Antibody Uptake Research Articles

Investigation of the Impact of the H310A FcRn Region Mutation on 89Zr-Immuno-PET Brain Imaging with a BBB-Shuttle Anti‑Amyloid Beta Antibody

PurposeIn the emerging field of antibody treatments for neurodegenerative diseases, reliable tools are needed to evaluate new therapeutics, diagnose and select patients, monitor disease progression, and assess therapy response. Immuno-PET combines the high affinity and exceptional specificity of monoclonal antibodies with the non-invasive imaging technique positron emission tomography (PET). Its application in neurodegenerative disease brain imaging has been limited due to the marginal uptake across the blood–brain barrier (BBB). The emergence of BBB-shuttle antibodies with enhanced uptake across the BBB extended immuno-PET to brain imaging. We recently reported about specific brain uptake of a bispecific aducanumab mTfR antibody in APP/PS1 TG mice using 89Zr-immuno-PET. However, a sufficient target-to-background ratio was reached at a relatively late scanning time point of 7 days post-injection. To investigate if a better target-to-background ratio could be achieved earlier, an aducanumab BBB-shuttle with a mutated Fc region for reduced FcRn affinity was evaluated.ProceduresAduH310A-8D3 and Adu-8D3 were modified with DFO*-NCS and subsequently radiolabeled with 89Zr. The potential influence of the H310A mutation, modification with DFO*-NCS, and subsequent radiolabeling on the in vitro binding to amyloid-beta and mTfR1 was investigated via amyloid-beta peptide ELISA and FACS analysis using mTfR1 transfected CHO-S cells. Blood kinetics, brain uptake, in vivo PET imaging and target engagement of radiolabeled AduH310A-8D3 were evaluated and compared to non-mutated Adu-8D3 in APP/PS1 TG mice and wild-type animals as controls.ResultsRadiolabeling was performed with sufficient radiochemical yields and radiochemical purity. In vitro binding to amyloid-beta and mTfR1 showed no impairment. [89Zr]Zr-AduH310A-8D3 showed faster blood clearance and earlier differentiation of amyloid-beta-related brain uptake compared to [89Zr]Zr-Adu-8D3. However, only half of the brain uptake was observed for [89Zr]Zr-AduH310A-8D3.ConclusionsAlthough a faster blood clearance of AduH310A-8D3 was observed, it was concluded that no beneficial effects for 89Zr-immuno-PET imaging of brain uptake were obtained.Graphical

PD-L1 has a heterogeneous and dynamic expression in gastric cancer with implications for immunoPET.

This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells. Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both in vitro and in vivo. Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment. Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by in vivo PET imaging and ex vivo biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation. This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.

Intravesical local administration of pembrolizumab for treatment of bladder cancer: A novel strategy to minimize toxicity.

