Human Anti-mouse Antibody Research Articles

8747 A Rare Receptor Resistance with Paradoxical Outcomes!

Abstract Disclosure: I. Patoli: None. S. Batool: None. A.S. Gill: None. A. Makdissi: None. Introduction: Resistance to thyroid hormone receptor is an uncommon cause of abnormal TFTs. (1) We present an interesting case of THRB resistance with cardiac arrhythmia (CA). Clinical Case: 46-year-old man with history of atrial fibrillation (Afib) was referred to endocrinology for evaluation of abnormal TFTs and thyroid nodule. He had a recent hospital admission for Afib with rapid ventricular response; 3 years ago, he was treated with amiodarone for 2 months. TFTs revealed normal TSH of 1.375, elevated FT4 of 2.76 (normal ranges 0.55-4.78 and 0.89-1.76, respectively). Neck CT scan showed large peripherally enhancing centrally necrotic mass with calcification encompassing nearly the entirety of left thyroid lobe measuring 2.2 x 3.6 x 4.6 cm. A 0.8 cm hypodensity within right thyroid lobe also visualized. Thyroid ultrasound showed nodules bilaterally with the largest one in the lower pole of left lobe. FNA biopsy showed Bethesda category- II benign, benign appearing follicular cells and abundant colloid. Repeat labs showed similar results with persistently elevated free T4 (2.56) with equilibrium dialysis, normal alpha subunit, normal TSH (2.14) with human anti-mouse antibody (HAMA) antibody was negative. Alpha subunit:TSH ratio was <1. He remained clinically euthyroid over the course of 1.5 year but repeat labs at that time indicated that TSH had increased to 17 with elevated free T4 (2.6) . MRI brain ruled out TSH secreting adenoma. He reported thyroid abnormalities in son and brother but was unable to provide details. He was referred for genetic screening which showed THRB with pathogenic variant: c.9628> G (p.Y321C). Conclusion: THRB resistance is an uncommonly diagnosed modality in endocrinology. The genes encoding thyroid hormone receptors are THRα gene and THRβ gene (2). Patients with inherited THRB pathogenic variants present with resistance to thyroid hormone and often enlarged thyroid gland. Interestingly, despite having receptor resistance, patients may manifest with CA. This is hypothesized to be due to elevated T4 and T3 concentration in the heart which primarily has higher expression of THRA (3). The diagnosis of thyroid hormone resistance is important to avoid treating asymptomatic patients where treatment is not warranted.

B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood

Abstract Background Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120). Conclusions The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.

Advances of monoclonal antibodies and analysis of marketed antibody drugs

In recent years, there has been a frequent occurrence of various epidemics worldwide such as COVID-19, monkeypox, influenza, and others additionally, there has been an increase in the number of new patients diagnosed with various types of tumors. Traditional drugs have limited effectiveness against emerging infectious diseases, tumors, and autoimmune diseases. However, with the emergence of hybridoma technology, monoclonal antibodies have achieved extensive applications and antibody drugs are playing an important role in modern medicine. Monoclonal antibodies have undergone various development stages, starting from mouse-derived antibodies to human-mouse chimeric antibodies, humanized antibodies, and ultimately human antibodies. Throughout this process, their immunogenicity has gradually decreased, while their safety for human use steadily increased. Fully human antibodies are currently the safest form of antibody, because their sequences all come from human sources and they do not induce human anti-murine antibody reactions. With the advance of genetic engineering technology, flow cytometry coupled to single B cell gene amplification technology has made it easier to construct and screen for fully human monoclonal antibodies. The development of antibody drugs has provided new opportunities, and the market for monoclonal antibody drugs will further expand. This article reviews the research progress of monoclonal antibodies and presents information on the 163 monoclonal antibody drugs approved by the United States Food and Drug Administration (FDA) as of Oct 1st, 2023. The aim is to offer new insights for the development and production of monoclonal antibodies in China.

Concordance between APOE genotyping and proteotyping using the novel Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays

AbstractBackgroundThe APOE gene has three different alleles that encode for three different ApoE protein isoforms (APOE2, APOE3, and APOE4), resulting in 6 different phenotypes. Nucleic acid amplification test remains the gold standard for APOE genotyping, however quantification of the ApoE proteins using immunoassays could provide another piece of information, related to the expression levels of the proteins. In this regard, ApoE as a protein biomarker can be used in research to determine its value alone or as part of a panel of plasma biomarkers.MethodThe LUMIPULSE G System is a chemiluminescent enzyme immunoassay platform enabling fully automated processing of samples using ready‐to‐use immunoreaction cartridges. Time to result in these single use test cartridges takes about 30 minutes. The analytical performance of the newly developed Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays was determined. Parameters analyzed were precision, sensitivity, linearity, prozone and potential impact of interfering substances. Proof of concept of these novel assays was verified with a set of 230 genotyped plasma samples.ResultThe total within‐lab variability of both assays was ≤8% CV confirming high precision of the LUMIPULSE G system. Using low ApoE concentrated plasma samples, the observed LoD and LoQ (@20% CV) was 0.072 µg/mL (Pan‐ApoE) and 0.152 µg/mL (ApoE4). Linearity was shown across the assay range (0 – 100 µg/mL) for both assays. Neither prozone effect nor significant impact (recovery within 100±10%) of commonly tested endogenous substances (bilirubin, haemoglobin, low and high Protein content, and human anti‐mouse antibodies (HAMA)) was observed. Potential interference was observed for triglycerides and rheumatoid factor (RF) with observed recovery within 100±20%. ApoE proteotyping based on the novel assays resulted in a 99.8% concordance with APOE genotyping (1 discordant result). Moreover, applying the ratio ApoE4/Pan‐ApoE with the measurement values of respective assays, classified correctly between hetero‐ and homozygous E4 carrier status.ConclusionThe analytical performance studies demonstrate low variability and high sensitivity enabling measurement of pan‐ApoE and ApoE4 in plasma. Pan‐apoE and apoE4 quantification can offer relevant insights into the assessment of subjects at risk for AD or adverse events following anti‐amyloid treatments and should be studied further.

