Human Growth Hormone Receptor Research Articles

Site-Selective Zwitterionic Poly(caprolactone-carboxybetaine)-Growth Hormone Receptor Antagonist Conjugate: Synthesis and Biological Evaluation.

Zwitterionic polymers have been found to be biocompatible alternatives to poly(ethylene glycol) (PEG) for conjugation to proteins. This work reports the site-selective conjugation of poly(caprolactone-carboxybetaine) (pCLZ) to human growth hormone receptor antagonist (GHA) B2036-alkyne and investigation of safety, activity, and pharmacokinetics. Azide-end-functionalized pCLZs were synthesized and conjugated to GHA B2036-alkyne via copper-catalyzed click reaction. The resulting inhibitory bioactivity concentration responses in Ba/F3-GHR cells were compared to those of PEGylated GHA B2036. IgG and IgM antibody production was tested in mice, and no measurable antibody or cytokine production was detected for the pCLZ conjugate. Using 18F-labeled PET/CT imaging, the pCLZ conjugate showed an increase in circulation time compared to that of GHA B2036. Acute toxicity of the polymer was investigated in vivo and found to be nontoxic. Ex vivo degradation of the polymer on the conjugate was investigated. The results suggest that pCLZ-GHA is a potentially safe alternative to PEG-GHA.

6459 A Novel Long-Acting GH Receptor Antagonist Optimizing Therapeutic Efficacy and Duration

Abstract Disclosure: C. Ku: None. C. Kang: None. J. Oh: None. E. Wang: None. H. Song: None. K. Park: None. B. Kim: None. K. Kim: None. S. You: None. S. Park: None. E. Lee: None. Background: Forty to sixty percent of acromegalic patients undergo medical treatment, utilizing somatostatin analogues, dopamine agonists, and GH receptor (GHR) antagonists. The GHR antagonist, pegvisomant exhibits the most robust efficacy in reducing IGF-1 levels in acromegalic patients. However, pegvisomant faces several clinical limitations, such as poor compliance due to the necessity of daily injections. This study aimed to develop a novel, long-acting fusion GHR antagonist with optimized therapeutic efficacy and duration. Method: Twelve mutant proteins with alterations in the C-terminal and N-terminal regions of GHR were generated to confirm the GHR binding affinity and inhibitory potency compared with pegvisomant, which was evaluated with STAT5b luciferase reporter assay. Several carriers were engineered into selected mutant GHRs to increase the therapeutic duration. For in vivo experiments, single or repetitive injection of GHR antagonists were applied to acromegalic mouse model (somatotroph specific Aip knock out (sAIPKO) mice). Pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated in New Zealand White (NZW) rabbits. Result: Four out of the 12 GHR mutants exhibited higher affinity and stronger inhibitory potency toward human GHR when compared to pegvisomant. Specifically, three GHR mutants significantly reduced serum IGF-1 levels in sAIPKO mice following daily injections of 20 mg/kg for 7 days (52.3±6.0 to 5.9±1.3, 50.3±3.5 to 32.3±2.3, and 49.8±3.7 to 13.9±4.3 ng/mL; all p < 0.05). Among them, S4020N exhibited the lowest EC50 value for GHR binding affinity in in vitro analysis containing human GHR (0.594 nM) and mouse GHR (0.891 nM). Following fusion with each long-acting carrier, S4020N with the Fc carrier protein (ALT-B5) demonstrated the highest binding affinity and the strongest inhibitory potency toward human GHR, surpassing pegvisomant by 14 and 25 times, respectively. Administration of ALT-B5 (80 mg/kg) and pegvisomant (24 mg/kg) at a 3-day interval revealed the superior efficacy of ALT-B5 over pegvisomant (1879±219.4 vs. 3800±355.9 ng*hr/mL IGF-1 AUC for 5 days in ALT-B5 and pegvisomant, respectively; p < 0.01). In PK/PD studies, ALT-B5 was administered at doses of 3 mg/kg and 10 mg/kg in NZW rabbits, and PK/PD parameters were compared with the same molar concentration of pegvisomant. ALT-B5 exhibited the longest therapeutic duration and the highest efficacy compared to pegvisomant. Conclusion: ALT-B5 exhibited the longest duration and highest effectiveness in lowering IGF-1 levels. ALT-B5 emerges as a potential novel therapeutic option for acromegaly. Presentation: 6/1/2024

Race/ethnicity and human epidermal growth hormone receptor 2–targeted therapy for breast cancer.

