p < 0.01. Scale bar: 50 μm.

The final dose was administered on day 35. Three days later, tumors were harvested for immunohistochemistry (IHC) staining (Figure 5C). Pathological analysis revealed that the combination of IN10018 and Enhertu significantly reduced the number of CAFs (Figure 5D) and increased tumor uptake of Trastuzumab (Figure 5E) and DXD (Figure 5F) compared to Enhertu monotherapy. Ki67 and TUNEL staining data confirmed that the dual regimen can induce stronger tumor growth inhibition versus either monotherapy (Figures 5G and 5H). These findings are consistent with previous results from the NIH/3T3-NCI-N87 co-inoculation model, suggesting that the mechanism can be generalized to a broader context.

In addition to the LU-01-1626 model, the combinational effects of Enhertu and IN10018 were explored in a CRC PDX model, LD1-0038-361855, and an ovarian cancer PDX model, LD2-0032-200651. Both models exhibited HER2 expression and an abundance of CAFs within the tumor microenvironment. LD1-0038-361855 is relatively sensitive to Enhertu treatment. Both the Enhertu monotherapy and Enhertu-IN10018 co-treatment groups showed tumor regression starting from day 14. The study was terminated on day 24, and the dual combination resulted in significant tumor growth inhibition compared to Enhertu monotherapy (Figures S5A and S5B). In the ovarian cancer PDX model LD2-0032-200651, the co-treatment with Enhertu and IN10018 consistently outperformed Enhertu monotherapy in terms of tumor growth inhibition throughout the experiment (Figures S5C and S5D).

IN10018 enhances the tumor growth inhibition of TROP2 ADCs across various cancer models

To investigate whether targeting FAK could augment the efficacy of antibody-based therapies beyond Enhertu, we established a human PDAC PDX model, PC-07-0067, to assess the combinatorial effects of IN10018 and the TROP2 ADC sacituzumab tubulysin. Monotherapy with IN10018 did not demonstrate significant tumor growth inhibition. However, the combination of IN10018 and TROP2 ADC exhibited superior anti-tumor efficacy compared to either monotherapy (Figure 6A). All animals tolerated the treatment regimen well (Figure 6B).

Figure 6.

IN10018 enhances the tumor growth inhibition of TROP2 ADCs across various cancer models

(A and B) The tumor growth curves and body weight changes for the efficacy study with pancreatic cancer PDX model PC-07-0067. IN10018 was dosed at 12.5 mg/kg once daily by oral gavage. TROP2 tubulysin was dosed once weekly by tail vein injection. For the 1^st week, the dose of the ADC was 3 mg/kg and then was adjusted to 6 mg/kg from week 2 to week 4. The tumor volumes and body weights were recorded twice a week. Scale bar: 100 μm.

(C) The representative images of the IHC staining with anti-FAP, anti-human IgG, and anti-tubulysin, anti-Ki67, Sirius red staining, and TUNEL assay for the tumors from the PC-07-0067 model.

(D–G) The data analysis for the Sirius red staining and the IHC staining with FAP antibody, anti-human IgG-positive cells, and anti-tubulysin of the tumors from the PC-07-0067 model.

(H–I) The Ki67 and TUNEL staining analysis of the tumors from PC-07-0067 model. The data from pathological assays were analyzed by ImageJ. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. ∗p < 0.05, ∗∗p < 0.01. Scale bar: 50 μm.

Histological analysis of tumor samples revealed that the combination treatment with IN10018 and TROP2 ADC effectively eliminated CAFs and collagen, as evidenced by FAP and Sirius red staining, in contrast to TROP2 ADC monotherapy (Figures 6C–6E). Additionally, the dual regimen enhanced the tumor uptake of sacituzumab (Figure 6F) and its payload (Figure 6G), indicating that targeting FAK can improve the delivery of TROP2 ADC within tumors, extending beyond the effects observed with Enhertu. More tumor cell elimination was observed in the combination group compared to each monotherapy by Ki67 and TUNEL staining assay, further confirming the results from tumor measurements (Figures 6H and 6I).

