[99mTc]-labelled anti-Programmed Death-Ligand 1 single-domain antibody SPECT/CT: a novel imaging biomarker for myocardial PD-L1 expression.

BACKGROUND: Myocardial programmed death-ligand 1 (PD-L1) expression is implicated in immune checkpoint inhibitor (ICI)-associated myocarditis. Measurement of myocardial PD-L1 expression may have potential use as a mechanistic and predictive biomarker. The aim of this study was to determine non-invasive assessment of myocardial PD-L1 expression using [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT. METHODS: Thoracic [99mTc]NM-01 SPECT/CT was performed in lung cancer patients (n = 10) at baseline and 9-weeks following anti-programmed cell death protein 1 (PD-1) therapy. Baseline and 9-week left ventricular and right ventricular to blood pool ratios (LVmax:BP) and (RVmax:BP) were measured. LVmax was compared to background skeletal muscle (musclemax). Intra-rater reliability was determined by intraclass correlation coefficient (ICC) and Bland-Altman analysis. RESULTS: Mean LVmax:BP values were 2.76 ± 0.67 at baseline vs 2.55 ± 0.77 at 9 weeks (p = 0.42). Mean RVmax:BP was 1.82 ± 0.32 at baseline vs 1.76 ± 0.45 at 9 weeks (p = 0.67). Myocardial PD-L1 expression was at least threefold greater than skeletal muscle at baseline for the LV (LVmax to musclemax 3.71 ± 0.77 vs 0.98 ± 0.20 (p < 0.001)) and at least twofold for the RV (LVmax to musclemax 2.49 ± 0.63 vs 0.98 ± 0.20 (p < 0.001)). There was excellent intra-rater reliability for LVmax:BP with ICC 0.99 (95% confidence interval 0.94-0.99, p < 0.001), mean bias -0.05 ± 0.14 (95% limits of agreement -0.32 to 0.21). There were no major adverse cardiovascular events or myocarditis during follow-up. CONCLUSION: This study is the first to report PD-L1 expression of the heart that can be quantified non-invasively without invasive myocardial biopsy, with high reliability and specificity. This technique can be applied to investigate myocardial PD-L1 expression in ICI-associated myocarditis and cardiomyopathies. Clinical trial registration PD-L1 Expression in Cancer (PECan) study (NCT04436406). https://clinicaltrials.gov/ct2/show/NCT04436406 June 18th, 2020.

Preclinical development and characterisation of 99mTc-NM-01 for SPECT/CT imaging of human PD-L1.

The level of expression of programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1) is a predictive biomarker for cancer immunotherapy, however, its detection remains challenging due to tumour heterogeneity and the influence from the binding of therapeutic agents. We recently developed [99mTc]-NM-01 as a companion diagnostic imaging agent for non-invasive molecular imaging of PD-L1 by single-photon emission computed tomography (SPECT). The aim of the study was to evaluate the [99mTc] radiolabelling of GMP graded NM-01 and its pharmacology, pharmacokinetics and toxicology. NM-01 bound specifically to human PD-L1 (Kd=0.8 nM) and did not interfere with the binding of the anti-PD-L1 antibody atezolizumab. NM-01 can bind various PD-L1-positive cancer cell lines and only interact with PD-L1 expressed on the cell surface. In SPECT/CT imaging, high [99mTc]-NM-01 accumulation was observed in the HCC827 mouse xenografted tumour model (30-min: 1.50 ± 0.27 %ID/g; 90-min: 1.23 ± 0.18 %ID/g), demonstrated a predominantly renal elimination (high uptake in bladder and kidney), while activity in the blood pool and other major organs remained low. The tumour-to-muscle and tumour-to-blood ratios were comparable with/without atezolizumab (P<0.04) but were significantly lowered when co-injected with excess NM-01 (P=0.04 and P=0.01, respectively.) The blood clearance of [99mTc]-NM-01 is bi-phasic; consisting of an initial fast washout phase with half-life of 2.1 min and a slower clearance phase with half-life of 25.4 min. In an intravenous extended single-dose toxicity study, no treatment-related changes were observed and the maximum tolerated dose of [99mTc]-NM-01 was 2.58 mg/kg. [99mTc]-NM-01 has suitable properties as a potential candidate for SPECT/CT imaging of PD-L1 assessment in cancer patients.

Theranostic Approach in Breast Cancer: A Treasured Tailor for Future Oncology.