91 Background: Breast cancer is the most common cancer in the US. Up to 25% of breast cancers are classified as human epidermal growth factor receptor 2 (HER2)-positive and associated with an aggressive tumor burden. HER2-targeting monoclonal antibodies improves survival in patients with HER2-positive breast cancer. Prior work has demonstrated disparities in access to oncologic therapies among older women. Our study seeks to examine racial/ethnic disparities in receiving HER2-targeted therapies for patients with HER2-positive breast cancer and trends in use over time. Methods: In the Surveillance, Epidemiology and End Results (SEER) Medicare-linked database we identified women diagnosed with breast cancer from 2010 to 2019. We included those >66 years old who survived 12 months after diagnosis, were enrolled in Medicare Parts A/B for 12 months pre and post-diagnosis, and had local or regional stage HER2-positive cancer. Our primary outcome was receipt of HER2-targeted therapies in the 12 months after diagnosis. Our independent variable was race/ethnicity as measured by the validated Research Triangle Institute race/ethnicity codes in Medicare. Due to small sample sizes, we focus our analysis on White, Black, or Hispanic women. We used multivariable Modified Poisson regression to evaluate changes in the probability of HER2-targeted therapy receipt by race/ethnicity over time, adjusted for sociodemographics, cancer factors, and medical comorbidities. Results: Our cohort comprised 13,887 patients including 8.7% Black, 7.5% Hispanic, and 83.8% White (mean age 74.9). Overall, 7,351 (52.9%) received HER2-targeted therapies in the 12 months post-diagnosis, with use increasing over time in all racial/ethnic groups (Table). Compared with White patients, Black and Hispanic patients had a 19% (adjusted risk ratio [aRR] 0.81, 95% CI:0.69-0.96) and 27% (aRR 0.73, 95% CI 0.61-0.89) lower likelihood, respectively, of receiving HER2-targeted therapies from 2010-2011 (Table). By 2018-2019, no significant differences in receiving HER2-targeted therapies were observed across racial/ethnic groups. Conclusions: In a national cohort of Medicare enrollees with HER2-positive breast cancer, we observed significant racial/ethnic disparities which narrowed over time. Overall utilization rates were low. Future work is needed to understand how improved access to breast cancer treatments impact downstream outcomes. Use of human epidermal growth hormone receptor 2–targeted therapy from 2010-2011 and 2018-2019 by race/ethnicity. Race and Ethnicity Unadjusted Rates Adjusted Risk Ratios (95% CI)* 2010-2011 2018-2019 2010-2011 2018-2019 Black 36% 62% 0.81 (0.69-0.96) 0.97 (0.87-1.08) Hispanic 31% 68% 0.73 (0.61-0.89) 1.01 (0.92-1.12) White 42% 64% Reference Reference *Adjusted for age, marital status, region, breast cancer stage and year of diagnosis, and medical comorbidities.

Unveiling the Molecular Mechanism of Trastuzumab Resistance in SKBR3 and BT474 Cell Lines for HER2 Positive Breast Cancer.

HER2-positive breast cancer is one of the most prevalent forms of cancer among women worldwide. Generally, the molecular characteristics of this breast cancer include activation of human epidermal growth factor receptor-2 (HER2) and hormone receptor activation. HER2-positive is associated with a higher death rate, which led to the development of a monoclonal antibody called trastuzumab, specifically targeting HER2. The success rate of HER2-positive breast cancer treatment has been increased; however, drug resistance remains a challenge. This fact motivated us to explore the underlying molecular mechanisms of trastuzumab resistance. For this purpose, a two-fold approach was taken by considering well-known breast cancer cell lines SKBR3 and BT474. In the first fold, trastuzumab treatment doses were optimized separately for both cell lines. This was done based on the proliferation rate of cells in response to a wide variety of medication dosages. Thereafter, each cell line was cultivated with a steady dosage of herceptin for several months. During this period, six time points were selected for further in vitro analysis, ranging from the untreated cell line at the beginning to a fully resistant cell line at the end of the experiment. In the second fold, nucleic acids were extracted for further high throughput-based microarray experiments of gene and microRNA expression. Such expression data were further analyzed in order to infer the molecular mechanisms involved in the underlying development of trastuzumab resistance. In the list of differentially expressed genes and miRNAs, multiple genes (e.g., BIRC5, E2F1, TFRC, and USP1) and miRNAs (e.g., hsa miR 574 3p, hsa miR 4530, and hsa miR 197 3p) responsible for trastuzumab resistance were found. Downstream analysis showed that TFRC, E2F1, and USP1 were also targeted by hsa-miR-8485. Moreover, it indicated that miR-4701-5p was highly expressed as compared to TFRC in the SKBR3 cell line. These results unveil key genes and miRNAs as molecular regulators for trastuzumab resistance.