e14687 Background: A major hallmark of bladder cancer (BCa) is a higher treatment success rate than prostate cancer following stimulation of innate anti-tumor immune response either by intravesical BCG or to FDA approved intravenous anti-PD1 checkpoint antibody (pembrolizumab) approved for BCG resistant BCa since 2021. However, injected pembrolizumab is characterized by a markedly higher incidence (66%) of serious adverse events than the median duration of complete response of ~40% for non-muscle invasive BCa (NMIBC) and 15-20% for invasive BCa. Since the toxicity (fatigue, Pneumonitis and diarrhea) of injected pembrolizumab is linked to systemic breakdown of T-cell mediated immune surveillance we enquired whether analogous to BCG, pembrolizumab can be administered intravesically to safely induce a localized anti-tumor response in mouse model of BCa. Methods: Six weeks old female mice of B6D2F1 strain (n=10) were ad libitum fed carcinogen, N-butyl-N-4-hydroxybutyl nitrosamine (BBN) in drinking water (0.05% w/v) for 12 weeks. The presence of BBN evoked tumor was ascertained by unenhanced T2 weighted MRI at week 11 prior to single intravesical dosing by 24G transurethral catheter under isoflurane anesthesia of 0.1mL vehicle (sterile water) with or without 0.3mg of Pembrolizumab (Lipella Pharmaceuticals). 6 weeks later, T2 weighted MRI was repeated together with T1 mapping by ICE-MRI prior to organ harvest. Results: BBN feeding evoked multiclonal tumors (red*) of comparable size on T2 weighted MRI at week 11 but repeat MRI 7 weeks after treatment revealed that tumor progression and perivesical invasion noted in vehicle group was arrested in pembrolizumab group. Longest dimension of BBN tumor in vehicle treated mice was ≥ 1mm longer (p<0.05) than in pembrolizumab treated mice. T1 mapping revealed thickened bladder wall missed by T2 MRI. Conclusions: These findings on single intravesical dose of pembrolizumab (10mg/kg) in mice are consistent with human study on multiple intravesical dosing of BCG plus pembrolizumab (1-2mg/kg) in BCG resistant NMIBC patients. Intravesical pembrolizumab mirrors the limited systemic uptake of radiolabelled antibodies together with substantially higher ingress into tumor foci. The tumor selective ingress for BCa immunotherapy without any toxicity will be enhanced with Lipella Pharmaceutical’s liposomal delivery of Pembrolizumab.

Development and Preclinical Evaluation of a Copper-64-Labeled Antibody Targeting Glycine-Alanine Dipeptides for PET Imaging of C9orf72-Associated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

Aggregating poly(glycine-alanine) (poly-GA) is derived from the unconventional translation of the pathogenic intronic hexanucleotide repeat expansion in the C9orf72 gene, which is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly-GA accumulates predominantly in neuronal cytoplasmic inclusions unique to C9orf72 ALS/FTD patients. Poly-GA is, therefore, a promising target for PET/CT imaging of FTD/ALS to monitor disease progression and therapeutic interventions. A novel 64Cu-labeled anti-GA antibody (mAb1A12) targeting the poly-GA protein was developed and evaluated in a transgenic mouse model. It was obtained with high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity, and showed high stability in vitro and ex vivo and specifically bound to poly-GA. The affinity of NODAGA-mAb1A12 for poly-GA was not affected by this modification. [64Cu]Cu-NODAGA-mAb1A12 was injected into transgenic mice expressing GFP-(GA)175 in excitatory neurons driven by Camk2a-Cre and in control littermates. PET/CT imaging was performed at 2, 20, and 40 h post-injection (p.i.) and revealed a higher accumulation in the cortex in transgenic mice than in wild-type mice, as reflected by higher standardized uptake value ratios (SUVR) using the cerebellum as the reference region. The organs were isolated for biodistribution and ex vivo autoradiography. Autoradiography revealed a higher cortex-to-cerebellum ratio in the transgenic mice than in the controls. Results from autoradiography were validated by immunohistochemistry and poly-GA immunoassays. Moreover, we confirmed antibody uptake in the CNS in a pharmacokinetic study of the perfused tissues. In summary, [64Cu]Cu-NODAGA-mAb1A12 demonstrated favorable in vitro characteristics and an increased relative binding in poly-GA transgenic mice compared to wild-type mice in vivo. Our results with this first-in-class radiotracer suggested that targeting poly-GA is a promising approach for PET/CT imaging in FTD/ALS.