We further evaluated the anti-cancer efficacy of the combination regimen using a clinical-stage TROP2 ADC, ESG401, with IN10018 in two additional pancreatic cancer PDX models (PC-07-0013 and PC-07-0041) and a breast cancer CDX model (MDA-MB-468). In the PC-07-0013 model, neither IN10018 nor ESG401 monotherapy demonstrated robust anti-cancer effects; however, the combination group exhibited substantial tumor growth suppression (Figures S6A and S6B). Similar findings were discovered in a study with PC-07-0041 model (Figures S6C and S6D). Consistent with the results from the PDX models mentioned earlier, the ESG401-IN10018 combination group also showed the most potent anti-tumor effects in the breast cancer MDA-MB-468 mouse model (Figures S6E and S6F).

In summary, targeting FAK with IN10018 enhances the anti-cancer efficacy of ADCs targeting either HER2 or TROP2 by reducing cancer-associated fibroblasts and increasing tumor uptake of the macromolecules, as evidenced by the more focal distribution of antibodies and payloads compared to ADC monotherapy (Figure 7A). These preclinical findings support the need for further clinical validation.

Figure 7.

The schematic figure of the combinational regimen of FAK inhibition and ADCs

Discussion

Conventional chemotherapies remain crucial in cancer treatment, yet their efficacy is often compromised by adverse effects.²⁰ ADCs have emerged as a promising alternative, integrating targeted therapy to mitigate these drawbacks. Each ADC comprises three core components: an antibody, a linker, and a payload. The antibody precisely directs the ADC to cancer cells by recognizing specific tumor antigens. The linker is crucial for controlling toxicity, designed to release the payload specifically within cancer cells or the tumor microenvironment. Unintended linker breakage can lead to off-target toxicities. Payloads are potent small molecules with high cytotoxic potential, often more toxic than conventional chemotherapies.²¹ The advent of ADCs gained significant traction following the approval of Enhertu, addressing unmet medical needs in treating HER2 low-expression/negative tumors. However, HER2 high-expression patients still exhibit superior clinical outcomes, indicating that antigen abundance remains a critical determinant of ADC efficacy.^4,5,22 Our studies on nine PDX models revealed that Enhertu-sensitive models exhibited higher HER2 expression levels compared to resistant models, though the difference was not statistically significant, suggesting additional factors influence ADC response.

Multiple facets contribute to ADC resistance. Generally, resistance can stem from the antibody/antigen interaction or the payload. Reduced or heterogeneous antigen expression can impair antibody binding, leading to resistance. Higher antibody affinity may limit tumor infiltration, restricting ADC access to deeper tumor regions.²³ Payload-related resistance involves potent chemotherapeutic agents, including microtubule inhibitors like auristatins and maytansine analogs, DNA damage inducers such as anthracyclines and calicheamicin, and RNA polymerase inhibitors like amanitin. These payloads share mechanisms of action and resistance with conventional chemotherapies. For instance, auristatins, similar to taxanes, are substrates for ATP-binding cassette (ABC) transporters, leading to drug efflux.²⁴ Upregulation of DNA repair pathways can render cells resistant to DNA-damage-inducing payloads, akin to platinum and alkylating agents.²⁵ Additionally, CAFs can impede ADC uptake, creating a physical barrier that protects cancer cells from therapeutic agents.¹⁴ In this study, we unveiled that CAFs also play an important role in determining the drug effects of ADCs. More excessive CAFs result in less tumor uptake of antibodies, which confers drug resistance of ADCs to the cancer cells. In that case, the agents that can remove the barrier are emergently needed for tackling the problem.

