Distinct Extracellular Domains Research Articles

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues.

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

Zanidatamab (zani), a HER2-targeted bispecific antibody, in combination with docetaxel as first-line therapy (1L) for patients (pts) with advanced HER2-positive breast cancer (BC): Updated results from a phase Ib/II study.

1 Background: Despite HER2-targeted agents improving outcomes in HER2-positive (+) BC, some pts develop resistance, relapse, or do not respond to current 1L therapies. Zani, also known as ZW25, is a novel HER2-targeted bispecific antibody that binds to two distinct extracellular domains of HER2. Preliminary results from this Phase Ib/II trial (NCT04276493) showed that zani plus docetaxel had a manageable safety profile and demonstrated promising antitumor activity in pts with advanced HER2+ BC; here we present the updated data following enrollment completion. Methods: Cohort 1 of this open-label study is evaluating zani in combination with docetaxel as a 1L therapy in adult females with advanced HER2+ BC who may have received prior neoadjuvant/adjuvant therapy. Cohort 1a pts received zani 30 mg/kg intravenously (IV), Cohort 1b pts received zani 1800 mg IV, both with docetaxel 75 mg/m2 IV every 3 weeks. The primary endpoints were safety and investigator (INV)-assessed objective response rate (ORR) per RECIST v1.1. Secondary endpoints included INV-assessed duration of response (DoR) and disease control rate (DCR). Results: As of Nov 22, 2022, 37 pts(median age 55.0 years [range: 33-80]) were assigned to Cohort 1a (n=10) or 1b (n=27). Median study follow-up was 15.5 months (range: 1.1-29.3); patients received a median of 13 treatment cycles (range 1-37) and 18 (48.6%) pts remained on treatment. Of the 33-efficacy evaluable (EE) pts, confirmed ORR was 90.9% (95% confidence interval [CI]: 75.7, 98.1). Efficacy data are summarized in the table. In total, 36 (97.3%) pts experienced ≥1 treatment-related adverse event (TRAE); 25 (67.6%) pts experienced ≥grade 3 TRAEs. The most common ≥grade 3 TRAEs were decreased neutrophil count, experienced by 18 (48.6%) pts, and decreased white blood cell count, experienced by 7 (18.9%) pts. Serious TRAEs occurred in 6 (16.2%) pts; no TRAEs led to death. Conclusions: Zani combined with docetaxel demonstrated promising antitumor activity as 1L therapy for advanced HER2+ BC, with a manageable safety profile. Clinical trial information: NCT04276493 . [Table: see text]

Zanidatamab (zani), a HER2-targeted bispecific antibody, in combination with docetaxel as first-line therapy (1L) for patients (pts) with advanced HER2-positive breast cancer (BC): Updated results from a phase 1b/2 study.

1044 Background: Despite HER2-targeted agents improving outcomes in HER2-positive (+) BC, some pts develop resistance, relapse, or do not respond to current 1L therapies. Zani, also known as ZW25, is a novel HER2-targeted bispecific antibody that binds to two distinct extracellular domains of HER2. Preliminary results from this phase 1b/2 trial (NCT04276493) showed that zani plus docetaxel had a manageable safety profile and demonstrated promising antitumor activity in pts with advanced HER2+ BC; here we present the updated data following enrollment completion. Methods: Cohort 1 of this open-label study is evaluating zani in combination with docetaxel as a 1L therapy in adult females with advanced HER2+ BC who may have received prior neoadjuvant/adjuvant therapy. Cohort 1a pts received zani 30 mg/kg intravenously (IV), Cohort 1b pts received zani 1800 mg IV, both with docetaxel 75 mg/m2 IV every 3 weeks. The primary endpoints were safety and investigator (INV)-assessed objective response rate (ORR) per RECIST v1.1. Secondary endpoints included INV-assessed duration of response (DoR) and disease control rate (DCR). Results: As of Nov 22, 2022, 37 pts (median age 55.0 years [range: 33-80]) were assigned to Cohort 1a (n=10) or 1b (n=27). Median study follow-up was 15.5 months (range: 1.1-29.3); patients received a median of 13 treatment cycles (range 1-37) and 18 (48.6%) pts remained on treatment. Of the 33 efficacy evaluable (EE) pts, confirmed ORR was 90.9% (95% confidence interval [CI]: 75.7, 98.1). Efficacy data are summarized in Table. In total, 36 (97.3%) pts experienced ≥1 treatment-related adverse event (TRAE); 25 (67.6%) pts experienced ≥grade 3 TRAEs. The most common ≥grade 3 TRAEs were decreased neutrophil count, experienced by 18 (48.6%) pts, and decreased white blood cell count, experienced by 7 (18.9%) pts. Serious TRAEs occurred in 6 (16.2%) pts; no TRAEs led to death. Conclusions: Zani combined with docetaxel demonstrated promising antitumor activity as 1L therapy for advanced HER2+ BC, with a manageable safety profile. Clinical trial information: NCT04276493 . [Table: see text]

