Dramatic activation of an antibody by a single amino acid change in framework

Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFβ2 and TGFβ3 (Sun et al. in Sci Transl Med, 2021. https://doi.org/10.1126/scitranslmed.abe0407). Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFβ2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFβ2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFβ2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 (Kabat numbering) of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

Dual Block HER2 Assessment Increased HER2 Immunohistochemistry Positive Rate in Resected Specimens of Gastric Cancer: A Prospective Multicenter Clinical Trial From China

Background Former single center studies indicated that HER2 assessment with two primary tumor blocks (dual block HER2 assessment) could be an efficient and practical approach to overcome the adverse impact of heterogeneity and acquire a HER2 positive rate in gastric cancer (GC). This multicenter prospective clinical trial (NCT 02843412) was launched to verify its value and generality. Methods A total of 3806 participants with primary GCs have been enrolled from 8 hospitals in China. Two primary tumor blocks were selected and recorded as block 1 and block 2 after histological evaluation. An HER2 (4B5) rabbit monoclonal antibody was used for the immunohistochemistry (IHC) analysis. In total patients, HER2 IHC positive (3+) rate with dual block assessment (9.4%) was higher than that with single block assessment (block 1: 7.8%, block 2: 7.8%) (P < 0.001). Compared with single-block assessment, dual-block assessment increased the positive rate by approximate 20%. Similarly, HER2 equivocal (2+) rate was increased in dual block assessment (25.8%), which was higher than that in single block assessment (block 1: 20.3%, block 2: 20.9%) (P < 0.001). Conversely, dual block assessment demonstrated a lower HER2 negative (0/1+) rate (64.8%) than single block assessment (block1: 71.9%, block 2: 71.3%) (P < 0.001). These findings were also confirmed in individual hospitals. Conclusions Dual block HER2 assessment effectively increased HER2 IHC positive rate in resected specimens of GC. We recommended dual block HER2 assessment be promoted in routine clinical practice in GC.

The Genomic Characteristics of RET Fusion Positive Tumors in Chinese Non-Small Cell Lung Cancer (NSCLC) Patients

Background: Approximately 1-2% of non-small cell lung cancer (NSCLC) patients harbor RET (rearranged during transfection) fusions. The oncogenic RET fusions could lead to constitutive kinase activation and oncogenesis.Methods: 1746 Chinese NSCLC patients were enrolled from in this study. Tumor tissues were collected during surgery, and were formalin fixed, paraffin-embedded (FFPE) and archived. Peripheral blood (PB) samples were also collected from each patient as control. In addition, we selected 17 of them for cfDNA NGS testing and 14 tumor samples for immunohistochemistry testing using PD-L1 rabbit monoclonal antibody, clones 28-8 (Abcam, Cambridge, UK).Results: Of the 1746 NSCLC cases, RET rearrangements were identified in 25 cases (1.43 %) with locally advanced or metastatic NSCLC, of which 20 (80%) were female. We found that 14 out of 25 patients had an KIF5B-RET fusion, with KIF5B exon15-RET exon12, KIF5B exon23-RET exon12, and KIF5B exon24-RET exon11 detected in 14, 3, and 1 patients respectively. We also identified one novel RET fusion partner PLCE1 and 4 intergenic-breakpoint fusions. Conclusion: In this study, using the hybrid capture based NGS (the next generation sequencing) techniques, we revealed the genomic profiling for the patients with RET fusion-positive NSCLC. To the best of our knowledge, this is the first study that exhibited the detailed breakpoints of Chinese NSCLC patients with RET rearrangement, and we found a novel new partner PLCE1. The results provided genomic information for patients with RET fusion which is significant for personalized clinical management in the era of precision medicine.

HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

Dramatic Activation of an Antibody by a Single Amino Acid Change in Framework

Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFβ2 and TGFβ31. Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFβ2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFβ2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFβ2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.

The Genomic Characteristics of RET Fusion Positive Tumors in Chinese Non-small Cell Lung Cancer (NSCLC) Patients

Background: Approximately 1-2% of non-small cell lung cancer (NSCLC) patients harbor RET (rearranged during transfection) fusions. The oncogenic RET fusions could lead to constitutive kinase activation and oncogenesis.Methods: 1746 Chinese NSCLC patients were enrolled from in this study. Tumor tissues were collected during surgery, and were formalin fixed, paraffin-embedded (FFPE) and archived. Peripheral blood (PB) samples were also collected from each patient as control. In addition, we selected 17 of them for cfDNA NGS testing and 14 tumor samples for immunohistochemistry testing using PD-L1 rabbit monoclonal antibody, clones 28-8 (Abcam, Cambridge, UK).Results: Of the 1746 NSCLC cases, RET rearrangements were identified in 25 cases (1.43 %) with locally advanced or metastatic NSCLC, of which 20 (80%) were female.We found that 14 out of 25 patients had an KIF5B-RET fusion, with KIF5B exon15-RET exon12, KIF5B exon23-RET exon12, and KIF5B exon24-RET exon11 detected in 14, 3, and 1 patients respectively. We also identified one novel RET fusion partner PLCE1 and 4 intergenic-breakpoint fusions. Conclusion: In this study, using the hybrid capture based NGS (the next generation sequencing) techniques, we revealed the genomic profiling for the patients with RET fusion-positive NSCLC. To the best of our knowledge, this is the first study that exhibited the detailed breakpoints of Chinese NSCLC patients with RET rearrangement, and we found a novel new partner PLCE1. This result provides genomic information for personalized clinical management for patients with RET fusion in the era of precision medicine.

Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein

To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2.