FAK signaling is pivotal in CAF formation and dynamics across various cancer types. Inhibition of FAK signaling alters fibrosis development and metabolism within the tumor microenvironment, affecting tumor progression.^26,27 In addition to genetic manipulation, the use of small molecule inhibitors to target FAK has been shown to effectively reduce CAFs,^18,19 thereby enhancing the efficacy of various therapeutic strategies. Previous studies have demonstrated that the reduction of stromal components following FAK inhibition is positively associated with the responsiveness of immune checkpoint inhibitors, likely due to increased infiltration of cytotoxic lymphocytes into the tumor microenvironment.²⁸ The desmoplastic stroma can also serve as a significant barrier to drug therapies that rely on direct interaction with cancer cells.²⁹ Here, we demonstrate that FAK signaling is crucial for maintaining the activity and viability of CAFs, as evidenced by the ability of cancer cells to transform normal fibroblasts into CAFs by upregulating the signature biomarkers of activated fibroblasts. Inhibition of FAK using IN10018 reduces the number of transformed fibroblasts, creating a more homogeneous tumor microenvironment that enhances the uptake of macromolecules such as antibodies and ADCs. This approach is applicable to ADC-based treatments targeting various biomarkers, as validated by tests using agents against HER2 and TROP2.

In addition to enhanced anti-tumor efficacy, the ability of patients to tolerate a clinical regimen is a critical factor for its success. Most ADCs exhibit on-target or off-target toxicity due to the specific nature of their antibody and payload components. For instance, Enhertu has been associated with interstitial lung disease (ILD), potentially due to the non-specific binding of the antibody to alveolar macrophages,³⁰ leading to payload release in lung tissue and subsequent DNA damage to normal lung cells, ultimately resulting in ILD.³¹ Additionally, TROP2, which is overexpressed in the salivary gland, has been linked to oral mucositis in patients treated with TROP2 ADCs.³² These treatment-related adverse effects (TRAEs) can limit patients' ability to continue receiving therapeutic benefits from ADCs. However, in our preclinical study evaluating the synergy between ADCs and the FAK inhibitor IN10018, we observed no abnormalities in the mice, suggesting that the animals tolerated the treatment well. IN10018 is a clinical-stage FAK inhibitor with reversible proteinuria as its most common adverse effect for which there is a practical management plan in clinical settings.³³ To date, IN10018 has not shown any overlapping TRAEs with emerging ADCs. Although there is no indication that the ADC-IN10018 dual regimen may cause undesirable toxicities, careful monitoring for any unforeseen adverse effects will be essential in clinical trials.

In conclusion, targeting FAK can enhance the tumor uptake of ADCs, leading to potent anti-cancer effects that can overcome drug resistance, particularly in desmoplastic stroma-rich environments. This dual regimen demonstrated no significant toxicity in animal studies. The preclinical findings support further investigation in clinical settings, particularly for cancers characterized by excessive CAFs.

Limitations of the study

In this study, we demonstrated that reducing CAFs through FAK targeting increases the uptake of macromolecules such as antibodies and ADCs, thereby enhancing their anti-tumor effects. However, this mechanism is not exhaustive; other modalities may also contribute to the enhanced efficacy. For example, FAK inhibition might regulate endothelial cell permeability, thereby increasing drug uptake in tumors.³⁴ Further research is necessary to elucidate the comprehensive mechanisms by which FAK inhibition enhances the anti-tumor efficacy of ADCs.

Resource availability

Lead contact

Further information and requests for resources and reagents should be contacted directly to and will be fulfilled by the lead contact, Baoyuan Zhang (baoyuan.zhang@inxmed.com).

Material availability

This study did not generate new unique reagents.

Data and code availability

• •Data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

The authors would like to thank Ping Zhang, Jingwen Cheng, Wanyu Xie, and Menglu Pu for their help in the experiment and data collection. This study was supported by InxMed.

Author contributions

Conceptualization, B.Y.Z., Z.X.Z., J.M.G., L.L., and Z.Q.W.; methodology, B.Y.Z., J.M.G., S.Q.L., and R.P.; investigation, B.Y.Z., Z.X.Z., and J.M.G.; format analysis, B.Y.Z., Z.X.Z., D.F.L., X.H., and N.Q.; writing—original draft, B.Y.Z.; writing—review & editing, B.Y.Z., Z.X.Z, and J.M.G.; funding acquisition, B.Y.Z. and Z.Q.W.; resources, B.Y.Z., L.L., and Z.Q.W.; supervision, B.Y.Z.