Abstract 2577: Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling

Abstract Understanding the mechanisms driving specific target engagement versus off-target nonspecific cellular uptake of antibodies can help inform design and/or delivery strategies that maximize therapeutic indices. Receptor binding kinetics, rates of receptor-mediated internalization and nonspecific pinocytosis are often measured through in vitro cell culture models, but there have been fewer efforts to collectively measure these cellular parameters in vivo. Single photon emission computed tomography (SPECT) imaging is a powerful method of estimating these parameters by quantifying both free/bound (intact) and internalized/catabolized antibody using non-residualizing halogen labels and residualizing radiometal-chelates, respectively. Modeling longitudinal in vivo imaging data of non-residualizing antibody can inform receptor occupancy (RO), while residualizing tracer kinetics can provide additional insights into catabolism due to pinocytosis and receptor-mediated internalization in the intracellular spaces. Here, we describe SPECT imaging of tumor-targeting trastuzumab (anti-HER2) and non-targeting anti-glycoprotein D (anti-gD) antibody, each labeled with non-residualizing ([125I]SIB) and residualizing radiometal-chelate (111In-DOTA) radionuclides in HER2+ xenograft-bearing mice. A non-linear compartmental model was fitted to SPECT imaging data to characterize RO of 10 mg/kg of anti-HER2 out to 7 days post dosing as well as to estimate rate constants of pinocytosis and receptor-mediated internalization of anti-HER2 in tumor. Longitudinal SPECT imaging demonstrated statistically significant differences in tumor drug exposure between 125I- and 111In-labeled anti-HER2 relative to anti-gD (p < 0.01). Tracer kinetics of anti-HER2 and associated RO simulations in tumor showed rapid extravasation from blood to tumor with peak RO of 25% at 13 hours post administration followed by a reduction in RO, reaching ~10% by day 7. Simulations for free, bound and catabolized tracer kinetics from our fitted model showed that anti-HER2 in the tumor interstitium was primarily receptor-bound with minimal pinocytosis of unbound anti-HER2 as well as gradual receptor-mediated catabolism, reaching a peak concentration of 113 µg/mL 7 days post dosing. Rate constants of nonspecific pinocytosis and HER2-mediated receptor internalization were estimated to be 0.0145 and 0.036 hours−1, respectively. Our model allowed us to successfully capture anti-HER2 antibody receptor binding and specific and nonspecific internalization over time in vivo. These data and analyses demonstrate the power of SPECT imaging using both non-residualizing and residualizing radiolabels combined with compartmental modeling to better characterize the different biological states (free, bound, and catabolized) of antibody within interstitial and intracellular compartments. Citation Format: Nicholas Cho, Phillip Spinosa, Danielle Mandikian, Geoffrey Del Rosario, Shang-Fan Yu, Jason Ho, Greg Ferl, Charles A. Boswell. Analyzing specific and nonspecific tissue uptake of antibodies by SPECT imaging and non-linear compartmental modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2577.

Abstract 3108: Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies

Abstract Introduction: There is a growing need in the immunotherapy research field for tools to rapidly screen novel therapeutic antibodies, whether looking for cells expressing IgG, target binding, or tracking internalization. Thermo Fisher Scientific has developed the next generation of Invitrogen™ Zenon™ reagents to enable screening of potential therapeutic antibodies, working across a variety of sample types including primary B cells and hybridomas, while being compatible with a wide range of platforms including flow cytometry, high content imaging, and in-incubator imaging systems. Methods: The Invitrogen™ Zenon™ Alexa Fluor™ Plus IgG Labeling Reagents to screen for antibody binding provide a fast, versatile, and reliable method to evaluate antibody binding to cell surface antigens. Zenon screening reagents are labeled with Invitrogen Alexa Fluor Plus 488, Alexa Fluor Plus 594, or Alexa Fluor Plus 647 dyes (i.e., labeling reagent). The labeling reagent is directed against the Fc portion of an intact mouse or human IgG primary antibody. The formation of the zenon-antibody complex is rapid and requires no purification steps. The Invitrogen™ Zenon™ pHrodo™ Deep Red Labeling Reagents for screening antibody internalization provides a highly sensitive method for monitoring antibody internalization. Zenon pHrodo Deep Red utilizes the same screening reagent as Zenon Alexa Fluor Plus reagents, replacing Alexa fluor dyes with the pH-sensitive dye, pHrodo Deep Red. The pHrodo Deep Red sensors have minimal fluorescence at neutral pH and only upon entry into the late endosome and lysosome do they brightly fluoresce. Zenon pHrodo Deep Red Labeling Reagents are perfectly suited for screening the endocytosis and degradation of potentially therapeutic antibodies including antibody-drug conjugates. Results: Live cells stained with Zenon-labeled antibodies show remarkable signal to background in both flow and imaging applications, while retaining their signal following fixation. Therapeutic antibodies such as anti-Her2 labeled with Zenon pHrodo Deep Red show as bright fluorescent puncta upon lysosomal degradation in the Alexa Fluor 647 channel and co-localize with lysosomal markers such as Invitrogen™ LysoTracker Red dye. Both Zenon Alexa Fluor Plus and Zenon phrodo Deep Red are ideal reagents for screening antibody panels of up to 96 different antibodies. Conclusions: Thermo Fisher Scientific has developed sensitive and rapid Zenon reagents to allow for high throughput therapeutic antibody screening. In addition to Zenon reagents, Thermo Fisher Scientific has developed additional tools that allow for specific monitoring of antibody and protein uptake into the lysosome for degradation. These tools not only greatly improve the workflow from our existing portfolio, but allow for increased sensitivity allowing the field to make decisive decisions when selecting candidate immunotherapies. Acknowledgments: I would like to thank the whole team that worked to develop these new labeling reagents including: Maria Olson and Chris Langsdorf. Citation Format: Ryan W. Holly. Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3108.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed “organoids.” Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Investigation of Antibody Pharmacokinetics in the Brain Following Intra-CNS Administration and Development of PBPK Model to Characterize the Data.