ABSTRACT: Breast cancer is the most frequent invasive malignancy and the second major cause of cancer death in female subjects mostly due to the considerable diagnostic delay and failure of therapeutic strategies. Thus, early diagnosis and possibility to monitor response to the treatment are of utmost importance. Identification of valid biomarkers, in particular new molecular therapeutic targets, that would allow screening, early patient identification, prediction of disease aggressiveness, and monitoring response to the therapeutic regimen has been in the focus of breast cancer research during recent decades. One of the intensively developing fields is nuclear medicine combining molecular diagnostic imaging and subsequent (radio)therapy in the light of theranostics. This review aimed to survey the current status of preclinical and clinical research using theranostic approach in breast cancer patients with potential to translate into conventional treatment strategies alone or in combination with other common treatments, especially in aggressive and resistant types of breast cancer. In addition, we present 5 patients with breast cancer who were refractory or relapsed after conventional therapy while presumably responded to the molecular radiotherapy with 177Lu-trastuzumab (Herceptin), 177Lu-DOTATATE, and 177Lu-FAPI-46.

HER2-directed antibodies, affibodies and nanobodies as drug-delivery vehicles in breast cancer with a specific focus on radioimmunotherapy and radioimmunoimaging.

PURPOSE: The aim of the present paper is to review the role of HER2 antibodies, affibodies and nanobodies as vehicles for imaging and therapy approaches in breast cancer, including a detailed look at recent clinical data from antibody drug conjugates and nanobodies as well as affibodies that are currently under development. RESULTS: Clinical and preclinical studies have shown that the use of monoclonal antibodies in molecular imaging is impaired by slow blood clearance, associated with slow and low tumor uptake and with limited tumor penetration potential. Antibody fragments, such as nanobodies, on the other hand, can be radiolabelled with short-lived radioisotopes and provide high-contrast images within a few hours after injection, allowing early diagnosis and reduced radiation exposure of patients. Even in therapy, the small radioactively labeled nanobodies prove to be superior to radioactively labeled monoclonal antibodies due to their higher specificity and their ability to penetrate the tumor. CONCLUSION: While monoclonal antibodies are well established drug delivery vehicles, the current literature on molecular imaging supports the notion that antibody fragments, such as affibodies or nanobodies, might be superior in this approach.

Early Phase I Study of a 99mTc-Labeled Anti-Programmed Death Ligand-1 (PD-L1) Single-Domain Antibody in SPECT/CT Assessment of PD-L1 Expression in Non-Small Cell Lung Cancer.

Immunotherapy with checkpoint inhibitor programmed cell death 1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies demonstrates improvements in treatment of advanced non-small cell lung cancer. Treatment stratification depends on immunohistochemical PD-L1 measurement of biopsy material, an invasive method that does not account for spatiotemporal heterogeneity. Using a single-domain antibody, NM-01, against PD-L1, radiolabeled site-specifically with 99mTc for SPECT imaging, we aimed to assess the safety, radiation dosimetry, and imaging characteristics of this radiopharmaceutical and correlate tumor uptake with PD-L1 immunohistochemistry results. Methods: Sixteen patients (mean age, 61.7 y; 11 men) with non-small cell lung cancer were recruited. Primary tumor PD-L1 expression was measured by immunohistochemistry. NM-01 was radiolabeled with [99mTc(OH2)3(CO)3]+ complex binding to its C-terminal hexahistidine tag. Administered activity was 3.8-10.4 MBq/kg, corresponding to 100 µg or 400 µg of NM-01. Whole-body planar and thoracic SPECT/CT scans were obtained at 1 and 2 h after injection in all patients, and 5 patients underwent additional imaging at 10 min, 3 h, and 24 h for radiation dosimetry calculations. All patients were monitored for adverse events. Results: No drug-related adverse events occurred in this study. The mean effective dose was 8.84 × 10-3 ± 9.33 × 10-4 mSv/MBq (3.59 ± 0.74 mSv per patient). Tracer uptake was observed in the kidneys, spleen, liver, and bone marrow. SPECT primary tumor-to-blood-pool ratios (T:BP) varied from 1.24 to 2.3 (mean, 1.79) at 1 h and 1.24 to 3.53 (mean, 2.22) at 2 h (P = 0.005). Two-hour primary T:BP ratios correlated with PD-L1 immunohistochemistry results (r = 0.68, P = 0.014). Two-hour T:BP was lower in tumors with ≤1% PD-L1 expression (1.89 vs. 2.49, P = 0.048). Nodal and bone metastases showed tracer uptake. Heterogeneity (>20%) between primary tumor and nodal T:BP was present in 4 of 13 patients. Conclusion: This first-in-human study demonstrates that 99mTc-labeled anti-PD-L1-single-domain antibody SPECT/CT imaging is safe and associated with acceptable dosimetry. Tumor uptake is readily visible against background tissues, particularly at 2 h when the T:BP ratio correlates with PD-L1 immunohistochemistry results.