Declaration of interests

B.Y.Z., R.P., L.L., and Z.Q.W. are co-inventors of a patent application of InxMed. B.Y.Z., J.M.G., S.Q.L., R.P., N.Q., L.L., and Z.Q.W. are employees of InxMed. Z.X.Z., D.F.L., and X.H. are employees of WuXi AppTec.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal HRP-conjugated anti-alpha Tubulin	Proteintech	Cat#HRP-66031; RRID: AB_2687491
Rabbit monoclonal anti-FAP	Abcam	Cat#AB207178; RRID: AB_2864720
Rabbit monoclonal anti-FAK	Cell Signaling Technology	Cat#3285S; RRID: AB_2269034
Rabbit polyclonal anti-Phospho FAK Y397	Thermo Fisher Scientific	Cat#44-624G; RRID: AB_2533701
Rabbit polyclonal anti-human IgG	Proteintech	Cat#SA00004-6; RRID: AB_2890947
Mouse monoclonal anti-DXD	Youke	Cat#YKMP18020
Rabbit polyclonal anti-Ki67	Proteintech	Cat#27309-1-AP; RRID: AB_2756525
Rabbit polyclonal anti-Tubulysin	Oncomatryx	N/A
Trastuzumab	Henlius	Cat#NMPNS20200019
Sacituzumab	Biointron	N/A
Trastuzumab Cy5.5	InxMed	N/A
Trastuzumab GFP	InxMed	N/A
Chemicals, peptides, and recombinant proteins
IN10018	InxMed	N/A
Enhertu	Daiichi Sankyo	N/A
ESG401	Escugen	N/A
Tubulysin	Oncomatryx	N/A
Critical commercial assays
BCA protein assay kit	Thermo Fisher Scientific	Cat#23225
Cy5.5 lightning-link kit	Abcam	Cat#ab269891
DeadEnd Fluorometric Tunel system	Promega	Cat#G3250
Picro Sirius red staining kit	Abcam	Cat#ab150681
ECL kit	Epizyme Biotech	Cat#SQ201L
Experimental models: Cell lines
Human: NCI-N87	Cobier	Cat#CBP60491
Human: MDA-MB-468	Cobier	Cat#CBP60387
Mouse: NIH/3T3	Cobier	Cat#CBP60317
Mouse: CT26	Cobier	Cat#CBP61189
Experimental models: Organisms/strains
BALB/c Nude mice	Beijing Vital River Laboratory Animal Technology Co., Ltd	N/A
NOD-SCID mice	Beijing Vital River Laboratory Animal Technology Co., Ltd	N/A
Software and algorithms
GraphPad Prism 8.0	GraphPad	http://www.graphpad-prism.cn/?c=i&a=prism
Excel	Microsoft	https://www.microsoft.com/zh-cn/microsoft-365/excel
ImageJ	National Institutes of Health	https://github.com/imagej

Experimental model and study participant details

Cell lines

NCI-N87, CT26, MDA-MB-468, and NIH/3T3 cell lines were purchased from Cobier Bioscience. The cells were cultured with RPMI1640 medium (Basalmedia) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Thermo Fisher Scientific). All cells were maintained and cultured at 37°C in a humidified incubator with 5% CO₂ (vol/vol). The mycoplasma contamination was regularly detected by InxMed.

Mice

6 to 8-week-old female NOD-SCID and BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Company. The mice were used for PDX or CDX model generation and they were fed in the specific pathogen-free animal room. All animal studies were reviewed and approved by the ethics committee of WuXi AppTec, Sixin, and Lide under IACUC protocol. All the procedures were conducted following AAALAC guidelines.

