BACKGROUND: Overexpression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) contributes to cancer cell proliferation, survival and migration, playing crucial roles in tumor development. ROR1 has been proposed as a potential therapeutic target for cancer treatment. This study aimed to develop novel humanized ROR1 monoclonal antibodies and investigate their anti-tumor effects.
METHODS: ROR1 expression in tumor tissues and cell lines was analyzed by immunohistochemistry and flow cytometry. Antibodies from mouse hybridomas were humanized by the complementarity-determining region (CDR) grafting technique. Surface plasmon resonance spectroscopy, ELISA assay and flow cytometry were employed to characterize humanized antibodies. In vitro cellular assay and in vivo mouse experiment were conducted to comprehensively evaluate anti-tumor activity of these antibodies.
RESULTS: ROR1 exhibited dramatically higher expression in lung adenocarcinoma, liver cancer and breast cancer, and targeting ROR1 by short-hairpin RNAs significantly inhibited proliferation and migration of cancer cells. Two humanized ROR1 monoclonal antibodies were successfully developed, named h1B8 and h6D4, with high specificity and affinity to ROR1 protein. Moreover, these two antibodies effectively suppressed tumor growth in the lung cancer xenograft mouse model, c-Myc/Alb-cre liver cancer transgenic mouse model and MMTV-PyMT breast cancer mouse model.
CONCLUSIONS: Two humanized monoclonal antibodies targeting ROR1, h1B8 and h6D4, were successfully developed and exhibited remarkable anti-tumor activity in vivo.

OBJECTIVE: The efficacy of chimeric antigen receptor (CAR)-T cell for solid tumors is limited partially because of the lack of tumor-specific antigens and off-target effects. Low molecular weight peptides allowed CAR T cell to display several antigen receptors to reduce off-target effects. Here, we develop a peptide-based bispecific CAR for EGFR and tumor stroma, which are expressed in a variety of tumor types.
RESULTS: The peptide-based CAR T cells show excellent proliferation, cytotoxicity activity and are only activated by tumor cells overexpressing EGFR instead of normal cells with low EGFR expressing. In mouse xenograft models, the peptide bispecific CAR T cells can be delivered into the inner of tumor masses and thus are effective in inhibiting tumor growth. Meanwhile, they show strong expansion capacity and the property of maintaining long-term function in vivo. During treatment, no off-tumor toxicity is observed on healthy organs expressing lower levels of EGFR.
CONCLUSIONS: Our findings demonstrate that peptide-based bispecific CAR T holds great potential in solid tumor therapy due to an excellent targeting ability towards tumors and tumor microenvironment.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or β-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

T-cell receptor therapy (TCR-T) has demonstrated efficacy, durability, and safety advantages in certain solid tumors (such as human papillomavirus-related tumors, synovial sarcoma, and melanoma). This study aimed to provide careful considerations for developing TCR-T for solid tumors. Therefore, in this review, we have summarized the current clinical application, advantage of TCR-T modalities and explored efficacy/safety-related parameters, particularly avidity, pharmacokinetics/pharmacodynamics, and indications, for solid tumors. Furthermore, we have investigated critical factors related to avidity, including antigen selection, T-cell receptor acquisition, optimization, and co-receptor engagement. Moreover, we have re-examined the expression of tumor antigens for a potentially higher coverage rate of solid tumors based on the current RNA-seq datasets. Finally, we have discussed the current limitations and future directions of TCR-Ts.

A fundamental mistake in receptor theory has led to an enduring misunderstanding of how to estimate the affinity and efficacy of an agonist. These properties are inextricably linked and cannot be easily separated in any case where the binding of a ligand induces a conformation change in its receptor. Consequently, binding curves and concentration-response relationships for receptor agonists have no straightforward interpretation. This problem-the affinity-efficacy problem-remains overlooked and misunderstood despite it being recognized in 1987. To avoid the further propagation of this misunderstanding, we propose in this review that the affinity-efficacy problem should be included in the core curricula for pharmacology undergraduates proposed by the British Pharmacological Society and the International Union of Basic and Clinical Pharmacology (IUPHAR).