SAT482 A Case of Falsely Elevated Free T4 and Free T3 Levels Caused by Human Anti Mouse Antibodies.

Abstract Disclosure: I.J. Romao: None. M.H. Shanik: None. Introduction: When there are clinical and biochemical discrepancies with thyroid function tests immunoassay interference should be considered (1). Human anti mouse antibodies (HAMA) are antibodies found in humans that react with immunoglobulins in mice and are used as diagnostic reagents for laboratory measurements. The presence of HAMA most commonly causes a false elevation of thyroid stimulating hormone but can also increase Free T4 and Free T3 levels. Case presentation: A 31 year old woman presented for evaluation of elevated Free T4 and Free T3 levels. For several weeks she complained of feeling neck soreness and palpitations. She had a recent infection and low-grade fever. She also felt pressure behind her eyes. She had a history of anxiety and took Xanax as needed, multivitamin and probiotic. Cousin had thyroid disease. On exam her blood pressure was 138/86, heart rate 80, respiratory rate 12, weight 149 lbs., height 5’5”. Her thyroid was firm and not tender. She had a thyroid ultrasound that showed a homogenous gland with no discrete nodules. TSH was 1.3 uIU/mL (0.27-4.2), Total T4 10.4 ug/dL (4.5 – 12), Free T4 4.6 ng/dL (0.9-1.7), Total T3 1.6 ng/mL (0.8-2.0), Free T3 10.1 pg/mL (2.0-4.4). C reactive protein, erythrocyte sedimentation and interleukin 6 levels were in the normal range. Thyroid peroxidase antibodies and thyroglobulin antibodies were elevated. The differential diagnosis included thyroiditis, pituitary tumor producing thyroid stimulating hormone and thyroid hormone resistance. She did not have a thyroid uptake and scan because she had small children at home. To rule out a pituitary adenoma she had a pituitary MRI that showed a normal small pituitary gland. The test for resistance to thyroid hormone mutation analysis was negative. Over a few months she had some clinical improvement, and her thyroid function tests remained the same which would not be consistent with thyroiditis. HAMA level was checked to evaluate for immunoassay interference with thyroid function tests. The HAMA level was elevated at greater than 600 ng/mL (0-188). Free T4 by equilibrium dialysis was normal at 1.6 ng/dL (1.1 – 2.4). Conclusion: HAMA can cause falsely elevated Free T4 and Free T3 levels. It is important to suspect immunoassay interference when there are inconsistencies in the clinical presentation and thyroid function tests. Identifying the presence of elevated HAMA can prevent misdiagnosis and inappropriate patient management. 1. Favresse J, Burlacu MC, Maiter D, Gruson D. Interferences with thyroid immunoassays: Clinical implications and detection algorithm. Endocrine Reviews. 2018. Pages 830-850. Presentation Date: Saturday, June 17, 2023

SAT546 Papillary Thyroid Cancer Surveillance: Thyroglobulin Levels Affected by HAMA