Clinical characteristics and prognostic factors of breast cancer patients with tumor deposits in the ipsilateral axillary region

Objective: To investigate the clinicopathologic features and prognostic factors of breast cancer patients with tumor deposits in the ipsilateral axillary region. Methods: We retrospectively analyzed the clinicopathologic data and follow-up results of 155 patients with breast cancer diagnosed for the first time and complicated with tumor deposits in the ipsilateral axillary region in the Department of Thyroid-Breast-Vascular Surgery of Xijing Hospital from January 2008 to September 2018. Kaplan-Meier method was used for survival analysis. Log rank test was used for the univariate analysis of prognostic factors, and Cox regression was used for multivariate analysis. Results: The median disease free survival (DFS), median distant metastasis free survival (DMFS), and median overall survival (OS) of the 155 patients were 52.0 months, 66.6 months, and 102.2 months, respectively. The 5-year and 10-year DFS rates were 45.7% and 23.1%, the 5-year and 10-year DMFS rates were 56.9% and 28.9%, and the 5-year and 10-year OS rates were 79.3% and 46.0%, respectively. Multivariate Cox regression analysis showed that family tumor history (HR=0.362, 95% CI: 0.140-0.937), clinical T stage (T3: HR=3.508, 95% CI: 1.380-8.918; T4: HR=2.220, 95% CI: 1.076-4.580), estrogen/progesterone receptor status (HR=0.476, 95% CI: 0.261-0.866), number of tumor deposits (HR=1.965, 95% CI:1.104-3.500) and neoadjuvant chemotherapy (HR=1.961, 95% CI: 1.032-3.725) were independent influencing factors for DFS. Molecular subtype [human epidermal growth factor receptor-2(HER-2) positive and hormone receptor negative: HR=7.862, 95% CI: 3.189-19.379], number of tumor deposits (HR=2.155, 95% CI: 1.103-4.212), neoadjuvant chemotherapy (HR=5.002, 95% CI: 2.300-10.880) and radiotherapy (HR=2.316, 95% CI: 1.005-5.341) were independent influencing factors of DMFS. Histological grade (HR=4.362, 95% CI: 1.932-9.849), estrogen/progesterone receptor expression (HR=0.399, 95% CI: 0.168-0.945), HER-2 expression (HR=2.535, 95% CI: 1.114-5.768) and neoadjuvant chemotherapy (HR=4.080, 95% CI: 1.679-9.913) were independent influencing factors of OS. Conclusions: The presence of tumor deposits weakens the influence of axillary lymph node status and distant metastases on the prognosis of breast cancer patients. Therefore, a clinicopathological staging system taking into account tumor deposits should be developed. Since the number of tumor deposits affects the risk of recurrence and metastasis of breast cancer patients, we recommend that the number of tumor deposits should be reported in detail in the pathological report after breast cancer surgery.

THU046 Repeated Treatment With AZP-3813, A Bicyclic, 16-Amino Acid Peptide Antagonist Of The Human Growth Hormone Receptor, Induces Enhanced Suppression Of IGF1 In Beagle Dogs