Understanding the agonist concentration-response curve (CRC) is the cornerstone in pharmacology. While CRC parameters, agonist potency (EC50) and efficacy (maximum response, Imax) are well-studied, the role of unliganded gating (minimum response, Imin) on CRC is often overlooked. This study explores the effect of unliganded gating on agonist response in muscle-type acetylcholine (ACh) receptors, focusing on the underexplored role of Imin in modulating EC50 and Imax. Three Gain-of-Function (GOF) mutations that increase, and two Loss-of-Function (LOF) mutations that decrease the unliganded gating equilibrium constant (L0) were studied using automated patch-clamp electrophysiology. GOF mutations enhanced agonist potency, whereas LOF mutations reduced it. The calculated CRC aligned well with empirical results, indicating that agonist CRC can be estimated from knowledge of L0. Reduction in agonist efficacy due to LOF mutations was calculated and subsequently validated using single-channel patch-clamp electrophysiology, a factor often obscured in normalized CRC. The study also evaluated the combined impact of mutations (L0) on CRC, confirming the predictive model. Further, no significant energetic coupling between distant residues (>15 Å) was found, indicating that the mutations\' effects are localized and do not alter overall agonist affinity. These findings substantiate the role of unliganded gating in modulating agonist responses and establishes a predictive model for estimating CRC parameters from known changes in L0. The study highlights the importance of intrinsic activity in receptor theory.

Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.

UNASSIGNED: Peach allergy is common food allergen. Allergen components-specific antibodies of different isotypes in peach-allergy patients are poorly studied. Factors other than Pru p 3-sIgE levels may be related to severe symptoms.
UNASSIGNED: To evaluated peach component-specific-IgE, IgG1, and IgG4 characteristics in individuals with and without peach allergy, and Pru p 3-sIgE affinity in patients with different clinical symptoms.
UNASSIGNED: Fifteen healthy controls and 32 peach-allergy patients were enrolled. sIgE, sIgG1, and sIgG4 to 5 Escherichia coli-expressed peach-allergen components were determined by enzyme-linked immunosorbent assays. Pru p 3-sIgE affinity was measured in Pru p 3-sIgE-positive patients, using immunoadsorbance.
UNASSIGNED: Patients were divided into oral allergy syndrome (OAS) and peach-induced anaphylaxis (PIA) groups. Serum Pru p 1-, Pru p 2-, Pru p 3-, Pru p 4-, and Pru p 7-sIgG1s were detected. Pru p 1- and Pru p 2-sIgG1 levels were higher in healthy controls, but Pru p 3-sIgG1 levels were significantly higher in peach-allergy patients. Pru p 1-, Pru p 3-, and Pru p 4-sIgG4-positivity was significantly greater among patients than among controls. Pru p 3 was the predominant allergen in peach-allergy patients. Allergen-sIgG1 and sIgG4 were similar between OAS and PIA patients. Pru p 3-sIgE levels were significantly higher in PIA patients, but Pru p 3-sIgE-positivity was similar in both groups. In Pru p 3-sIgE-positive patients, Pru p 3-sIgE affinity was significantly higher in PIA than OAS patients.
UNASSIGNED: Allergen-sIgG1 was associated with allergen exposure. Both Pru p 3-sIgE levels and affinity are key factors in severe peach-allergy patients.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

The variability of the Conditioned Pain Modulation (CPM) effect can be attributed to conditioning stimulus (CS) characteristics, such as intensity, duration, unpleasantness, or affinity. This study investigates the impact of affinity and unpleasantness variables on the CPM effect using two protocols (cold water and ischemia) in the same healthy individuals (n = 54). Additional variables were also examined for their potential influence on the CPM effect. The main results are as follows: (1) a higher level of affinity and a lower level of unpleasantness for the stimuli used resulted in a stronger CPM effect; (2) significant differences were observed in the extreme categories (high and low) of both variables, whereas the \'indifferent\' group did not show a clear trend; (3) within-subject analysis demonstrated that affinity for the CS had a clear impact on the CPM effect; (4) no correlations were found between the CPM effect and the additional variables, except for the extraversion variable with the CPM effect of the ischemia protocol, and CS duration variable with CPM effect in the cold water protocol; and (5) only the affinity variable explained the CPM effect in both protocols in the multiple linear regression analysis. The affinity variable was found to influence the CPM effects significantly, indicating its important role in our perception and response to pain.