Abstract OT2-16-04: KN026 in combination with docetaxel as neoadjuvant treatment for HER2-positive early or locally advanced breast cancer: A single arm, multicenter, phase 2 study

Abstract Background: Despite the use of targeted therapy has revolutionized the treatment in the neoadjuvant setting for early, locally advanced, HER2-positive breast cancer, these approaches still have limited efficacy, which calls for persistent exploration for optimized treatment strategy. KN026 is a bispecific monoclonal antibody that targets the distinct extra-cellular domains II (Pertuzumab binding site) and IV (Trastuzumab binding site) of HER2. KN026 has better anti-tumor activity than either Trastuzumab or Pertuzumab used alone, and also aimed to demonstrate similar or better anti-tumor response than Trastuzumab in combination with Pertuzumab. Here we report the preliminary results of KN026 and docetaxel as neoadjuvant treatment in patients with HER2-positive early or locally advanced breast cancer (LABC). Methods: Treatment naive patients with HER2-positive early (T1c or 2, N1, M0; T2 or 3, N0, M0) or locally advanced breast cancer (T1c or 2 or 3, N2, M0; T3N1M0; T1c or 2 or 3, N3a or 3b, M0) were enrolled to receive 4 cycles of KN026 (30mg/kg, ivgtt d1, q3w) and docetaxel (75 mg/m2 ivgtt d1, q3w) neoadjuvant treatment. The primary endpoint was total pCR rate (tpCR; defined as absence of any residual invasive cancer in the breast and lymph nodes) [ypT0/is, ypN0]). Secondary endpoints were pCR in the breast (bpCR, defined as absence of any residual invasive cancer in the breast [ypT0/is]), ORR (objective response rate), safety, PK (pharmacokinetics) and immunogenicity. The study is still ongoing, and the planned enrollment number is 30. This study is registered in ClinicalTrials.gov, number NCT04881929. The data cutoff date was April 15, 2022. Results: Between August 8, 2021, and April 15, 2022, a total of 15 patients were enrolled from 5 sites. 7 (46.7%) patients were stage II, and 8 (53.3%) patients were stage III; 12 (80%) patients with lymph node metastases, and 3 (20%) patients without lymph node metastases; 7 (46.7%) patients were hormone receptor positive, and 8 (53.3%) patients were hormone receptor negative. As of April 15, 2022, 4 patients are still under neoadjuvant therapy, 1 patient withdrew from the study earlier due to AE during neoadjuvant treatment period, and 10 patients completed the surgery. Of the 10 patients who completed surgery, tpCR rate were 50% (5/10, 95% CI: 18.7%-81.3%), bpCR rate were 50% (5/10, 95% CI:18.7%-81.3%), and ORR were 100% (10/10, 95% CI: 69.2%-100%). The incidence of TRAE and Grade ≥3 TRAEs were 100% (15/15) and 53.3% (8/15) respectively. The most common TRAE were alopecia (14/15, 93.3%), white blood cell decreased (10/15, 66.7%), anemia (10/15, 66.7%), diarrhea (9/15, 60.0%), neutrophil count decreased (9/15, 60.0%), alanine aminotransferase increased (8/15, 53.3%) and hypoalbuminemia (8/15, 53.3%). The Grade ≥3 TRAE include neutrophil count decreased (8/15, 53.3%), white blood cell decreased (5/15, 33.3%), alanine aminotransferase increased (1/15, 6.7%), and lymphocyte count decreased (1/15, 6.7%). No Grade 5 TRAEs occurred. 3 TRAEs (Alanine aminotransferase increased, blood bilirubin increased and neutrophil count decreased) leading to KN026 interruption, and no TRAE leading to KN026 discontinuation. 1 SAE (hepatitis E-not TRAE; outcome: recovered) occurred in 1 patient, without other SAE or death throughout the study. Conclusions: KN026 and docetaxel as neoadjuvant treatment has shown promising clinical benefit for patients with HER2-positive early or locally advanced breast cancer with an acceptable and manageable safety profile. Further validation in a large-scale randomized controlled trial is warranted. Citation Format: Jiong Wu, Benlong Yang, Linxiaoxi Ma, Mingliang Zhang, Kun Wang, Yiding Chen, Zhimin Fan, Jing Zhang, Summer Xia. KN026 in combination with docetaxel as neoadjuvant treatment for HER2-positive early or locally advanced breast cancer: A single arm, multicenter, phase 2 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-16-04.