Despite the promising potential of direct central nervous system (CNS) antibody administration to enhance brain exposure, there remains a significant gap in understanding the disposition of antibodies following different intra-CNS injection routes. To bridge this knowledge gap, this study quantitatively investigated the brain pharmacokinetics (PK) of antibodies following intra-CNS administration. The microdialysis samples from the striatum (ST), cerebrospinal fluid (CSF) samples through cisterna magna (CM) puncture, plasma, and brain homogenate samples were collected to characterize the pharmacokinetics (PK) profiles of a non-targeting antibody, trastuzumab, following intracerebroventricular (ICV), intracisternal (ICM), and intrastriatal (IST) administration. For a comprehensive analysis, these intra-CNS injection datasets were juxtaposed against our previously acquired intravenous (IV) injection data obtained under analogous experimental conditions. Our findings highlighted that direct CSF injections, either through ICV or ICM, resulted in ~ 5-6-fold higher interstitial fluid (ISF) drug exposure than IV administration. Additionally, the low bioavailability observed following IST administration indicates the existence of a local degradation process for antibody elimination in the brain ISF along with the ISF bulk flow. The study further refined a physiologically based pharmacokinetic (PBPK) model based on new observations by adding the perivascular compartments, oscillated CSF flow, and the nonspecific uptake and degradation of antibodies by brain parenchymal cells. The updated model can well characterize the antibody PK following systemic and intra-CNS administration. Thus, our research offers quantitative insight into antibody brain disposition pathways and paves the way for determining optimal dosing and administration strategies for antibodies targeting CNS disorders.

First exploration of the on-treatment changes in tumor and organ uptake of a radiolabeled anti PD-L1 antibody during chemoradiotherapy in patients with non-small cell lung cancer using whole body PET

BackgroundIn patients with locally advanced unresectable non-small cell lung cancer (NSCLC), durvalumab, an anti-programmed cell death ligand-1 (PD-L1) antibody, has shown improved overall survival when used as consolidation therapy following...