Abstract Disclosure: J. Sanchez Perez: None. M. Obeid: None. M. Fariduddin: None. D.J. Toft: None. Introduction: Thyroglobulin (TG) measurement is a major means of detecting recurrence of previously treated differentiated thyroid cancers. Human-Anti Mouse Antibodies (HAMA) can interfere with lab measurements of TG levels and affect clinical decisions and treatment for these cancers.Clinical Case: A 65-year-old female was diagnosed with papillary thyroid cancer (PTC). Surgical pathology following total thyroidectomy demonstrated a tumor of 3.5 cm in its greatest dimension, unifocal, without invasion of locoregional tissue classified as Stage I (pT2N0M0) per AJCC 8th edition. Radioiodine whole body scan (WBS) demonstrated iodine uptake in the neck and 126.5 mCi of I-131 was given. In surveillance 5 years later, TSH was found to be 0.17 mcIU/ml (ref range: 0.35 - 4.00) TG 64.7 ng/ml (ref range: 1.3 - 31.8 ng/mL), and anti-Tg antibodies were undetectable. Neck US consistently showed nonspecific findings of stable small nodes with normal morphology in both supraclavicular regions. Given elevated TG levels, 250 mCi of I-131 was given. Follow-up TG levels ranged from 27.8 to 36.4 ng/ml. A repeat WBS did not reveal any uptake, but as TG remained elevated, I-131 (238 mCi) was again given. Subsequent TG throughout the following 15 years ranged from 2.8 - 58.6 ng/ml. Repeat Neck US only showed benign-appearing lymph nodes. WBS did not have any focal activity to suggest recurrent disease and PET/CT did not show any abnormal FDG uptake. The persistently high TG levels without clinical evidence of tumor recurrence raised the suspicion of lab interference. The serum sample was treated with a blocking reagent that contained mouse immunoglobulin after which the TG level decreased to 2.7 ng/ml compared to untreated value of 20.5 ng/ml.Conclusion: HAMA are human antibodies against mouse antibodies which are believed to develop in humans from direct contact with rodents, including mice, and from the recent increase in the use of mouse monoclonal antibodies for diagnostic and therapeutic purposes. These antibodies implicate an unexpected source of interference in immunoassays, as seen in our patient, which can confound laboratory results and create a clinical dilemma. This patient with PTC had a complete radiological response after surgery, but an incomplete biochemical response which caused the patient to be given a total of 614 mCi of I-131. This case highlights the importance of recognizing the possible presence of HAMA when analyzing TG levels, particularly when the TG levels are used for treatment decisions. Presentation Date: Saturday, June 17, 2023

FRI532 Thyroid Hormone Resistance: A Possible New Variant In The THRB Gene

Abstract Disclosure: A. Siddiqui: None. Z. Khan: None. T. Ahmed: None. K. Selk: None. Background: Resistance to the Thyroid Hormone (RTH) is caused by reduced target tissue response to thyroid hormone, resulting in the loss of the normal feedback mechanism that suppresses secretion of thyrotropin (TSH). Most cases of RTH are familial, usually inherited in an autosomal dominant manner, with over 100 different pathogenic variants having been described. Clinical Case: A 45-year-old male with history of attention deficit disorder presented for management of hypothyroidism. The patient was Anti-TPO positive, consistent with Hashimoto's thyroiditis and he was treated with varying doses of levothyroxine. Labs on 100 mcg levothyroxine showed a TSH of 7.07 (0.35-4.94 uIU/mL), free t4 1.86 (0.7-1.48 ng/dL), and free t3 4.41 (2.3-4.2 pg/mL). This initially prompted concern for a TSHoma. An alpha subunit level was found to be 0.3 ng/dL and pituitary MRI was within normal limits. Given continued persistent thyroid hormone elevations, further testing for human anti-mouse antibody interference was performed and also unrevealing. The patient continued to report feeling tired and fatigued, with multiple joint pains and inability to focus or concentrate. His levothyroxine was increased to 125 mcg per day. However, even after the adjustment, he still did not notice any change in his symptoms. A trial of addition of liothyronine 5mcg daily was also performed but he self-discontinued this as he did note any improvement. Overall, his findings were highly suspicious for thyroid hormone resistance. Genetic testing for a thyroid hormone resistance beta (THRB) mutation was performed. Direct sequence analysis of the coding region and splice-sites of the THRB gene identified one copy of the c.1368G>T (p.Leu456Phe) variant of uncertain significance. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. However, the THRB c.1368G>T (p.Leu456Phe) variant has not been described in online databases or published literature. It also has not been reported in large, multi-ethnic general populations. Conclusion: This is the first case to our knowledge demonstrating the THRB c.1368G>T (p.Leu456Phe) variant gene as likely giving rise to RTH. Further testing of family members has been recommended to help clarify the clinical significance of this finding. Presentation: Friday, June 16, 2023

Semi-quantitative analysis with 99mTc-Besilesomab in musculoskeletal system infections

ObjectivesTo evaluate the usefulness of 99mTc-Besilesomab to diagnose infectious processes by using monoclonal antibodies BW 250/183 in a semi-quantitative analysis, and to determine the effect on diagnostic capacity of different thresholds for the difference between counts in early versus delayed images. MethodsThe study included 77 patients with suspected osteomyelitis who underwent scintigraphy with 99mTc-Besilesomab. After confirming the absence of human anti-mouse antibodies in all patients, early and delayed static images were acquired at 4 and 24 h post-injection, respectively. Visual and semi-quantitative analyses were conducted of regions of interest (ROIs) in areas suspected of infection on early and delayed images. Findings were considered positive when the ratio between counts in delayed and early images exceeded a given threshold after correction for decay. The definitive diagnosis was obtained by clinical follow-up, microbiological culture, or response to medical and/or surgical treatment. ResultsThe optimal threshold was 1.02 (i.e., positive result = count increase of >2 % in delayed image), obtaining a sensitivity of 0.864, specificity of 0.858, positive predictive value of 0.708, negative predictive value of 0.940, and accuracy of 0.860. Application of the usual threshold of 1.10 (10 %) reduced the sensitivity to 0.734. ConclusionSemi-quantitative analysis of studies with 99mTc-Besilesomab is a useful technique for the diagnosis of musculoskeletal system infections and contributes to the definitive diagnosis when visual assessments are doubtful or non-conclusive.