Zanidatamab (zani), a HER2-targeted bispecific antibody, in combination with docetaxel as first-line (1L) therapy for patients (pts) with advanced HER2-positive breast cancer: Preliminary results from a phase 1b/2 study.

1031 Background: HER2-targeted agents have improved outcomes in HER2-positive breast cancer, but some pts develop resistance, relapse, or do not respond to current 1L therapies. Zani, also known as ZW25, is a novel HER2-targeted bispecific antibody that binds to two distinct extracellular domains of HER2. In a Phase 1 trial (NCT02892123) zani was well tolerated and demonstrated preliminary antitumor activity as monotherapy/with chemotherapy in pts with pre-treated advanced HER2+ breast cancer. Methods: Cohort 1 of this ongoing open-label, Phase 1b/2 study (NCT04276493) is evaluating zani in combination with docetaxel as a 1L therapy in adult females with advanced HER2+ breast cancer who may have received prior neoadjuvant/adjuvant treatment. Cohort A pts received zani 30 mg/kg IV, Cohort B pts received zani 1800 mg IV, both with docetaxel 75 mg/m2 IV Q3W. Primary endpoints were safety and investigator (INV)-assessed objective response rate (ORR) per RECIST v1.1. Secondary endpoints included INV-assessed duration of response (DoR), disease control rate (DCR) and progression-free survival (PFS). Results: As of Nov 26, 2021, 25 pts with a median age of 57.0 years (range: 33.0–80.0) were assigned to Cohort A (n=11) or B (n=14). Median study follow-up was 7.0 months (range: 1.1–17.4) and the median number of treatment cycles was 10 (range: 2–20), 16 (64.0%) pts remained on treatment. Of the 22-efficacy evaluable (EE) pts, confirmed ORR was 86.4% (95% CI: 65.1, 97.1). The 6 months PFS rate was 90.9% (95% CI: 68.3, 97.7). Efficacy data are summarized in Table 1. All pts experienced ≥ 1 treatment emergent adverse event (TEAE) and 17 (68.0%) pts experienced ≥ Grade 3 TEAEs. In total, 23 (92.0%) pts experienced treatment related TEAEs (trTEAEs), and 17 (68.0%) pts experienced ≥ Grade 3 trTEAEs. The most common trTEAEs were diarrhea (56.0%) and decreased neutrophil count (52.0%). Serious trTEAEs occurred in two (8.0%) pts, trTEAEs leading to treatment discontinuation occurred in one (4.0%) pt and no trTEAEs led to death. Conclusions: Zani and docetaxel combination demonstrated antitumor activity in 1L therapy for advanced HER2+ breast cancer, with a manageable safety profile. Clinical trial information: NCT04276493. [Table: see text]

Zanidatamab (zani), a HER2-targeted bispecific antibody, in combination with chemotherapy (chemo) and tislelizumab (TIS) as first-line (1L) therapy for patients (pts) with advanced HER2-positive gastric/gastroesophageal junction adenocarcinoma (G/GEJC): Preliminary results from a phase 1b/2 study.