Abstract B044: Intracellular sclerostin plays a vital role in tumor progression and metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited treatment options. It is essential to continue advancing well-defined molecular targets in TNBC. Sclerostin (SOST) is mostly concerned as an osteocyte secreted protein and a drug target for osteoporosis. Recently, sclerostin was reported to be expressed in a majority of clinical TNBC tissues. Upregulated SOST expression was reported to be associated with worse prognosis in breast cancer patients. This study aimed to investigate the role of sclerostin in TNBC. Methods: Cell proliferative ability was evaluated by colony formation assay. Cell migratory and invasive abilities were evaluated by transwell assay. The tumor growth and metastasis were evaluated in 4T1 orthotopic and metastatic mouse models. The cellular uptake of antibody and aptamer was evaluated by fluorescence microscopy. Results: It was found that sclerostin knockout demonstrated inhibitory effects on proliferation and migration/invasion in both human (MDA-MB-231) and murine (4T1) TNBC cell lines in vitro. Surprisingly, no tumor growth and metastasis were found in 4T1-sost KO inoculated orthotopic and metastatic mouse models compared to their wildtype counterparts. It suggested the vital role of sclerostin in TNBC. Anti-sclerostin antibody (Scl-Ab) could not be internalized by TNBC cells and demonstrated no effects on proliferation and migration/invasion in vitro. On the other hand, no difference in proliferation and migration/invasion was also observed between TNBC cells with and without supplementing sclerostin in vitro. Genetically, there was no difference in tumor growth between 4T1 inoculated WT mice and SOST -/− mice. Pharmacologically, no difference was also observed in tumor growth between Scl-Ab treatment and IgG control in 4T1 inoculated mice. It suggested that the role of extracellular and systemic sclerostin could be excluded in TNBC. Aptamers are single-strand nucleic acids which bind to specific targets. It was found that sclerostin-specific aptamer (Scl-Ap) could be internalized by TNBC cells and demonstrated inhibitory effects on proliferation and migration in vitro. Consistently, Scl-Ap demonstrated inhibitory effects on tumor growth in 4T1 inoculated mice. It suggested that intracellular sclerostin might play a vital role in TNBC. Conclusions: Intracellular sclerostin plays a vital role in TNBC progression and metastasis. Citation Format: Meiheng SUN, Shuangying Qiao, Hang Luo, Xin Yang, Liu Yang, Huarui Zhang, Zefeng Chen, Luyao Wang, Xiaohui Tao, Yuanyuan Yu, Yuan Ma, Fenglai Yuan, Daqing Ma, Qianjun Chen, Aiping Lu, Bao-ting Zhang, Ge Zhang, Fangfei Li. Intracellular sclerostin plays a vital role in tumor progression and metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B044.

Intranasal Nose-to-Brain Drug Delivery via the Olfactory Region in Mice: Two In-Depth Protocols for Region-Specific Intranasal Application of Antibodies and for Expression Analysis of Fc Receptors via In Situ Hybridization in the Nasal Mucosa.

A region-specific catheter-based intranasal administration method was successfully developed, established, and validated as reported previously. By using this method, drugs can be applicated specifically to the olfactory region. Thereby, intranasally administered drugs could be delivered via neuronal connections to the central nervous system. Here, we present a detailed protocol with a step-by-step procedure for nose-to-brain delivery via the olfactory mucosa.Fc receptors such as the neonatal Fc receptor (FcRn) and potentially Fcγ receptor IIb (FcγRIIb) are involved in the uptake and transport of antibodies via the olfactory nasal mucosa. To better characterize their expression levels and their role in CNS drug delivery via the nose, an in situ hybridization (ISH) protocol was adapted for nasal mucosa samples and described in abundant details.

Antibodies internalization mechanisms by dendritic cells and their role in therapeutic antibody immunogenicity.

Internalization and processing by antigen-presenting cells such as dendritic cells (DCs) are critical steps for initiating a T-cell response to therapeutic antibodies. Consequences are the production of neutralizing antidrug antibodies altering the clinical response, the presence of immune complexes, and, in some rare cases, hypersensitivity reactions. In recent years, significant progress has been made in the knowledge of cellular uptake mechanisms of antibodies in DCs. The uptake of antibodies could be directly related to their immunogenicity by regulating the quantity of materials entering the DCs in relation to antibody structure. Here, we summarize the latest insights into cellular uptake mechanisms and pathways in DCs. We highlight the approaches to study endocytosis, the impact of endocytosis routes on T-cell response, and discuss the link between how DCs internalize therapeutic antibodies and the potential mechanisms that could give rise to immunogenicity. Understanding these processes could help in developing assays to evaluate the immunogenicity potential of biotherapeutics.