Anti-ricin toxin human neutralizing antibodies and DMAbs protection against ricin toxin poisoning

DNA-encoded monoclonal antibodies (DMAbs) and in vivo expression of antibody therapeutics presents an innovative alternative to conventional delivery methods. Therefore, in order to prevent the lethal dose of ricin toxin (RT) and to avoid human anti-mouse antibody (HAMA) reaction, we developed the human neutralizing antibody 4–4E against RT and constructed DMAb-4–4E. The human neutralizing antibody 4–4E could neutralize RT in vitro and in vivo, while the mice in RT group all died. Using intramuscular electroporation (IM EP), antibodies were rapidly expressed in vivo within 7 days and were enriched in intestine and gastrocnemius muscle mostly. Besides, we found that DMAbs have shown a broad protective efficacy of RT poisoning prophylaxis. Driven by plasmids for IgG expression, mice were survived and the blood glucose level of mice in DMAb-IgG group returned to normal at 72 h post RT challenge, and the RT group died within 48 h. Furthermore, hindrance of protein disulfide isomerase (PDI) and accumulation of RT in endosomes were found in IgG-protected cells, revealing the possible mechanism of neutralization details. These data support the further study of RT-neutralizing monoclonal antibodies (mAbs) in the development.

Improved radioimmunodetection of carcinomas with a re-injection of monoclonal antibodies after formation of anti-mouse antibodies.

Scintigraphic imaging was satisfactory in animal experiments, i.e., in the radioimmunodetection with 125J anti-tissue polypeptide antigen monoclonal antibodies and implanted HELA cell carcinomas. Unlabeled anti-mouse antibodies (AMAB), in a surplus of 40:1, 200:1 and 4000:1 compared to the radioactive antibody, were administered five days after administering the 125I anti-TPA antibody (RAAB). In immunoscintigraphies, radioactivity accumulated in the liver immediately after administering the secondary antibody, and the tumor's imaging worsened. It can be expected that imunoscintigraphic imaging might improve when radioimmunodetection is re-performed after the formation of human anti-mouse antibodies (AMAB) and when the ratio of the primary to the secondary antibody is nearly equivalent because, in this ratio, the formation of immune complexes might be accelerated. It is possible to measure the quantity of formed anti-mouse antibodies (AMAB) with immunography measurements. A second administration of diagnostic or therapeutic monoclonal antibodies might lead to the formation of immune complexes if the quantities of the monoclonal antibodies and the anti-mouse antibodies have an equivalent ratio. A second performance of the radioimmunodetection four to eight weeks after the first radioimmunodetection can achieve better tumor imaging because human anti-mouse antibodies (AMAB) can be formed. Immune complexes of the radioactive antibody and the human anti-mouse antibody (AMAB) can be formed to concentrate radioactivity in the tumor.

Abstract #1402838: Thyroid Stimulating Hormone-Secreting Adenoma: A Rare Case of Hyperthyroidism

TSH-secreting adenomas account for only 0.5 to 3 % of all functioning pituitary tumors. We present this case report to highlight the importance of early diagnosis and prevention of severe complications. A 48-year-old postmenopausal female with a history of hypothyroidism was referred to our clinic for a second opinion. The patient had been stable on levothyroxine for over 10 years. Routine blood work was significant for elevated TSH and free T4. Despite adjustments to her levothyroxine, TSH and free T4 remained elevated. Her prior endocrinologist discontinued the levothyroxine and ordered an MRI brain, which showed an 8mm pituitary adenoma. The patient was referred to a neurosurgeon for evaluation, which then prompted the second endocrinology evaluation. Patient denied any palpitations, weight loss, heat intolerance, headaches, vision changes, or galactorrhea. Lab work repeated off of thyroid supplementation showed persistent elevated TSH at 6.5 mIU/L and free T4 at 2.1 ng/dL. Differential diagnosis included thyroid hormone resistance versus interference to thyroid assays versus TSHoma. The patient tested negative for the thyroid hormone resistance beta mutation, deeming thyroid hormone resistance unlikely. She tested negative for the human anti-mouse antibody, making interference to thyroid assays unlikely. Her alpha subunit level was normal, after which TSHoma was not a certainty. Since the diagnosis was unclear, patient was advised to follow up closely. Repeat MRI showed the tumor was now 1.2cm. Due to the increase in size, she was advised to proceed with trans-sphenoidal surgery (TSS). Pathology stained for TSH, confirming it was a TSH-secreting adenoma. After TSS, TSH normalized and free T4 was low, requiring the patient to be restarted on levothyroxine. TSH-secreting adenomas are quite rare, accounting for less than 1% of all cases of hyperthyroidism. Common clinical features include goiter, visual field defects, headache, menstrual disturbances, and galactorrhea. The presence of high free T4 and T3 and measurable serum TSH in the presence of a macroadenoma on MRI present strong evidence of a TSHoma. Definitive therapy includes transsphenoidal resection. Long-acting somatostatin analogs or dopamine agonists are used to restore euthyroidism prior to surgery. Long-term antithyroid drug therapy or thyroid ablation is not recommended because prolonged decreases in thyroid hormone secretion can stimulate TSH secretion and tumor growth. If TSHomas are left untreated, they can grow aggressively and impinge on nearby structures. Complications include visual field defects due to optic nerve compression, galactorrhea due to pituitary stalk compression, hypopituitarism leading to blood pressure dysregulation and decreased sexual function, and cardiac arrythmias leading to blood clots, stroke, and heart failure. Therefore, when hyperthyroxinemia is present with an elevated TSH, TSHoma should be considered. Even when alpha subunit levels are low or normal, TSHoma is still a possibility. Further investigation and close monitoring should be done to confirm diagnosis and prevent negative outcomes.

