Clinical Study on the Application of PET Probes Targeting DDR2 in the Diagnosis of Interstitial Lung Disease With Cognitive Impairment

Clinical Study on the Application of Positron Emission Tomography (PET) Probes Targeting DDR2 in the Diagnosis of Interstitial Lung Disease and Interstitial Lung Disease With Cognitive Impairment

According to statistics, 45% of human disease-related deaths are associated with organ fibrosis, among which pulmonary fibrosis poses a severe threat to patients' lives. In recent years, the application of novel therapeutic approaches (such as tumor immunotherapy and organ transplantation) and COVID-19 infections have further expanded the clinical demand for the diagnosis and treatment of pulmonary fibrosis. Currently, the two small-molecule drugs approved for idiopathic pulmonary fibrosis (IPF) (pirotinib and nintedanib) can only slow the decline in lung function and fail to improve patient mortality . Therefore, early diagnosis and early treatment of pulmonary fibrosis have become a clinical consensus , urgently requiring the emergence of innovative technologies and methods.

Recent studies have demonstrated that collagen is not only a product of fibrosis but also a driving factor in its sustained progression . Therefore, identifying key molecular targets that promote collagen-driven fibrotic progression represents a critical direction for anti-fibrotic therapeutic research. Human collagen receptors identified include the discoidin domain receptor (DDR) family (including DDR1 and DDR2) and the integrin family (including α1β1, α2β1, α10β1, and α11β1). Extensive literature and preliminary research by various groups have established that DDR2 is the collagen receptor with the most significantly elevated expression level in the lung tissue of IPF patients. Unlike the "fast-on, fast-off" activation pattern of cytokine receptor tyrosine kinases (RTKs), the tyrosine phosphorylation of DDR1 and DDR2 requires the binding of large ligand molecules such as collagen for several hours before induction and can persist for dozens of hours, exhibiting a unique "slow-on, slow-off" pattern. This activation characteristic suggests that such molecular mechanisms may underlie the enduring biological effects mediated by DDRs in the progression of chronic fibrotic diseases.

According to statistics, 45% of human disease-related deaths are associated with organ fibrosis, among which pulmonary fibrosis poses a severe threat to patients' lives. In recent years, the application of novel therapeutic approaches (such as tumor immunotherapy and organ transplantation) and COVID-19 infections have further expanded the clinical demand for the diagnosis and treatment of pulmonary fibrosis. Currently, the two small-molecule drugs approved for idiopathic pulmonary fibrosis (IPF) (pirotinib and nintedanib) can only slow the decline in lung function and fail to improve patient mortality . Therefore, early diagnosis and early treatment of pulmonary fibrosis have become a clinical consensus , urgently requiring the emergence of innovative technologies and methods.

Recent studies have demonstrated that collagen is not only a product of fibrosis but also a driving factor in its sustained progression . Therefore, identifying key molecular targets that promote collagen-driven fibrotic progression represents an important direction for anti-fibrotic therapeutic research. Human collagen receptors identified include the discoidin domain receptor (DDR) family (including DDR1 and DDR2) and the integrin family (including α1β1, α2β1, α10β1, and α11β1). Extensive literature and preliminary research by various groups have shown that DDR2 is the collagen receptor with the most significantly elevated expression level in the lung tissue of IPF patients. Unlike the "fast-on, fast-off" activation pattern of cytokine receptor tyrosine kinases (RTKs), the tyrosine phosphorylation of DDR1 and DDR2 requires the binding of large ligand molecules such as collagen for several hours before induction and can persist for dozens of hours, exhibiting a unique "slow-on, slow-off" pattern. This activation characteristic suggests that it may represent the molecular biological basis for the sustained biological effects mediated by DDRs in the progression of chronic fibrotic diseases .

Using tissue samples from dozens of lung transplantation procedures in IPF patients, the applicants discovered that the expression levels and phosphorylation of the collagen receptor DDR2 were significantly higher in both IPF and non-IPF lung tissues compared to healthy donor lungs, while the expression of the other five collagen receptors showed no change or insignificant variation. In 20 IPF lung tissues, the mRNA expression of DDR2 was on average upregulated by approximately 100-fold, suggesting that this molecule may be the primary receptor driving the sustained progression of collagen signal-mediated pulmonary fibrosis . DDR2 is not expressed in quiescent cells but is predominantly expressed in activated fibroblasts. Our research team further confirmed, using single-cell transcriptome data from IPF patients, that DDR2 is specifically highly expressed in five groups of activated fibroblasts in IPF lung tissues, with its abundance exceeding that of the classical activated fibroblast marker FAP (fibroblast activation protein) .