4032 Background: Zani, also known as ZW25, is a novel HER2-targeted bispecific antibody that targets two distinct extracellular domains of HER2. Zani has shown preliminary antitumor activity and tolerability in pts with HER2+ gastroesophageal adenocarcinoma as monotherapy/with chemo in Phase 1/2 studies (NCT02892123, NCT03929666). TIS, an anti-PD-1 antibody, has demonstrated antitumor activity in pts with advanced solid tumors. Combining anti-HER2 therapy with anti-PD-1 therapy and chemo increased tumor response in G/GEJC in a Phase 3 clinical trial. Methods: Cohort 2 of this ongoing open-label, Phase 1b/2 study was in pts with untreated locally advanced/metastatic HER2+ G/GEJC (NCT04276493). Cohort A received zani 30 mg/kg IV, Cohort B received zani 1800 mg IV (weight < 70 kg) or 2400 mg IV (weight ≥ 70 kg), both with TIS 200 mg IV and capecitabine/oxaliplatin (CAPOX) Q3W. Primary endpoints were safety and investigator (INV)-assessed objective response rate (ORR) per RECIST v1.1. Secondary endpoints included INV-assessed duration of response (DoR), disease control rate (DCR) and progression-free survival (PFS). Results: As of Nov 26, 2021, 33 pts with a median age of 64.0 years (range: 29.0–80.0) were assigned to Cohort A (n=19) or B (n=14). Median study follow-up was 7.7 months (range: 2.1–19.0) and the median number of treatment cycles was 10 (range: 1–28), 20 (60.6%) pts remained on treatment. All pts were efficacy evaluable ([EE], n=33), confirmed ORR was 72.7% (95% CI: 54.5, 86.7). Median PFS was 10.9 months (95% CI: 6.9, NE). Efficacy data are summarized in the Table. All pts experienced ≥ 1 treatment emergent adverse event (TEAE), and 24 (72.7%) pts experienced ≥ Grade 3 TEAEs. All pts experienced treatment related TEAEs (trTEAEs), 20 (60.6%) pts experienced ≥ Grade 3 trTEAEs and trTEAEs leading to death occurred in two (6.1%) pts. Immune-mediated AEs (imAEs) occurred in nine (27.3%) pts, of which seven (21.2%) pts experienced ≥ Grade 3 imAEs. Conclusions: Zani, TIS and CAPOX combination demonstrated a manageable safety profile and antitumor activity as 1L therapy for pts with HER2+ G/GEJC. Clinical trial information: NCT04276493. [Table: see text]

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

BackgroundHypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy.MethodsParental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system.ResultsBased on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity.ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

Zanidatamab, an anti-HER2 bispecific antibody, plus chemotherapy with/without tislelizumab as first-line treatment for patients with advanced HER2-positive breast cancer or gastric/gastroesophageal junction adenocarcinoma: A phase 1B/2 trial-in-progress.