Commentary on Severe Hypophosphatemia: The Hidden Truth.

Abnormal phosphate results are common in daily practice, but often neglected by laboratorians and clinicians. This case of severe hypophosphatemia highlights the critical implications that a delayed diagnostic workup of the underlying cause can have. Early diagnosis and treatment could have prevented years of continuous bone loss and immobility. Therefore, severe hypophosphatemia should always trigger additional investigations to find the underlying cause. Determination of the tubular maximum reabsorption of phosphate for glomerular filtration rate was pivotal for establishing renal phosphate wasting and for guiding further diagnostic measures. This rarely used parameter can easily be determined in most laboratories, but pathologists are often unaware of its diagnostic value. Consequently, many laboratories limit themselves to the measurement of timed urinary phosphate excretion, despite the fact that a wide reference interval limits its diagnostic utility. In adult patients with renal phosphate wasting, measurement of fibroblast growth factor-23 (FGF-23) helps to further narrow the spectrum of potential causes. Elevated FGF-23 concentrations are typical in tumor-induced osteomalacia, but normal values can occur when the underlying tumor secretes other phosphatonins or in the presence of interfering heterophile antibodies or human antimouse antibodies that disturb the assay (1).

PDGFRα monoclonal antibody: Assessment of toxicity in juvenile mice administered a murine surrogate antibody of olaratumab.

Olaratumab (Lartruvo™) is a recombinant human IgG1 monoclonal antibody that specifically binds PDGFRα. In order to support use of Lartruvo in pediatric patients, a definitive juvenile animal study in neonatal mice was conducted with a human anti-mouse PDGFRα antibody analog of olaratumab (LSN3338786). A pilot study was used to set doses for the definitive juvenile mouse study. In the definitive study, juvenile mice were administered vehicle, 50, 100, or 150 mg/kg LSN3338786 by subcutaneous (SC) injection every 3 days between postnatal days (PND) 1 and 49, for a total of 17 doses. Blood samples were collected on PND 49 for antibody analysis and toxicokinetic evaluation. Tissues were collected on PND 52 for histopathologic examination. Results of the pilot study indicated that dosing neonatal mice starting on PND 1 via SC administration every 3 days was logistically feasible, produced exposures consistent with prior animal studies, and the selected dose levels were well tolerated by juvenile mice. In the definitive juvenile study, there were no LSN3338786-related deaths, clinical findings, and no effects on mean body weights, body weight gains, or food consumption. Additionally, there were no adverse LSN3338786-related hematology findings, and no macroscopic, organ weight, or microscopic findings of note. The highest dose evaluated, 150 mg/kg, was considered the NOAEL for juvenile toxicity. In conclusion, the juvenile animal studies did not identify any new toxicities or increased sensitivities for the intended pediatric patient population. The use of the surrogate antibody approach in a standard rodent model enabled the de-risking of theoretical concerns for toxicity in pediatric patients due to disruption of the PDGFRα pathway during early human development, such as pulmonary development.

Safety and efficacy of autologous and allogeneic humanized CD19-targeted CAR-T cell therapy for patients with relapsed/refractory B-ALL

BackgroundMurine chimeric antigen receptor T (CAR-T) cell therapy has demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, the potential immunogenicity of the murine single-chain...

Prevalence and pathogenesis of macro-TSH in neonates: Analysis of umbilical cord blood from 939 neonates and their mothers.

Background: Macro-thyroid stimulating hormone (macro-TSH) is a large molecular weight TSH that causes elevated serum TSH concentrations due to its slow clearance. It is primarily a complex of TSH and anti-TSH autoantibodies. The aims of this study were to examine the prevalence and nature of macro-TSH in neonates and to determine how to cope with macro-TSH in neonates suspected to have congenital hypothyroidism through neonatal mass screening. Methods: The presence of macro-TSH was examined using polyethylene glycol (PEG), gel filtration chromatography (GFC), and 125I-TSH binding studies in 939 umbilical cord blood samples from neonates and their mothers. Results: Among 138 serum samples with a PEG precipitation ratio of TSH > 68.9% (mean + SD), human anti-mouse antibodies were found in nine samples. The presence of macro-TSH was examined in the remaining 129 serum samples using a 125I-TSH binding study and GFC. The 125I-TSH binding study revealed that four babies (0.43%) had significantly high ratios of 125I-TSH binding to their sera. Two of the babies were siblings, and their mother and the other two mothers also showed significantly high binding ratios. The 125I-TSH binding was displaced by a large amount (1 µg) of unlabeled human TSH in a similar way between babies and their mothers in all cases, suggesting the presence of anti-TSH autoantibodies in their sera. Further characterization of the autoantibodies in one baby and its mother showed a low affinity and high specificity to human TSH, and the nature was very similar between them. These findings may indicate that the anti-TSH autoantibodies that developed in the mother were transferred to the baby through the placenta and formed macro-TSH by binding to neonatal TSH. GFC revealed macro-TSH in only one baby and its mother, probably because of the dissociation of TSH from autoantibodies during the analytical procedure. Conclusions: Macro-TSH was found in 0.43% of neonates and their mothers all had macro-TSH as well. We recommend that if a baby's serum TSH concentration is high enough to consider levothyroxine treatment suspecting congenital hypothyroidism but the fT4 level is normal, their mother's macro-TSH should be checked.