Mucosal absorption of antibody drugs enhanced by cell-penetrating peptides anchored to a platform of polysaccharides

Our previous studies demonstrated that L-octaarginine grafted onto hyaluronic acid via a tetraglycine spacer significantly enhanced intranasal absorption of protein drugs with a molecular weight (Mw) of 22 kDa or less. The present study focused on its potential as an absorption enhancer for antibody drugs with a larger Mw and the enhancement mechanism. When ranibizumab (48 kDa) alone was intranasally administered in mice, its absolute bioavailability was 0.67% on average. The mean bioavailability elevated to 6.2% under coadministration with tetraglycine-L-octaarginine-linked hyaluronic acid. A similar result was observed under substitution of ranibizumab with certolizumab pegol (91 kDa), although bioavailability itself decreased with the Mw increase, irrespective of coadministration with the hyaluronic acid derivative. Rat experiments also revealed that coadministration with the polysaccharide derivative resulted in significant enhancement of intranasal absorption of trastuzumab (148 kDa). In vitro studies using gene-knocked down cells indicated that syndecan-4-induced macropinocytosis played a crucial role on acceleration of antibody uptake into epithelial cells on the nasal mucosa, irrespective of their Mw. It appeared that neither clathrin heavy chain nor caveolin-1 involved in cellular uptake of antibodies. Tetraglycine-L-octaarginine-linked hyaluronic acid was concluded to be a promising delivery tool that possessed universal absorption-enhancing abilities independent to Mw of biologics.

SAT495 Persistent Euthyroid State in a Patient with Biochemical and Imaging Evidence of Graves' Disease

Abstract Disclosure: M. Antony: None. S. Gundlapally: None. M. Joglekar: None. B. Fritz: None. S. Patel: None. V. Verma: None. R. Kant: None. INTRODUCTION: Elevated TSI antibody and/or diffuse uptake on radioactive Iodine-123 scan is diagnostic of Graves’ disease. Thyromegaly with increased blood flow on color doppler seen on thyroid USG can also aid in the diagnosis. Rare presence of positive TSI antibody without hyperthyroidism has been reported1. Rare co-existence of both Graves’ disease and Hashimoto’s thyroiditis in a patient causing a challenging clinical course has been reported2. Presence of a neutralizing or blocking antibody against the TSH receptor1 or a false positive TSI have been implicated in such rare cases. CLINICAL CASE: A 37-year-old Caucasian Female was seen at our endocrinology clinic for further evaluation of thyroid disorder. Patient initially experienced goiter during 1st pregnancy at age 18, followed by hypothyroidism during last pregnancy needing levothyroxine treatment. Due to goiter history, patient underwent work-up by her primary physician in 2021. Thyroid ultrasound (usg) showed bilateral hyperemia, heterogeneous echotexture, and 0.2 cm cystic nodule in the right upper lobe. Labs showed normal TSH 1.33 uIU/ml (0.3-4.0 uIU/ml), normal Free T4 0.99 ng/dl (0.8-1.8 ng/dl), normal total T3 140 ng/dl (71-180 ng/dl). Patient underwent further work-up by endocrinology in 2022. Thyroid ultrasound again revealed bilateral hyperemia with other similar findings. Labs showed normal TSH 2.02uUI/ml, normal free T4 0.93 ng/dl, normal total T3 138 ng/dl. Interestingly, TSI antibody and TPO antibody were significantly elevated at 66.70 IU/L (0.0-0.55 IU/L) and 517 IU/ml (0-34 IU/ml), respectively. Repeat labs in April 2022, again showed normal TSH 1.77 uIU/ml, normal free T4 0.90 ng/dl and normal free T3 2.94 (2.0-4.40 pg/ml). Thyroid uptake scan was not performed due to persistently normal TSH values. Patient denied the use of biotin supplementation. Patient continues to remain clinically and biochemically euthyroid without treatment. CONCLUSION: Our case supports the rare presence of TSI antibody without hyperthyroidism. Close monitoring is warranted due to possibility of future progression to overt hyperthyroidism. REFERENCE: 1. Alvin Mathew A, Papaly R, Maliakal A, et al.(November 10, 2021)Elevated Graves’ Disease-Specific Thyroid-Stimulating Immunoglobulin and Thyroid Stimulating Hormone Receptor Antibody in a Patient With Subacute Thyroiditis. Cureus 13(11): e19448. doi:10.7759/cureus.194482. Vipin Verma, MD, Mc Anto Antony, MD, PSAT346 Persistent Fluctuation Between Hypothyroidism and Hyperthyroidism in a Patient with Both Hashimoto's Thyroiditis and Graves’ Disease: Challenges in Diagnosis and Treatment Approach, Journal of the Endocrine Society, Volume 6, Issue Supplement_1, November-December 2022, Page A833, https://doi.org/10.1210/jendso/bvac150.1723 Presentation Date: Saturday, June 17, 2023