Efficacy study with animal models

For the cell line-derived xenograft (CDX) models, NCI-N87 (5 x 106), NIH/3T3-NCI-N87 (5 x 105)-(5 x 106) and MDA-MB-468 (1 x 107) were injected into the right flank of each NOD-SCID mouse for model establishment. For the patient-derived xenograft (PDX) models, the seed tumors were sliced into pieces (around 30 mm3 for each) and implanted into the right flanks of the mice. The PDX models were established on nude mice or NOD-SCID mice. The solvent for IN10018 was 0.5% Natrosol 250 HX in distilled water and it was dosed through oral gavage at 12.5 or 25 mg/kg once a day. Enhertu and TROP2 ADC were prepared using 0.9% saline and administered at indicated doses by tail vein injection. The tumor volumes and body weight changes were recorded twice a week. Tumor volumes were measured by caliper and estimated with a formula of 0.5 x long diameter x short diameter2. Dosing began when the tumor volume reached 50–300 mm3 in the subcutaneous models. At the end of the study, the tumors were collected for further pathological analysis. In the PDX study depicted in Figure 1A, Enhertu was administered at a single dose of 3 mg/kg by tail vein injection on day 0, with treatments continuing for 20/21 days. For the study with ST-02-0103 and ST-02-0103R models, Enhertu was dosed at 1 mg/kg once weekly for 28 days. In the efficacy evaluation studies with the dual regimen of IN10018 and Enhertu, for the LU-01-1626 PDX model, IN10018 was administered orally at 25 mg/kg (days 0–3) and 12.5 mg/kg (days 4 to end) once daily. Enhertu was administered at 10 mg/kg (day 0), 3 mg/kg (day 7), and 6 mg/kg (day 14 to end) once weekly. For the animal tests in Figure S5, IN10018 was dosed at 12.5 mg/kg once daily, yet Enhertu was dosed at 3 mg/kg once on day 9 for the LD1-0038-361855 PDX model. As for the LD2-0032-200651, IN10018 was dosed at 25 mg/kg once daily and Enhertu was dosed at 3 mg/kg once a week on Day 3, 10, and 17. In the animal studies for evaluating the combination regimen of IN10018 and TROP2 ADCs, for the PC-07-0067 PDX model, IN10018 was administered orally at 12.5 mg/kg once daily. Sacituzumab Tubulysin was administered by IV at 3 mg/kg twice weekly during the first week, with the dose increased to 6 mg/kg twice weekly from the second week until the end. Regarding the animal experiments in Figure S6, IN10018 was administered at 25 mg/kg per oral once daily. The TROP2 ADC ESG401 was dosed at 1 mg/kg (Day 0–10)/3 mg/kg (Day 11–28) for the PC-07-0041 model and 4.5 mg/kg for the PC-07-0013 model and 1 mg/kg for the MDA-MB-468 model via tail vein injection twice weekly. For the animal study in Figures 2A–2B, NCI-N87 alone and NIH/3T3-NCI-N87 co-inoculation models were established, and one dose of 3 mg/kg Enhertu was injected via tail vein at day 0. For the animal study in Figures 4H and 4I, NIH/3T3-NCI-N87 co-inoculated model was generated, IN10018 25 mg/kg was dosed orally once daily from day 0. 3 mg/kg Enhertu was dosed twice on day 9 and day 28, respectively via tail vein injection.

In vivo imaging study with animal models

For the study with Trastuzumab Cy5.5 in Figure 2, the NCI-N87 alone model and NIH/3T3-NCI-N87 co-inoculation model were generated, and 3 mg/kg of Trastuzumab Cy5.5 was dosed to the mice once. 24 h later the tumors were collected for further analysis. Regarding the animal study in Figure 4, NIH/3T3-NCI-N87 model was generated. IN10018 25 mg/kg was dosed orally once daily since the tumors grew to around 200 mm3. 10 days later, one dose of the 3 mg/kg Trastuzumab Cy5.5 was given to the mice. The fluorescent signal was detected through the IVIS Spectrum In vivo imaging system (PerkinElmer).

Method details

Clonogenic assay

The CT26 or NCI-N87 cancer cells were cocultured with NIH/3T3 cells using a 12-well transwell system (Corning). 1000 of the cancer cells were cultured in the upper chambers and 1000 of the NIH/3T3 cells were plated onto each bottom well. 3 μM IN10018 or DMSO control was added to the wells for treatment. 10 days later, the upper chambers were removed and the NIH/3T3 cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min. Then, the cells were stained with 0.5% crystal violet staining solution (Beyotime) for 30 min. Finally, the excessive dye was rinsed off with tap water. The morphology of the cells attached to the wells was recorded by camera and the colony amounts were evaluated by ImageJ software. In brief, the colony area function module in ImageJ was used for the analysis following the protocol provided in the software.³⁵ The colony images were converted into grayscale (8-bit) and cell colonies were recognized by processing with “ColonyArea” in the plugins menu. Then “Colony measurer” tool was run for the measurement of colony numbers in each well.