Analytical performance of the Lumipulse® G β‐Amyloid 1‐40 Plasma and Lumipulse® G β‐Amyloid 1‐42 Plasma RUO assays

AbstractBackgroundCurrent research indicates that plasma β‐amyloid1‐42/β‐amyloid1‐40 ratio has the potential for application in clinical settings and clinical trials to predict brain β‐amyloid burden. It could reduce the number of lumbar punctures (for CSF biomarker testing) or Aβ‐PET scans in light of clinical trial recruitment and could serve as a tool to evaluate target engagement and efficacy of disease‐modifying drugs. The aim of the current study was to demonstrate the analytical performance of the newly developed Lumipulse G β‐Amyloid 1‐40 Plasma and Lumipulse G β‐Amyloid 1‐42 Plasma RUO assays.MethodThe LUMIPULSE G system is a chemiluminescent enzyme immunoassay platform enabling fully automated processing of samples using ready‐to‐use immunoreaction cartridges. Time to result in these single use test cartridges takes about 30 minutes. The analytical performance of the newly developed Lumipulse G β‐Amyloid 1‐40 Plasma and Lumipulse G β‐Amyloid 1‐42 Plasma RUO assays (using respectively 2G3 and 21F12 antibody for capturing and 3D6 antibody ALP‐conjugate for detection; two‐step immunoassay method) was determined. Parameters analysed were precision, sensitivity, linearity, and potential impact of interfering substances. The studies included a series of buffer‐ and K2EDTA plasma‐based samples at different concentration levels, each tested in several replicates (70 µL/replicate (Aβ1‐40); 110 µL/replicate (Aβ1‐42)) sized to the different parameters tested.ResultThe total within‐lab variability of both assays was ≤6 % CV confirming high precision of the LUMIPULSE G system. Using low β‐amyloid concentrated plasma samples, the observed LoD was 0.44 pg/mL (Aβ1‐40) and 0.37 pg/mL (Aβ1‐42), and the LoQ was 0.44 pg/mL (Aβ1‐40) and 0.43 pg/mL (Aβ1‐42). Linearity was shown across the assay range (0 – 5000 pg/mL (Aβ1‐40); 0 – 1000 pg/mL (Aβ1‐42)). No significant impact of commonly tested endogenous substances (bilirubin, chyle, triglycerides, rheumatoid factor (RF) and human anti‐mouse antibodies (HAMA)) was observed, except for haemoglobin.ConclusionThe analytical performance studies demonstrate – amongst other characteristics – low variability and high sensitivity enabling measurement of Aβ1‐40 and Aβ1‐42 in plasma. The Lumipulse G β‐Amyloid 1‐40 Plasma and Lumipulse G β‐Amyloid 1‐42 Plasma RUO assays are now ready to be explored further for clinical utility in various contexts of use.

Updated Results and Correlative Analysis: Autologous CD30.CAR-T-Cell Therapy in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma (CHARIOT Trial)