Abstract Disclosure: G. Ravel: None. C. Chalmey: None. C. Berardet: None. D. Duracher: None. H. Kurasaki: None. T. Tomiyama: None. P. Reid: None. M.D. Culler: None. Medical treatment of acromegaly is based on either suppressing pituitary growth hormone (GH) secretion or inhibiting GH action by preventing interaction with its receptor in order to suppress the elevated levels of insulin-like growth factor 1 (IGF1). AZP-3813 is a 16-amino acid, bicyclic peptide antagonist of the GH receptor (GHR) derived from peptide sequences discovered using a unique, cell-free in vitro transcription-translation system screened against the human GHR, and optimized by rational design to increase binding affinity, solubility and half-life. The KD of AZP-3813 for the human GHR is 1.9nM. In previous studies, AZP-3813 was demonstrated to suppress IGF1 secretion in juvenile rats in a dose-related manner, and to maintain IGF1 suppression when given daily for extended periods. Recently, we demonstrated that AZP-3813 is also very effective in suppressing IGF1 in normal Beagle dogs. Dogs receiving a single 0.1, 1 and 10 mg/kg subcutaneous injection of AZP-3813 responded with a dose-related 2.2 + 7.1, 21.4 + 2.7 and 27.8 + 1.5% decrease in IGF1 after 24 hours, respectively, and the decrease in IGF1 attained with a single 10 mg/kg dose was maintained for over 72 hours. The relatively small further increase in IGF1 suppression with 10 versus 1 mg/kg suggests that 10 mg/kg AZP-3813 may induce near maximal GHR antagonism. To examine the effect of repeated and higher dose treatment of dogs with AZP-3813, young adult, non-fasted, Beagle dogs were injected subcutaneously at rotated dorsal sites with AZP-3813 at doses of 10, 30 or 60 mg/kg (n=2, one male and one female) daily for seven days. Blood samples, collected prior to dosing to establish baseline IGF1 levels and 24 hour after the final, seventh AZP-3813 injection, were assayed for total IGF1 content by radioimmunoassay. Baseline IGF1 levels were lower in the female dogs (136.7 + 36.1 ng/ml) than in the male dogs (197.0 + 11.3 ng/ml); however, the percent IGF1 suppression resulting from AZP-3813 treatment was similar for both sexes. AZP-3813, injected for seven days at 10, 30 and 60 mg/kg, induced highly significant 69.0 + 4.0, 70.5 + 9.5 and 75.0 + 1.0% decreases in IGF1, respectively. The similar magnitude of IGF1 suppression attained with the three doses of AZP-3813 implies that maximal GH antagonism is essentially achieved with the 10 mg/kg dose. The substantial increase in IGF1 suppression achieved by repeated 10 mg/kg injection of AZP-3813, compared to the previously observed suppression after a single 10 mg/kg injection, likely represents compound accumulation (under investigation) as well as a progressive effect of maximal GH antagonism on IGF1 production. These results demonstrate that the potent GHR antagonist activity of AZP-3813 translates to a highly effective suppression of IGF1 in normal dogs and further support the development of AZP-3813 as a potential therapy for acromegaly. Presentation: Thursday, June 15, 2023

THU048 Determination Of Receptor Binding Affinity Of Growth Hormone Analogues To The Growth Hormone Receptor In A Drug Screening Context

Abstract Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023

Dose-dense paclitaxel plus carboplatin in combination with trastuzumab neoadjuvant versus standard adjuvant therapy in human epidermal growth factor receptor-2 positive and hormone receptor negative breast cancer: a prospective cohort study

Objective: To provide survival evidence of anthracycline-free neoadjuvant chemotherapy for patients with stages Ⅱ-Ⅲ human epidermal growth factor receptor-2 (HER-2) positive and hormone receptor (HR) negative breast cancer. Methods: The prospective cohort study was conducted at the Department of Medical Oncology of Cancer Hospital, Chinese Academy of Medical Sciences. Patients with HER-2 positive and HR negative breast cancer in stages Ⅱ-Ⅲ were enrolled to receive neoadjuvant therapy (NAT) of dose-dense paclitaxel (175 mg/m(2)) plus carboplatin (AUC=4.0) biweekly for 6 cycles in combination with trastuzumab (PCbH), and matched patients who received standard adjuvant therapy of physicians' choice were recruited for survival and safety comparison. Results: From July 2013 to November 2019, 166 patients were included (neoadjuvant 51, adjuvant 115). Compared with those who received adjuvant therapy, patients receiving NAT were younger (<35 years: 19.6% vs 5.2%, P=0.014), had larger tumors (T3: 62.7% vs 7.8%, P<0.001) and more advanced diseases (stage ⅡA: 2.0% vs 41.7%, P<0.001). Patients in the neoadjuvant group all received surgery, and 96 (83.5%) in the adjuvant group received anthracycline-and-taxane-containing regimens. A total of 98 patients (49 pairs) were matched, and the covariates between the two groups were acceptably balanced. Within a median follow-up of 46.5 (range, 14-87) months, the 4-year recurrence-free survival (RFS) rate among patients who received NAT was 73.3% (95% CI: 59.0%-87.6%), versus 80.6% (95% CI: 67.9%-93.3%) among those in the adjuvant group without statistical difference (P=0.418). A similar result was observed for the 4-year overall survival (OS) [neoadjuvant versus adjuvant: 91.5% (95% CI: 81.7%-100.0%) vs 97.8% (95% CI: 93.5%-100.0%), P=0.314]. Compared with standard adjuvant therapy, PCbH was related to less neutropenia and better cardiac safety. Conclusions: These results support the consideration of anthracycline-free neoadjuvant chemotherapy combined with anti-HER-2 therapy for patients with stages Ⅱ-Ⅲ HER-2-positive and HR-negative breast cancer. Optimized regimens with both efficacy and safety are needed and to be further investigated.