Western blot

5 x 104 of the NIH/3T3 cells were cultured with/without 5 x 104 of the CT26 or NCI-N87 cancer cells for 3 or 5 days in a 12-well transwell system. The fibrotic cells were plated onto the bottom well and the cancer cells were plated in the upper well. Protein samples from NIH/3T3 cells were lysed from the testing system by the RIPA lysis buffer (Rockland). The BCA assay (Thermo Fisher Scientific) was performed to check the protein amounts. Then, the protein samples were mixed with 4 x laemmli blue loading buffer (Bio-Rad). The samples were boiled at 95°C for 10 min. The SDS-PAGE gel was implemented for the electrophoresis of the protein samples. After that, wet transfer was performed to transfer the protein from gels to PVDF membranes (Bio-Rad). The membranes were incubated with primary antibodies overnight at 4°C. The secondary antibodies were incubated with the samples at RT for 1 h. Finally, the samples were imaged by a ChemiDoc MP kit (Bio-Rad) upon the exposure process with an ECL kit (Epizyme biomedical).

Immunofluorescence assay

The CT26 or NCI-N87 cells were plated onto glass layers and cocultured with NIH/3T3 cells with the 12-wells transwell system described in the clonogenic assay section. Briefly, the coculture system was maintained for 5 days with/without the treatment of IN10018. Then, the glass layers with cancer cells were removed from the system and the cells were fixed with 4% paraformaldehyde for 15 min. The samples were sealed with fluorescence mounting medium (DAKO) and covered by coverslips. The samples were imaged with confocal microscope (Zeiss).

Immunohistochemistry assay

The tumor tissues were acquired from animal models and fixed with 4% paraformaldehyde (Beyotime) and prepared into formalin-fixed paraffin-embedded (FFPE) blocks. For the IHC staining, the pathology slides were stained with primary and secondary antibodies for Immunohistochemistry to detect the region of interest. Sirius red staining was performed using a kit from Abcam, the procedures were conducted following the protocol. KF-PRO-120 scanner (KFBIO) was used to scan the pathology slides. ImageJ was used for the analysis of the images.

Tunel assay

The pathology slides for tumor tissues were permeabilized in 0.2% Triton X-100 (Beyotime) containing PBS for 8 min, then washed and blocked in 20% normal goat serum (Yeasen) for another 1 h at 37°C. The Tunel reagents produced by Promega were added to the slides and the procedures were performed as the protocol said. The slides were counterstained with DAPI and mounted with mounting medium (DAKO). The percentage of TUNEL positive area was measured by ImageJ system.

Generation of Trastuzumab Cy5.5

Trastuzumab was purchased from Henlius and the concentration is 7.44 mg/mL (50.27 μM). The naked antibody was conjugated by Cy5.5 lighting-link from Abcam. All the procedures followed the protocol. The Dye/Protein ratio was 2.01 after conjugation. The dose of Trastuzumab Cy5.5 was 3 mg/kg for each mouse in the animal study.

Conjugation of sacituzumab and tubulysin

Sacituzumab was obtained from Biointron. The antibody was further conjugated to Tubulysin (Oncomatryx) through Cys-based nondirected conjugation in the presence of TCEP reagent (Thermo Fisher Scientific). In brief, the mixture of Sacituzumab and TCEP regent was stirred by an agitator for 2 h. Then, Tubulysin was added. The system was stirred overnight. Ultimately, the conjugated ADC was purified by a 30 KD ultrafiltration centrifugal tube (Millipore). Before the use of the ADC, the quality was checked and confirmed by SEC-HPLC (Agilent) and HIC-HPLC (Agilent).

Quantification and statistical analysis

Each data point displayed is represented as Means ± SEM. The statistical significance of differences between the tested groups was determined using an unpaired two-tailed Student’s t test. Correlation between FAP positive area in tumors and TGIs was performed using Pearson r analysis. Throughout the whole study, the statistical analyses were performed with GraphPad Prism 8.0 software. p values less than 0.05 were considered to be significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Published: December 6, 2024