scholarly journals Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

2008 ◽  
Vol 17 (7) ◽  
pp. 1175-1187 ◽  
Author(s):  
Valentina Tereshko ◽  
Serdar Uysal ◽  
Akiko Koide ◽  
Katrina Margalef ◽  
Shohei Koide ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Crystal Packing ◽  
Single Domain ◽  
Single Domain Antibody ◽  
X Ray ◽  
Domain Antibody

scholarly journals Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer

2021 ◽  
Vol 9 (6) ◽  
pp. e002131
Author(s):  
Tianhang Zhai ◽  
Chao Wang ◽  
Yifeng Xu ◽  
Weifeng Huang ◽  
Zhijun Yuan ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Single Domain ◽  
Single Domain Antibody ◽  
X Ray ◽  
X Ray Crystallography ◽  
Domain Antibody ◽  
Cell Assays

BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%–30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).MethodsTo determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.ResultsPM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.ConclusionsA unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy

2021 ◽  
Vol 22 (11) ◽  
pp. 5501
Author(s):  
Yutong Xing ◽  
Keyuan Xu ◽  
Shixiong Li ◽  
Li Cao ◽  
Yue Nan ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Animal Studies ◽  
Single Domain ◽  
Soluble Form ◽  
Therapeutic Window ◽  
Single Domain Antibody ◽  
Pseudomonas Exotoxin A ◽  
Simple Strategy ◽  
Domain Antibody ◽  
Recombinant Immunotoxin

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris

2006 ◽  
Vol 43 (5) ◽  
pp. 426-435 ◽  
Author(s):  
Fatemeh Rahbarizadeh ◽  
Mohammad J. Rasaee ◽  
Mehdi Forouzandeh ◽  
Abdol-Amir Allameh
Keyword(s):  
Pichia Pastoris ◽  
Antibody Fragments ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Yeast Pichia Pastoris ◽  
Over Expression ◽  
Domain Antibody

scholarly journals Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e115405 ◽  
Author(s):  
Dan Zabetakis ◽  
Mark A. Olson ◽  
George P. Anderson ◽  
Patricia M. Legler ◽  
Ellen R. Goldman
Keyword(s):  
Thermal Stability ◽  
Disulfide Bond ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Thermal Stability Of
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