Growth hormone receptor agonists and antagonists: from protein expression and purification to long-acting formulations.

Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarise methodologies and approaches used for making and purifying recombinant GH and GHA protein, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed. This article is protected by copyright. All rights reserved.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates

Human growth hormone (hGH) is a pituitary derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently there is a single FDA approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost three decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions - dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific poly-ethylene-glycol (PEG) conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and PRLR. Moreover, these novel hGHR antagonists display distinct antagonism of GH induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anti-cancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH associated pathologies.

AZP-3813, a bicyclic, 16-amino acid peptide antagonist of the human growth hormone receptor, effectively suppresses IGF1 in beagle dogs

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract 967: Real-world transcriptomic biomarkers as replacement for immunohistochemistry and FISH studies in breast cancer

Abstract Background: Human epidermal growth factor receptor-2 (HER2) and hormone receptors are typically used as binary biomarkers for selecting breast cancer therapy. There is a need to explore the clinical relevance of these biomarkers as continuous variables. This is particularly relevant for the new class of antibody-drug conjugates (ADC), in which a relatively low HER2 expression level is adequate for targeting tumor cells. We explored the potential of RNA profiling, determined by next generation sequencing (NGS), to provide more flexible clinical biomarkers as compared with immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH). Methods: Clinical and laboratory data from 57 breast cancers collected by the COTA real-world data company were used to study biomarker levels as detected by routine clinical transcriptomic tests. HER2 (ERBB2), estrogen receptor alpha (ESR1), and androgen receptor (AR) mRNA levels were compared with reported HER2 and estrogen receptor (ER) IHC and FISH results. Results: RNA levels accurately reflected and predicted HER2 amplification (see table below). Importantly, RNA data showed significant variation and overlap in the levels of ERBB2 mRNA between cases scored by IHC as zero, 1+, and 2+. This variation correlated with progression-free survival (PFS). Similarly, the ESR1 RNA levels accurately reflected ER status and demonstrated significant variation between positive cases. RNA data also showed significant variations in the AR levels. Patients wssith high AR mRNA levels had significantly better PFS (P=0.05). Patients expressing high ER and AR levels had significantly better PFS than those expressing low ESR1 and AR levels (P=0.03). Conclusions: These findings suggest that RNA analysis using NGS is an alternative to IHC and FISH. RNA provides continuous data that can determine cut-off points predicting response to therapy and should be explored in predicting ADC response. ERBB2 mRNA levels (FPKM) in various HER2 IHC groupss IHC Score Valid N Mean Median Minimum Maximum Quartile Range Std.Dev. Zero 19 231 243 63 537 170 110 Zero vs one 3 151 155 45 253 208 104 Zero to one vs one 18 397 302 172 845 365 222 One Vs two 13 495 423 159 1094 273 284 Two vs three 4 7523 7649 937 13859 7183 5310 Citation Format: Maher Albitar, Andre Goy, Andrew Pecora, Deena Graham, Donna Donna McNamara, Ahmad Ahmad Charifa, Andrew IP, Wanlong Ma, Stanley Waintraub. Real-world transcriptomic biomarkers as replacement for immunohistochemistry and FISH studies in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 967.

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding.

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

Clinical significance and prognostic value of receptor conversion after neoadjuvant chemotherapy in breast cancer patients.