Introduction Early results from the Pilot segment of a Phase 2 trial of autologous CD30.CAR-T in patients with relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL) (NCT#04268706) demonstrated a favorable safety profile and excellent anti-tumor responses of CD30.CAR-T in patients with heavily pretreated r/r cHL (Ahmed et al., 2021). We report here updated clinical data and exploratory biomarker analysis from the Pilot segment. Methods This is a Phase 2 single arm, multi-center study, enrolling patients (12-75 years) with cHL progression after at least 3 lines of therapy, including chemotherapy, brentuximab vedotin, and anti-programmed cell death (PD)-1 antibodies. The pilot segment of this study has fully enrolled with safety as the primary endpoint and overall response rate (ORR) as assessed centrally according to Lugano Classification Revised Response System for malignant lymphoma (Cheson et al., 2014) as a secondary endpoint. The patients were treated with CD30.CAR-T cells, after lymphodepletion chemotherapy using bendamustine and fludarabine, with an allowable dose range of 2.0 to 2.7 × 108 CD30.CAR-T cells per m2. Second CD30.CAR-T infusion is allowed if the patients show progression after initial response to first CD30.CAR-T infusion. Exploratory objectives include assessment of expansion and persistence of autologous CD30.CAR-T, cytokine profiling, immunological parameters, circulating tumor DNA (ctDNA), and other biomarkers in blood and tumor tissue. Results As of 22 July 2022, 17 patients were screened and 15 patients enrolled in the Pilot segment (median age: 35 years [21-57], 66.7% male). The median number of prior therapies was 6 (range: 3-18). All 15 patients were treated with CD30.CAR-T single infusion, and 7 patients received a second CD30.CAR-T infusion. ORR after CD30.CAR-T single infusion was 73.3% (n=11) with complete response (CR) rate of 60% (n=9). ORR in 5 patients with available response assessment after the second infusion was 100%, with CR of 60% (n= 3). CD30.CAR-T treatment was well tolerated with one grade 1 Cytokine Release Syndrome (CRS) and no neurotoxicity. CD30.CAR-T cells in peripheral blood peaked at 7 days after first CAR-T infusion with median Cmax (peak exposure) of 6964.7 copies/µg DNA.There was no correlation observed between response and Cmax, or AUC (area under the curve) 0-42 days, 0-90 days, 0-180 days after the first CD30.CAR-T infusion. Biopsies obtained at the time of screening and relapse were reviewed by an independent pathologist with 100% concordance of CD30 positivity between local and central testing. HAMA (Human Anti-Mouse Antibody) in serum was negative after first and secondCD30.CAR-T infusion in all patients. An increase of the homeostatic cytokines interleukin (IL)-7 and IL-15 was detected after lymphodepletion chemotherapy. Serum CCL17 (Thymus and Activation Regulated Chemokine; TARC), a predictive marker of early response assessment in HL (Guidetti et al., 2017) was elevated before CD30.CAR-T infusion and had a dramatic decrease after treatment in responding patients. Conclusions Overall, CD30.CAR-T therapy was well tolerated with no unexpected safety signal and showed excellent anti-tumor efficacy with an ORR of 73.3% in heavily pretreated r/r cHL patients. The CD30.CAR-T cells showed good expansion and persistence after infusion. The efficacy, safety and exploratory biomarkers of CD30.CAR-T will be further evaluated in the Pivotal segment of this Phase 2 study.

RF35 | PSAT304 A Case of COVID-19 Infection; COVID-19 mRNA-Based Vaccine Induced Human Anti-Mouse Antibodies (HAMA) Interfering With TSH Testing

Abstract A 71-year-old male presented to the endocrine clinic for evaluation of hypothyroidism which has recently been difficult for his primary care physician to control. He initially developed hypothyroidism in 1990 after neck radiation for squamous cell carcinoma of his sternocleidomastoid muscle. He was previously controlled for many years on levothyroxine 150mcg daily with TSH readings in the normal reference range. Patient was infected with COVID-19 in 2020 and was hospitalized for 10 days. He was treated with 5 days of Remdesivir and 10 days of Decadron. 6 months after the infection he received mRNA COVID-19 vaccination. Upon his first visit to the endocrine clinic post COVID-19 infection and vaccination, his TSH was elevated at 90 uIU/mL with a free thyroxine that was elevated at 1.8 ng/dL even after multiple attempts by his primary care physician to increase his Levothyroxine which was up to 225 mcg daily before he was referred to endocrinology. He is compliant with his levothyroxine and takes it on an empty stomach first thing in the morning without any of his other medications. He is not taking iron supplements, biotin or any other supplements or new medications. TSH values had been normal 1 month prior to his COVID-19 infection and was first noted to be abnormal 8 months after his COVID-19 infection and 2 months after his mRNA COVID-19 vaccine. There was no TSH drawn between when he had COVID-19 and when he received his vaccine. It was suspected that he may have a factitious elevation in his TSH due to antibody interference. Labs were drawn for a TSH with human anti-mouse antibodies (HAMA) treatment which resulted with a markedly suppressed TSH at 0.007 uIU/mL with an elevated free thyroxine of 1.94 ng/dL. HAMA antibodies came back elevated at 552 ng/mL. Reference Klee, G., 2000. Human Anti-Mouse Antibodies. Archives of Pathology & Laboratory Medicine, 124(6), pp.921-923. Koshida, S., Asanuma, K., Kuribayashi, K., Goto, M., Tsuji, N., Kobayashi, D., Tanaka, M. and Watanabe, N., 2010. Prevalence of human anti-mouse antibodies (HAMAs) in routine examinations. Clinica Chimica Acta, 411(5-6), pp.391-394. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 1:00 p.m. - 1:05 p.m.

RF23 | PSAT300 The Curious Case of Abnormal Thyroid Function Tests: Think Streptavidin Interference

