The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant ...stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.
The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant ...stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF- beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re- expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.
Purpose
Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid ...cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies.
Methods
FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in
in vivo
imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule–based FAP-targeting agent.
Results
Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of
68
Ga-FAP-2286,
111
In-FAP-2286, and
177
Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues.
177
Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46.
Conclusion
In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of
68
Ga-FAP-2286 for imaging and
177
Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.
Purpose
FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in ...clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (
177
Lu) as a monotherapy and in combination with a PD-1 targeting antibody.
Methods
C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with
177
Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of
177
Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses.
Results
177
Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors,
177
Lu-FAP-2287 increased CD8
+
T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8
+
T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86.
Conclusion
In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8
+
T cells. These findings provide a rationale for clinical studies of combined
177
Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.
Abstract
Background: FAP is a membrane-bound protease with limited expression in normal tissues but high expression on cancer-associated fibroblasts abundant in the stroma of most tumors. FAP-2286 is ...a potent and selective FAP-targeted peptide linked to the chelator DOTA that allows for attachment of radionuclides for therapeutic and imaging applications. Assessing patterns of FAP expression in different tumor types and correlating expression with FAP-2286 uptake can help guide tumor selection for FAP-2286 therapy. Methods: FAP immunohistochemistry (IHC) was performed on FFPE tissue specimens from 16 tumor types using the SP325 antibody. Overall and tumor/stroma-specific H-scores were calculated using Visiopharm and HALO analysis, respectively. Autoradiography with 111In-FAP-2286 was performed on a subset of matched frozen tissue sections. Results: Gene expression analysis across The Cancer Genome Atlas data set revealed elevated FAP mRNA expression in multiple tumor types. A pan-tumor IHC screen confirmed that ≥30% of samples in various indications (eg, sarcoma, pancreatic adenocarcinoma, mesothelioma, head and neck squamous cell carcinoma) had high FAP expression (H-score ≥30). While in most tumor types FAP was predominantly localized to the stroma, FAP expression was also observed in tumor cells, especially in tumors of mesenchymal origin, eg, sarcoma and mesothelioma. High FAP expression was independent of tumor stage or grade and detected in both primary and metastatic samples. Multiple sarcoma and mesothelioma subtypes demonstrated high FAP H-scores, suggesting that FAP expression is not limited to a specific subtype. There was significant correlation between FAP expression observed by IHC and FAP-2286 binding as assessed by autoradiography in matched frozen tissues (Pearson r=0.73; p<0.01). Conclusions: Our IHC screen identified high FAP expression in various tumor types that correlated with in vitro FAP-2286 binding, suggesting that FAP is an attractive target across a broad range of tumor types for peptide-targeted radionuclide therapy. Accordingly, the phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications.
Citation Format: Tanya T. Kwan, Minh Nguyen, Dirk Zboralski, Anne Schumann, Anne Bredenbeck, Matthias Paschke, Christian Haase, Aileen Hoehne, Ulrich Reineke, Christiane Smerling, Frank Osterkamp, Jim Xiao, Andrew D. Simmons, Thomas C. Harding, Kevin L. Lin. Pan-cancer analysis of fibroblast activation protein alpha (FAP) expression to guide tumor selection for the peptide-targeted radionuclide therapy FAP-2286 abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA032.
Abstract
Background: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high expression in tumors but limited expression in normal tissues. FAP-2286 is a ...FAP-targeted radiopharmaceutical in clinical development for multiple solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes for imaging and therapeutic use. Preclinically, we evaluated the potential modulation of the immune response and antitumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the β-emitting radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a programmed cell death protein 1-targeting antibody (anti-PD-1).
Methods: C57BL/6 mice bearing MCA205 fibrosarcoma mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu-FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses.
Results: 177Lu-FAP-2287 accumulated in MCA205-mFAP tumors with 8.2 %ID/g uptake at 3 hours post-injection (pi), which was maintained at 2.9 %ID/g by 24 hours pi. Treatment with 177Lu-FAP-2287 demonstrated significant tumor growth inhibition (TGI, 74%; 267 mm3; P < 0.01) vs vehicle control (750 mm3). Significant TGI was also observed from anti-PD-1 (57%; 373 mm³; P < 0.01) and the combination (92%; 145 mm³; P < 0.001) vs vehicle control. No significant differences in TGI were observed between the three treatment groups. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration by 2-fold on day 8 pi, alone or in combination with anti-PD-1 (16.9% and 18.0% of leukocytes, respectively), compared with 7.5% for vehicle control (P < 0.01). In contrast, anti-PD-1 alone had a nonsignificant increase in CD8+ T cell infiltration of 10.8% vs vehicle control (P > 0.05). On day 13 pi, the combination had durable higher levels of CD8+ T cells, with 16.3% of leukocytes vs 5.0% for vehicle control (P < 0.01). Monotherapy 177Lu-FAP-2287 also displayed a trend towards higher levels of CD8+ T cells at Day 13 vs vehicle (11.9% of leukocytes; P > 0.05). The increase in CD8+ T cells was accompanied by changes in co-stimulatory molecules such as CD86, resulting in greater activation by antigen-presenting cells. Additional immune cell characterization will be presented.
Conclusions: FAP-targeted radiopharmaceutical enhances PD-1-antibody-mediated TGI by increasing recruitment of tumor-infiltrating CD8+ T cells, which is enhanced and maintained in the presence of anti-PD-1. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint blockade in FAP-positive tumors.
Citation Format: Dirk Zboralski, Aileen Höhne, Esben Christensen, Matthias Paschke, Liliane Robillard, Andrew D. Simmons, Frank Osterkamp, Thomas C. Harding, Minh Nguyen. Fibroblast activation protein (FAP)-targeted radiotherapy increases tumor CD8+ T cell infiltration and enhances response to PD-1 immune checkpoint blockade abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2835.
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