In addition to typical interstitial lung diseases, neurodegenerative disorders represented by Alzheimer's disease (AD) are also closely associated with collagen-fibrosis deposition. The research team previously investigated the clinical phenomenon of "a significant increase in the proportion of cognitive impairment among patients with interstitial lung disease." Preliminary studies demonstrated that astrocyte DDR2 overexpression leads to perivascular fibrosis, impairs fluid transport, and exacerbates cognitive decline. Therefore, DDR2-based PET tracers may serve as potential comorbidity biomarkers for interstitial lung disease and neurodegenerative disorders accompanied by cognitive impairment.

Nanobodies (Nb) are natural antibodies derived from camelids and cartilaginous fish (e.g., alpacas and sharks) that lack light chains and bind antigens solely through the variable regions of the heavy chain. Monodomain antibodies composed solely of the heavy chain variable region are only one-tenth the size of traditional antibodies, with a molecular weight of approximately 15 kDa and a diameter <4 nm, hence termed nanobodies . In recent years, with the continuous advancement of immunoprecipitation positron emission tomography (immunoPET) technology , Nb has emerged as an excellent radionuclide probe carrier, particularly in PET/CT imaging, demonstrating a series of unique advantages over traditional monoclonal antibodies and being hailed as the "magic bullet" in immunimaging .

In summary, targeting DDR2 can achieve broader screening for early diagnosis of fibrotic effect cells. Our research group has long been engaged in the structural and functional studies of the collagen receptor DDR2, and based on this, we have screened and identified the nanobody 1A12, which targets the extracellular region of DDR2 and competes for collagen binding, enabling precise targeting of lesions. A series of research tasks have been completed, including preparation, sterility and pyrogen-free testing, targeting assays, formulation of related reagents, and screening of associated components. Preclinical and clinical pharmacological and toxicological data have been retrieved and organized. Based on the 1A12 precursor, our team designed a positron emission tomography (PET) probe labeled with the radionuclide 68Ga: 68Ga-1A12, as a technical tool for interstitial lung diseases and interstitial lung diseases combined with neurodegenerative disorders, to improve diagnostic accuracy and sensitivity, guide individualized treatment, and enhance therapeutic efficacy.

Inclusion Criteria:

• No gender restriction, age ≥18 years (inclusive);
• Patients with interstitial lung disease who meet one of the following diagnoses: connective tissue disease-associated interstitial lung disease (CTD-ILD), idiopathic nonspecific interstitial pneumonia (iNSIP), chronic allergic pneumonia, sarcoidosis, environmental/occupational lung disease, or unclassifiable idiopathic interstitial disease (IIP);
• Screening subjects who were definitively diagnosed with interstitial lung disease (ILD) by high-resolution computed tomography (HRCT) within the preceding 6 months;
• In the first 3 months prior to screening, the carbon monoxide diffusion capacity (DLCO) (Hb-corrected) was within 30% to 80% (inclusive) of the predicted value; the forced vital capacity (FVC) was within 40% to 70% (inclusive) of the predicted value;
• The chief complaint is memory decline or other symptoms of cognitive impairment, with a disease course of ≥3 months;
• meets the diagnostic criteria for cognitive impairment caused by Alzheimer's disease (AD), mild cognitive impairment (MCI), or other related neurodegenerative diseases;
• The scores on the cognitive function screening scale meet the criteria for cognitive impairment (e.g., Mini-Mental State Examination (MMSE) <24 points, or Montreal Cognitive Assessment (MoCA) <26 points);
• Exclusion of cognitive dysfunction caused by severe depression, brain trauma, brain tumors, cerebrovascular accidents, etc.

Exclusion Criteria:

• patients in critical condition requiring emergency care;
• Individuals with drug and/or alcohol abuse, or those with allergic predisposition;
• women of childbearing potential, pregnant and lactating women;
• bacterial, viral or fungal infections that require systemic treatment;
• The study excluded participants deemed unsuitable by the investigators.