Abstract Interpretation of abnormal thyroid function tests (TFTs) is routine in endocrine practice. However, when there is a discrepancy between biochemical parameters and the clinical picture, sleuthing is necessary to provide appropriate care.A 34-year-old woman was evaluated by an outside endocrine clinic for abnormal TFTs in the setting of worsening anxiety, night sweats, and menstrual irregularities. These showed elevated free thyroxine (FT4) levels (7.3; ref: 0.9-1.7 ng/dL), free triiodothyronine (FT3) levels (13.9; ref: 2.3-4.1 pg/mL) and total triiodothyronine (T3) (186; ref: 94-170 ng/dL) with normal TSH (0.65; ref: 0.27-4.20 mIU/L), negative microsomal and thyroid receptor antibodies. She denied use of contraceptives, biotin, lithium, glucocorticoids, prior radiation, or recent intravenous contrast. Mother and maternal aunt had unspecified thyroid disease. No physical signs of overt hyperthyroidism/hypothyroidism were noted. Our workup also showed elevated FT4 (1.8 ng/dL), FT3 (13.3 pg/mL), total thyroxine (T4) (24.8; ref: 5.5-10.2 µg/dL), T3 (>651; ref: 79-165 ng/dL), normal TSH (0.71; ref: 0.4-5.5 mIU/L), and normal gonadotropin alpha subunit; ultrasound showed no nodules nor evidence of thyroiditis. FT4 by equilibrium dialysis and liquid chromatography-mass spectrometry was normal (1.3; ref: 0.8-2.0 ng/dL). Human anti-mouse antibodies (HAMA) treated TSH was within normal limits (1.4; ref: 0.4-4.5 mIU/L; Siemens). Leading differential diagnosis was initially thyroid hormone resistance; however subsequent labs, done by PCP for non-specific symptoms, revealed a suppressed parathyroid hormone (PTH) (<6; ref 15-65 pg/mL) with normal serum calcium levels. Given no clinical correlate, lab interference was considered. All the testing, with the exception of the FT4 by mass spectrometry and HAMA TSH, were analyzed utilizing Roche Diagnostics (electrochemiluminescent immunoassays). To evaluate vendor-specific interferences, labs were analyzed utilizing Siemens (chemiluminescent immunoassay) which showed normal T4, T3, FT3, and PTH levels. Unlike the Siemens assays, Roche depends on the formation of a biotin-streptavidin complex during measurement and a ruthenium complex to produce a signal. HAMA and polyethylene glycol treatment also returned differing results from untreated samples possibly indicating the interference was a heterophilic antibody to an unknown target. An investigation by Roche Diagnostics showed evidence of streptavidin antibodies. Streptavidin is a common reagent in endocrine assays. The endogenous streptavidin antibody produces an unintended competition between streptavidin-biotin and streptavidin-antibody in the assay; resulting in falsely-low results for sandwich assays (e.g. PTH) and falsely-high results for competitive assays (e.g. FT3 and T4). The impact to the individual analyte is variable due to assay formulation (e.g. TSH).Interference from anti-streptavidin antibodies is a rare phenomenon and may cause unnecessary diagnostics or treatment. Analytical interference should be considered when clinically suspicious of results, and lab consultation is recommended. The patient was advised to obtain a Medic-Alert bracelet. She was also provided a letter detailing the findings so that providers may consult the laboratory prior to testing. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m.

PSAT329 A Delayed Diagnosis of Subclinical Hypothyroidism Due to Interferences with Thyroid Function Immunoassays

Abstract Introduction Automated immunoassays used to evaluate thyroid function tests (TFTs) are susceptible to various forms of interference that can lead to inaccurate results and misdiagnosis. Case Description A 55-year-old male was diagnosed with abnormal TFTs since 2017 [TSH 7.32 mIU/L (range 0.4-4.5), FT3 5.1 pg/mL (range 2.3-4.2), and FT4 1.2 ng/dL (range 0.9-2.2)]. Thyroid peroxidase antibody and thyroglobulin antibody were positive. He was on levothyroxine 50 mcg daily since 2017, and he did not feel significantly different despite treatment. He was referred to endocrine clinic in 2021. He had no symptoms of hypothyroidism or hyperthyroidism except occasional palpitations and headache. He reported blurred vision due to cataracts. He denied thyroid diseases in first degree relatives. He denied taking biotin or desiccated thyroid extract. Physical examination was unremarkable. Differentials included TSH-producing adenoma, assay interference, exogenous T3 use, T3 thyrotoxicosis with interference of TSH assay, and thyroid hormone resistance. Levothyroxine was discontinued in May 2021. Other pituitary hormones including alpha-subunit and MRI pituitary were unremarkable, which ruled out TSH-producing adenoma. Human anti mouse antibody (HAMA) was normal. In June 2021, prior to seeing genetics, FT4, FT3 and Total T3 were checked with equilibrium dialysis method and values were 1.2 ng/dL (range 0.9-2.2), 238 pg/dL (range 210-440) and 105 ng/dL (range 76-181), respectively and TSH 9.13 mIU/L (range 0.40-4.50), thyroglobulin 0.6 ng/ml (range 2.8-40.9), reverse T3 LC/MS/MS 12 ng/dL (range 8-25). Labs were therefore consistent with subclinical hypothyroidism, and he was started back on levothyroxine 75 mcg every morning. Discussion TFTs are usually measured by automated immunoassays, and this will have accurate results in most cases. However, immunoassays are vulnerable to interferences that can affect clinical decisions. It is important to consider assay interference such as this and to confirm with equilibrium dialysis method before pursuing further aggressive workup. Conclusion Potential interferences in TFTs should always be suspected whenever discrepancies arise. Awareness of these conditions is critical to the institution of appropriate therapy and preventing misdiagnosis. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.