The hormone receptor (HR) status and human epidermal growth hormone receptor 2 (HER2) status of patients with breast cancer may change following neoadjuvant chemotherapy (NAC). We retrospectively analyzed the clinical data of 294 patients with stage II/III breast cancer to evaluate the clinical significance and prognostic value of receptor transformation after NAC in breast cancer patients. Pathological complete response after NAC was achieved in 10.7% of patients. HR, estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67 conversion rates were 9.2%, 6.5%, 13.0%, 4.4%, and 33.7%, respectively. Patients with stable HR (P = 0.01) and HER2 (P = 0.048) expression had more favorable overall survival (OS). Low or reduced Ki-67 expression was associated with better disease-free survival (DFS) (P < 0.001) and OS (P < 0.01). Multivariate analysis showed that the number of lymph nodes after NAC, HR conversion, and radiotherapy were independent prognostic factors for overall survival. HR conversion implied a higher risk of death [hazard ratio, 2.56 (95% confidence interval: 1.19-5.51); P = 0.016]. Patients with HR conversion after NAC who received endocrine therapy had better DFS (P = 0.674) and OS (P = 0.363) than those who did not receive endocrine therapy, even if the HR changed from positive to negative. In conclusion, pathological testing should be performed before and after NAC, and even patients with HR conversion after NAC might benefit from endocrine therapy.

ODP353 Sustained Suppression of IGF1 with AZP-3813, a Bicyclic 16-Amino Acid Peptide Antagonist of the Human Growth Hormone Receptor and a Potential New Treatment for Acromegaly

Abstract Medical treatment of acromegaly is based on either suppressing pituitary GH secretion or inhibiting GH action by preventing interaction with its receptor in order to suppress the elevated levels of IGF1. AZP-3813 is a 16-amino acid, bicyclic peptide antagonist of the GH receptor (GHR) with a KD of 1.9nM for the human GHR, and 18.5nM for the rat GHR. The circulating half-life of AZP-3813 in the rat is 11.2 hours. In previous studies, AZP-3813 was demonstrated to suppress IGF1 secretion in juvenile rats in a dose-related manner, and to maintain IGF1 suppression when given over 4 days. To examine the ability of chronic AZP-3813 treatment to maintain suppression of IGF1 levels, we injected normal, 5-week old (∼150g), male Sprague Dawley rats subcutaneously either with vehicle or with AZP-3813 at doses of either 10 or 30mg/kg QD for 19 days (n=7/group). Blood samples were collected immediately prior to either vehicle or AZP-3813 injection on days 1, 2, 3, 7, 10, 15 and 19, and 24h after the last injection (day 20). Samples were assayed for total IGF1 content by radioimmunoassay, and samples collected on days 2, 10 and 19 were also analyzed for AZP-3813 content by LC-MS/MS. At the initiation of treatment, the average body weight of the rats was 150.3 + 3.0g, and over the 20 days of the study animals treated with vehicle gained an average of 158.4 + 4.9g. In contrast, rats treated QD with 10 or 30mg/kg AZP-3813 gained 23.6 + 2.1 and 27.9 + 2.2% less weight than the vehicle-treated controls, respectively. Accumulation of AZP-3813 levels in the blood was observed with repeated daily injections of both 10 and 30mg/kg, increasing from day 2 levels by 107.1 + 12.5 and 125.2 + 36.3%, respectively, by day 19. In the vehicle-treated control rats, IGF1 levels increased from an initial 395.1 +16.3ng/ml to 674.4 + 19.8ng/ml by day 20. Twenty-four hours after the first injections of 10 and 30mg/kg AZP-3813, IGF1 levels were suppressed by 27.6 + 3.4 and 29.2 + 3.3%, respectively, versus vehicle-treated control levels, and, with continued treatment, a similar magnitude of IGF1 suppression was maintained through day 20 despite the progressively increasing levels of IGF1 observed in the vehicle-treated rats. At study termination on day 20, animals treated with 10 and 30mg/kg AZP-3813 were observed to have 4.1 and 4.6% shorter femur length, 1.1 and 3.6% shorter body length, and 7.7 and 13.3% less liver weight, respectively, as compared with vehicle-treated control rats. These results demonstrate that with continued treatment, the potent GHR antagonist activity exhibited by AZP-3813 translates to sustained in vivo suppression of IGF1 levels and associated parameters, and support its development as a potential therapy for acromegaly. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:05 p.m. - 1:10 p.m.