Purified anti-CD3 × anti-HER2 bispecific antibody potentiates cytokine-induced killer cells of poor spontaneous cytotoxicity against breast cancer cells.

BACKGROUND: Chemical crosslinking is the most straightforward method to produce bispecific antibodies (BsAb) for arming ex vivo activated cytotoxic T lymphocytes. However, heterogeneous polymers are produced by chemical crosslinking. Currently, it is not known under what circumstances or to what extent further purification is needed. RESULTS: In this study, we purified Traut's Reagent-Sulfo-SMCC crosslinked anti-CD3 × anti-HER2 by size-exclusion column chromatography and compared the capacity of the crude and the purified forms of the BsAb in enhancing cytokine-induced killer (CIK) cell-mediated cytotoxicity in vitro. We found that the purified BsAb assisted CIK cells more efficiently than the crude form only when the spontaneous cytotoxicity of the CIK cells was relatively low; otherwise, the two forms performed almost identically. CONCLUSIONS: For the CIK cells of low spontaneous cytotoxicity, purified BsAb is a more powerful substitute for crude BsAb in enhancing their killing efficacy. However, that purification of BsAb is not necessary for robust CIK cells. This phenomenon also corroborates that CIK-mediated cytotoxicity is highly dependent on cell contact.

Preclinical assessment of anti-cancer drugs by using RP215 monoclonal antibody.

RP215 monoclonal antibody was originally generated against OC-3-VGH ovarian cancer cells. It was shown to recognize specifically a carbohydrate-associated epitope(s) of cancer cell-expressed immunoglobulin heavy chains designated as CA215. Previous studies suggest that CA215 is expressed by all human cancer cell lines and tissues in both membrane bound and secreted forms. It may be an ideal target for therapeutic treatments of human cancers with humanized RP215-related antibodies. Based on the results of large scale immunohistochemical studies (50-100 cases each), the following types of cancers revealed high percentage(s) of positive staining with RP215: esophagus (76%), stomach (50%), colon (44%), ovary (64%), breast (32%), lung (31%), cervix (84%) and endometrium (78%). Nude mouse experiments were performed to determine if RP215 has any inhibitory effect on the growth of cancer cells in vivo. Following injections of a single dose (10 mg/kg) of I(131)-labeled RP215 (specific activity, 12.5 muCi/mg), the tumor size (OC-3-VGH ovarian cancer cells) was reduced to 30% of the untreated control within two weeks. By injecting the same dose, the unlabeled RP215 also reduced the tumor size to 50% of the control during the same period. The antibody treatments were found to have little effect on the body weight as well as apparent toxicity of these animals. To proceed with clinical trial studies of RP215-based anti-cancer drugs, chimeric form of this monoclonal antibody was generated and characterized. Through our effort, the "proof of concept" of anti-cancer drugs development was clearly established for the next stages of clinical trial studies.

Molecular identity of a pan cancer marker, CA215.

The molecular nature of cancer-associated antigen, CA215 which reacts with RP215 monoclonal antibody and its unique epitope(s)was characterized. RP215 was initially selected and produced from one of 3,000 hybridomas which were generated from mice immunized with the cell extract of OC-3-VGH ovarian cancer cells. This cancer-associated antigen from various sources including cancer cell extract, shed culture medium and affinity-purified forms was analyzed by MALDI-TOF MS (Matrix Adsorption Laser Desorption Ionization-Time of Flight Mass Spectrometry), Western blot, carbohydrate profiling as well as enzyme immunoassays. The results of this study showed that CA215 is homologous to the heavy chains of human immunoglobulins with molecular sizes ranging from 50 to 70 KDa, when probed with RP215 or anti-human immunoglobulin G, A or M. Treatments of cancer cells with NaIO(4) drastically reduce RP215 binding to the carbohydrate-associated epitope(s) of CA215 located on the variable domain of the human immunoglobulin heavy chains. Further studies indicated that CA215 is predominantly expressed by cancer cells in both secreted and membrane-bound monomeric forms. The carbohydrate-associated epitope(s) with pH-sensitive immunoactivity appear to be present only in cancer cell-derived immunoglobulins, but not in normal human immunoglobulins. Compared to normal immunoglobulin G, CA215 contains a significantly higher percentage of N-acetyl and N-glycoyl neuraminic acid (28% vs. 8%) in the O-linked glycans, but a lower content of N-acetylglucosamine (28% vs. 41%) in the N-linked ones. It was concluded from this study that RP215 reacts specifically with carbohydrate-associated epitope(s) of immunoglobulin heavy chains expressed by various human cancer cells.