Enhanced Therapeutic Effects of 177Lu-DOTA-M5A in Combination with Heat Shock Protein 90 Inhibitor Onalespib in Colorectal Cancer Xenografts.

Carcinoembryonic antigen (CEA) has emerged as an attractive target for theranostic applications in colorectal cancers (CRCs). In the present study, the humanized anti-CEA antibody hT84.66-M5A (M5A) was labeled with 177Lu for potential CRC therapy. Moreover, the novel combination of 177Lu-DOTA-M5A with the heat shock protein 90 inhibitor onalespib, suggested to mediate radiosensitizing properties, was assessed in vivo for the first time. M5A antibody uptake and therapeutic effects, alone or in combination with onalespib, were assessed in human CRC xenografts and visualized using SPECT/CT imaging. Although both 177Lu-DOTA-M5A and onalespib monotherapies effectively reduced tumor growth rates, the combination therapy demonstrated the most substantial impact, achieving a fourfold reduction in tumor growth compared to the control group. Median survival increased by 33% compared to 177Lu-DOTA-M5A alone, and tripled compared to control and onalespib groups. Importantly, combination therapy yielded comparable or superior effects to the double dose of 177Lu-DOTA-M5A monotherapy. 177Lu-DOTA-M5A increased apoptotic cell levels, indicating its potential to induce tumor cell death. These findings show promise for 177Lu-DOTA-M5A as a CRC therapeutic agent, and its combination with onalespib could significantly enhance treatment efficacy. Further in vivo studies are warranted to validate these findings fully and explore the treatment's potential for clinical use.

Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau.

Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging. We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [18F]SFB as the prosthetic group, all four 18F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18F]SFB in acetonitrile/0.1 M borate buffer solution (final pH ~ 8.5) with an incubation of 20 min at room temperature, followed by purification on a PD MiniTrap G-25 size exclusion gravity column. Based on both in vitro and ex vivo evaluation, the bispecific antibodies displayed much higher brain concentrations than the unmodified antibody, supporting our subsequent F18-radiolabeling. [18F]SFB was produced in high yields in 60 min (decay-corrected radiochemical yield (RCY) 46.7 ± 5.4) with radiochemical purities of >95%, confirmed by analytical high performance liquid chromatography (HPLC) and radio-TLC. Conjugation of [18F]SFB and bispecific antibodies showed a 65%-83% conversion efficiency with radiochemical purities of 95%-99% by radio-TLC. We successfully labeled four novel and specifically engineered bispecific antibodies with [18F]SFB under mild conditions with a high RCY and purities. This study provides strategies to create brain-penetrable F-18 radiolabeled antibody probes for the selective detection of tau and Aβ aggregates in the brain of transgenic AD mice via in vivo PET imaging.