TPS2656 Background: Zanidatamab is a novel HER2-targeted antibody that binds two distinct extracellular domains of HER2, allowing for multiple mechanisms of action including enhanced binding, clustering, receptor internalization and downregulation; this results in inhibition of ligand-dependent and -independent proliferation and potent activation of antibody-dependent cellular cytotoxicity. Zanidatamab monotherapy is well tolerated and has shown promising anti-tumor activity in patients (pts) with pre-treated advanced HER2-positive cancers, and was well tolerated in a Phase I trial (NCT02892123). Tislelizumab is an investigational anti-programmed death-1 (PD-1) antibody engineered to minimize binding of FcγR on macrophages in order to abrogate antibody-dependent phagocytosis, which is a potential mechanism of T-cell clearance and resistance to anti-PD-1 therapy. Tislelizumab is well tolerated and has anti-tumor activity alone and in combination with chemotherapy in pts with advanced solid tumors. The highly immunogenic nature of HER2 tumors has led to the development of therapies combining anti-HER2 therapies with immune checkpoint blockade. Methods: This open-label, two cohort Phase 1B/2 study (NCT04276493) is designed to evaluate zanidatamab as a first-line therapy with chemotherapy in pts with HER2-positive metastatic breast cancer (mBC; cohort 1) or with chemotherapy + tislelizumab in pts with HER2-positive advanced gastric/gastroesophageal junction adenocarcinoma (GC/GEJC; cohort 2). Weight-based dosing (cohorts 1a and 2a) and flat dosing (cohorts 1b and 2b) regimens of zanidatamab are being investigated. In cohort 1 (n = 20), pts with treatment-naïve HER2-positive (IHC3+ or ISH amplified) mBC will receive intravenous (IV) zanidatamab 30 mg/kg (cohort 1a) or 1800 mg (cohort 1b), plus IV docetaxel 75 mg/m2 once every 3 weeks (Q3W). In cohort 2 (n = 30), treatment-naïve pts with HER2-positive (IHC3+ or IHC2+ with ISH amplification) advanced GC/GEJC will receive IV zanidatamab 30 mg/kg (cohort 2a), or 1800 mg (pts < 70kg; cohort 2b) or 2400 mg (pts ≥ 70kg; cohort 2b), plus IV tislelizumab 200 mg and chemotherapy (CAPOX regimen: oral capecitabine 1000 mg/m2 twice daily [days 1–14] and IV oxaliplatin 130 mg/m2 [day 1]) Q3W. For cohort 2 there is a six pt safety lead-in phase, followed by dose expansion after approval by the safety monitoring committee. Primary endpoints are the safety profile and objective response rate. Secondary endpoints include duration of response, time to response, progression-free survival, disease control rate, and overall survival. Clinical trial information: NCT02892123.

PMEPA1 Gene Isoforms: A Potential Biomarker and Therapeutic Target in Prostate Cancer

The identification of prostate transmembrane protein androgen induced 1 (PMEPA1), an androgen responsive gene, came initially from the studies of androgen regulatory gene networks in prostate cancer. It was soon followed by the documentation of the expression and functional analysis of transmembrane prostate androgen-induced protein (TMEPAI)/PMEPA1 in other solid tumors including renal, colon, breast, lung, and ovarian cancers. Further elucidation of PMEPA1 gene expression and sequence analysis revealed the presence of five isoforms with distinct extracellular domains (isoforms a, b, c, d, and e). Notably, the predicted amino acid sequences of PMEPA1 isoforms show differences at the N-termini, a conserved membrane spanning and cytoplasmic domains. PMEPA1 serves as an essential regulator of multiple signaling pathways including androgen and TGF-β signaling in solid tumors. Structure-function studies indicate that specific motifs present in the cytoplasmic domain (PY, SIM, SH3, and WW binding domains) are utilized to mediate isoform-specific functions through interactions with other proteins. The understanding of the “division of labor” paradigm exhibited by PMEPA1 isoforms further expands our knowledge of gene’s multiple functions in tumorigenesis. In this review, we aim to summarize the most recent advances in understanding of PMEPA1 isoform-specific functions and their associations with prostate cancer progression, highlighting the potentials as biomarker and therapeutic target in prostate cancer.

ZW25, an anti-HER2 bispecific antibody, plus chemotherapy with/without tislelizumab as first-line treatment for patients with advanced HER2-positive breast cancer or gastric/gastroesophageal junction adenocarcinoma: A phase 1B/2 trial-in-progress.