Treatment patterns and medical costs of metastatic breast cancer care in the United States.

e18834 Background: Substantial advances in treatment of metastatic breast cancer (mBC) have led to changes in clinical practice and treatment costs in the United States in the past decade. We aimed to identify treatment patterns in mBC patients by subgroup and year after mBC diagnosis and associated costs from payer and societal perspectives. Methods: This study used the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database (1/2011-5/2021). We excluded cases that were male, had &lt; 6 months of follow up, no treatment data, no structured data within 90 days of diagnosis, participated in clinical trials, had non-breast invasive cancers, received therapies not used in mBC, had no data on human epidermal growth factor (HER2) and hormone receptor (HR) status. We classified patients into four subgroups: HER2-HR+, HER2+HR+, HER2+HR-, and Triple Negative (TNBC). We calculated anti-cancer and supportive drug costs at the patient level and mean costs by year after diagnosis for each subgroup. We used average wholesale prices (AWP, from McKesson Corporation) for payer perspective and Medicare prices (part B payment limit data, Part D Drug Spending Dashboard) for societal perspective. We estimated costs up to year 5 after diagnosis of mBC (&lt; 8% received treatment after year 5). Results: 15,215 patients met the criteria (64.41% non-Hispanic White, 66.85% HER2-HR+, 18.30% HER2+HR+, 5.27% HER2+HR-, 9.58% TNBC). AWP and Medicare cost estimates by year after diagnosis and subgroup are shown in Table. 334,812 for HER2+HR+, $284,609 for HER2+HR-, $104,774 for HER2-HR+, and $54,355 for the TNBC. We observed an increase in annual costs between years 2011-2021 in most subgroups. This increase was most substantial for HER2-HR+ patients. From 2011 to 2019 (most recent complete year 1 data is for patients diagnosed in 2019), annual Medicare treatment costs in year 1 increased from $12,986 to $80,563 for HER2-HR+, $99,997 to $156,712 for HER2+HR+, and $31,397 to $53,775 for TNBC. There was no increase in costs of treating HER2+HR- in year 1, but an increase in year 2 (from $90,427 to $129,690 between 2011-2019). Conclusions: mBC treatment costs vary significantly by receptor subtype and years after diagnosis. Annual costs increased over time. Costs were greatest for HER2+ and lowest for TNBC.[Table: see text]

AZP-3813, a bicyclic 16-amino acid peptide antagonist of the human growth hormone receptor as a potential new treatment for acromegaly

AZP-3813, a bicyclic 16-amino acid peptide antagonist of the human growth hormone receptor as a potential new treatment for acromegaly

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression.

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Sex-specific phenotypic effects and evolutionary history of an ancient polymorphic deletion of the human growth hormone receptor.

The common deletion of the third exon of the growth hormone receptor gene (GHRd3) in humans is associated with birth weight, growth after birth, and time of puberty. However, its evolutionary history and the molecular mechanisms through which it affects phenotypes remain unresolved. We present evidence that this deletion was nearly fixed in the ancestral population of anatomically modern humans and Neanderthals but underwent a recent adaptive reduction in frequency in East Asia. We documented that GHRd3 is associated with protection from severe malnutrition. Using a novel mouse model, we found that, under calorie restriction, Ghrd3 leads to the female-like gene expression in male livers and the disappearance of sexual dimorphism in weight. The sex- and diet-dependent effects of GHRd3 in our mouse model are consistent with a model in which the allele frequency of GHRd3 varies throughout human evolution as a response to fluctuations in resource availability.

Order and disorder—An integrative structure of the full-length human growth hormone receptor

Because of its small size (70 kilodalton) and large content of structural disorder (>50%), the human growth hormone receptor (hGHR) falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small-angle x-ray scattering (SAXS) as the foundation. We develop an approach that combines SAXS, x-ray diffraction, and NMR spectroscopy data obtained on individual domains and integrate these through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The hGHR domains reorient freely, resulting in a broad structural ensemble, emphasizing the need to take an ensemble view on signaling of relevance to disease states. The structure provides the first experimental model of any full-length cytokine receptor in a lipid membrane and exemplifies how integrating experimental data from several techniques computationally may access structures of membrane proteins with long, disordered regions, a widespread phenomenon in biology.