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 μM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

The cardioprotective role of sirtuins is mediated in part by regulating KATP channel surface expression.

Sirtuins are NAD+-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases, and cardiac ischemia. Since ATP-sensitive K+ (KATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD+ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. KATP channels were studied with patch clamping, biochemistry techniques, and antibody uptake experiments. NMN led to an increase in intracellular NAD+ levels and an increase in the KATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of KATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased KATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2) and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a KATP channel-dependent manner. Overall, our data draw a link between intracellular NAD+, sirtuin activation, KATP channel surface expression, and cardiac protection against ischemic damage.

Validation of simplified uptake measures against dynamic Patlak Ki for quantification of lesional 89Zr-Immuno-PET antibody uptake

PurposePositron emission tomography imaging of zirconium-89-labelled monoclonal antibodies (89Zr-Immuno-PET) allows for visualisation and quantification of antibody uptake in tumours in vivo. Patlak linearization provides distribution volume (VT) and nett influx rate (Ki) values, representing reversible and irreversible uptake, respectively. Standardised uptake value (SUV) and tumour-to-plasma/tumour-to-blood ratio (TPR/TBR) are often used, but their validity depends on the comparability of plasma kinetics and clearances. This study assesses the validity of SUV, TPR and TBR against Patlak Ki for quantifying irreversible 89Zr-Immuno-PET uptake in tumours.MethodsTen patients received 37 MBq 10 mg 89Zr-anti-EGFR with 500 mg/m2 unlabelled mAbs. Five patients received two doses of 37 MBq 89Zr-anti-HER3: 8–24 mg for the first administration and 24 mg–30 mg/kg for the second. Seven tumours from four patients showed 89Zr-anti-EGFR uptake, and 18 tumours from five patients showed 89Zr-anti-HER3 uptake. SUVpeak, TPRpeak and TBRpeak values were obtained from one to six days p.i. Patlak linearization was applied to tumour time activity curves and plasma samples to obtain Ki.ResultsFor 89Zr-anti-EGFR, there was a small variability along the linear regression line between SUV (− 0.51–0.57), TPR (− 0.06‒0.11) and TBR (− 0.13‒0.16) on day 6 versus Ki. Similar doses of 89Zr-anti-HER3 showed similar variability for SUV (− 1.3‒1.0), TPR (− 1.1‒0.53) and TBR (− 1.5‒0.72) on day 5 versus Ki. However, for the second administration of 89Zr-anti-HER3 with a large variability in administered mass doses, SUV showed a larger variability (− 1.4‒2.3) along the regression line with Ki, which improved when using TPR (− 0.38–0.32) or TBR (− 0.56‒0.46).ConclusionSUV, TPR and TBR at late time points were valid for quantifying irreversible lesional 89Zr-Immuno-PET uptake when constant mass doses were administered. However, for variable mass doses, only TPR and TBR provided reliable values for irreversible uptake, but not SUV, because SUV does not take patient and mass dose-specific plasma clearance into account.

Targeting the Brain with Single-Domain Antibodies: Greater Potential Than Stated So Far?

Treatments for central nervous system diseases with therapeutic antibodies have been increasingly investigated over the last decades, leading to some approved monoclonal antibodies for brain disease therapies. The detection of biomarkers for diagnosis purposes with non-invasive antibody-based imaging approaches has also been explored in brain cancers. However, antibodies generally display a low capability of reaching the brain, as they do not efficiently cross the blood-brain barrier. As an alternative, recent studies have focused on single-domain antibodies (sdAbs) that correspond to the antigen-binding fragment. While some reports indicate that the brain uptake of these small antibodies is still low, the number of studies reporting brain-penetrating sdAbs is increasing. In this review, we provide an overview of methods used to assess or evaluate brain penetration of sdAbs and discuss the pros and cons that could affect the identification of brain-penetrating sdAbs of therapeutic or diagnostic interest.