TPS3145 Background: ZW25 is a novel HER2-targeted antibody that binds two distinct extracellular domains of HER2, allowing for multiple mechanisms of action, including activation of ADCC and inhibition of ligand-dependent and -independent cellular growth. ZW25 is well tolerated and showed single-agent antitumor activity in patients (pts) with advanced HER2-positive cancers. Previous reports suggested that tislelizumab, an investigational anti-PD-1 antibody engineered to minimize binding of FcgR on macrophages in order to abrogate antibody-dependent phagocytosis, was generally well tolerated and had antitumor activity alone and in combination with chemotherapy in pts with advanced solid tumors. Combining HER2-targeted agents with chemotherapy has resulted in improved survival; the highly immunogenic nature of HER2 tumors has led to the development of therapies combining anti-HER2 therapies with immune checkpoint blockade. Methods: This open-label, two cohort phase 1B/2 study is designed to evaluate ZW25 plus chemotherapy ± tislelizumab as first-line therapy in pts (n≈50) with HER2-positive metastatic breast cancer (mBC; cohort 1) or advanced gastric/gastroesophageal junction adenocarcinoma (GC/GEJC; cohort 2). In cohort 1, pts with HER2-positive (IHC3+ or ISH amplified) mBC must be treatment-naïve for metastatic disease and will receive intravenous (IV) ZW25 30 mg/kg plus docetaxel 75 mg/m2 IV once every 3 weeks (Q3W). In cohort 2, treatment-naïve pts with HER2-positive (IHC3+ or IHC2+ with ISH amplification) advanced GC/GEJC will receive ZW25 30 mg/kg plus tislelizumab 200 mg IV and chemotherapy (CAPOX regimen: capecitabine 1000 mg/m2 twice daily and oxaliplatin 130 mg/m2 IV) Q3W. A safety lead-in phase is designed for the first six pts in cohort 2, followed by dose expansion after a safety monitoring committee review. Primary endpoints are the safety/tolerability profile and objective response rate; secondary endpoints include duration of response, time to response, progression-free survival, disease control rate, and overall survival. Clinical trial information: Registered, NCT number pending will provide as soon as available .

EMS1 and BRI1 control separate biological processes via extracellular domain diversity and intracellular domain conservation

In flowering plants, EMS1 (Excess Microsporocytes 1) perceives TPD1 (Tapetum Determinant 1) to specify tapeta, the last somatic cell layer nurturing pollen development. However, the signaling components downstream of EMS1 are relatively unknown. Here, we use a molecular complementation approach to investigate the downstream components in EMS1 signaling. We show that the EMS1 intracellular domain is functionally interchangeable with that of the brassinosteroid receptor BRI1 (Brassinosteroid Insensitive 1). Furthermore, expressing EMS1 together with TPD1 in the BRI1 expression domain could partially rescue bri1 phenotypes, and led to the dephosphorylation of BES1, a hallmark of active BRI1 signaling. Conversely, expressing BRI1 in the EMS1 expression domain could partially rescue ems1 phenotypes. We further show that PpEMS1 and PpTPD1 from the early land plant Physcomitrella patens could completely rescue ems1 and tpd1 phenotypes, respectively. We propose that EMS1 and BRI1 have evolved distinct extracellular domains to control different biological processes but can act via a common intracellular signaling pathway.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Astroglial Vesicular Trafficking in Neurodegenerative Diseases.

The neocortex represents one of the largest estates of the human brain. This structure comprises ~30-40 billions of neurones and even more of non-neuronal cells. Astrocytes, highly heterogeneous homoeostatic glial cells, are fundamental for housekeeping of the brain and contribute to information processing in neuronal networks. Gray matter astrocytes tightly enwrap synapses, contact blood vessels and, naturally, are also in contact with the extracellular space, where convection of fluid takes place. Thus astrocytes receive signals from several distinct extracellular domains and can get excited by numerous mechanisms, which regulate cytosolic concentration of second messengers, such as Ca2+ and cAMP. Excited astrocytes often secrete diverse substances (generally referred to as gliosignalling molecules) that include classical neurotransmitters such as glutamate and ATP or neuromodulators such as D-serine or neuropeptides. Astrocytic secretion occurs through several mechanisms: by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release of gliosignalling molecules appears fundamentally similar to that operating in neurones, since it depends on the SNARE proteins-dependent merger of the vesicle membrane with the plasmalemma. However, the coupling between the stimulus and astroglial vesicular secretion is at least one order of magnitude slower than that in neurones. Here we review mechanisms of astrocytic excitability and the molecular, anatomical and physiological properties of vesicular apparatus mediating the release of gliosignalling molecules in health and in the neurodegenerative pathology.

Domain structure of growth signalobodies critically affects the outcome of antibody library selection.

Wide applications of antibodies have demanded rapid and easy methods for isolating high-affinity antibodies. We recently developed an antibody screening system in mammalian cells using a growth signalobody, which is a single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a target oligomeric antigen. However, we have never investigated how the domain structure of signalobodies affects the outcome of library screening. In this study, we screened naïve scFv library-inserted signalobodies having distinct extracellular and transmembrane (TM) domains. Although the previously constructed signalobody with the extracellular D1/D2 domains of erythropoietin receptor had recovered the clones with high affinity against a target antigen and with low background cell growth, its D1/D2-deficient variant which was tested in this study recovered the clones with low affinity against a target antigen and with considerable background cell growth. In addition, mutagenesis in the TM domain lowered the level of the background cell growth. These results suggest that the D1/D2 domains increase a threshold to activate signalobodies, thereby selecting clones with high affinity against a target antigen and that the TM domain could be engineered to minimize background growth signalling.

ROR1, an embryonic protein with an emerging role in cancer biology.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR family consisting of ROR1 and ROR2. RORs contain two distinct extracellular cysteine-rich domains and one transmembrane domain. Within the intracellular portion, ROR1 possesses a tyrosine kinase domain, two serine/threonine-rich domains and a proline-rich domain. RORs have been studied in the context of embryonic patterning and neurogenesis through a variety of homologs. These physiologic functions are dichotomous based on the requirement of the kinase domain. A growing literature has established ROR1 as a marker for cancer, such as in CLL and other blood malignancies. In addition, ROR1 is critically involved in progression of a number of blood and solid malignancies. ROR1 has been shown to inhibit apoptosis, potentiate EGFR signaling, and induce epithelial-mesenchymal transition (EMT). Importantly, ROR1 is only detectable in embryonic tissue and generally absent in adult tissue, making the protein an ideal drug target for cancer therapy.

Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells

Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non- N-methyl- d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.

2Bs or not 2Bs: that is the question

2Bs or not 2Bs: that is the question

Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of “target-guided proximity labeling,” such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100 Å from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 Å. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 × 10 12 pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, “target-guided proximity labeling” methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.

Evolution of the class C GPCR Venus flytrap modules involved positive selected functional divergence

BackgroundClass C G protein-coupled receptors (GPCRs) represent a distinct group of the GPCR family, which structurally possess a characteristically distinct extracellular domain inclusive of the Venus flytrap module (VFTM). The VFTMs of the class C GPCRs is responsible for ligand recognition and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs). An extensive phylogenetic investigation of the VFTMs was conducted by analyzing for functional divergence and testing for positive selection for five typical groups of the class C GPCRs. The altered selective constraints were determined to identify the sites that had undergone functional divergence via positive selection. In order to structurally demonstrate the pattern changes during the evolutionary process, three-dimensional (3D) structures of the GPCR VFTMs were modelled and reconstructed from ancestral VFTMs.ResultsOur results show that the altered selective constraints in the VFTMs of class C GPCRs are statistically significant. This implies that functional divergence played a key role in characterizing the functions of the VFTMs after gene duplication events. Meanwhile, positive selection is involved in the evolutionary process and drove the functional divergence of the VFTMs. Our results also reveal that three continuous duplication events occurred in order to shape the evolutionary topology of class C GPCRs. The five groups of the class C GPCRs have essentially different sites involved in functional divergence, which would have shaped the specific structures and functions of the VFTMs.ConclusionTaken together, our results show that functional divergence involved positive selection and is partially responsible for the evolutionary patterns of the class C GPCR VFTMs. The sites involved in functional divergence will provide more clues and candidates for further research on structural-function relationships of these modules as well as shedding light on the activation mechanism of the class C GPCRs.