<?xml version="1.0" encoding="UTF-8"?><rss version="2.0" xmlns:content="http://purl.org/rss/1.0/modules/content/" xmlns:wfw="http://wellformedweb.org/CommentAPI/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:atom="http://www.w3.org/2005/Atom" xmlns:sy="http://purl.org/rss/1.0/modules/syndication/" xmlns:slash="http://purl.org/rss/1.0/modules/slash/" > <channel> <title>Creative Biolabs Single Domain Antibody Blog</title> <atom:link href="https://www.creative-biolabs.com/blog/sdab/feed/" rel="self" type="application/rss+xml" /> <link>https://www.creative-biolabs.com/blog/sdab</link> <description></description> <lastBuildDate>Sat, 02 Dec 2023 07:20:00 +0000</lastBuildDate> <language>en-US</language> <sy:updatePeriod> hourly </sy:updatePeriod> <sy:updateFrequency> 1 </sy:updateFrequency> <generator>https://wordpress.org/?v=6.3.1</generator> <image> <url>https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/01/favicon-1-150x150.png</url> <title>Creative Biolabs Single Domain Antibody Blog</title> <link>https://www.creative-biolabs.com/blog/sdab</link> <width>32</width> <height>32</height> </image> <item> <title>Research progress on the application of HER2-targeting single-domain antibodies in tumor imaging</title> <link>https://www.creative-biolabs.com/blog/sdab/biological-knowledge/research-progress-on-the-application-of-her2-targeting-single-domain-antibodies-in-tumor-imaging/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Sat, 02 Dec 2023 06:51:11 +0000</pubDate> <category><![CDATA[Biological Knowledge]]></category> <category><![CDATA[HER2]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[tumor imaging]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/sdab/?p=163</guid> <description><![CDATA[Introduction to HER2 targets Human epidermal growth factor receptor 2 (HER2), also known as ERBB2, is a receptor tyrosine kinase that belongs to the ERBB receptor tyrosine kinase family together with HER1<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/biological-knowledge/research-progress-on-the-application-of-her2-targeting-single-domain-antibodies-in-tumor-imaging/">Read More...</a>]]></description> <content:encoded><![CDATA[<ol> <li>Introduction to HER2 targets</li> </ol> <p>Human epidermal growth factor receptor 2 (HER2), also known as ERBB2, is a receptor tyrosine kinase that belongs to the ERBB receptor tyrosine kinase family together with HER1 (EGFR), HER3, and HER4. HER receptors control the growth, differentiation, and survival of epithelial cells. In addition to HER2, other members of the EGFR family activate their activity through ligand binding, but no ligand has been found that directly binds to the extracellular domain of the HER2 receptor. HER2 activates cellular pathways via dimerization of transmembrane receptors. HER2 gene amplification or protein overexpression is closely related to adverse pathological changes and poor prognosis, such as increased cell proliferation, tumorigenesis, and invasion through the induction of downstream pathways such as PI3K/Akt. Reports indicate that HER2 gene amplification or HER2 protein overexpression has been detected in breast cancer, gastric cancer, lung cancer, colorectal cancer, and ovarian cancer.</p> <p>The specific detection of HER2 receptor protein using targeted drugs is very important for the diagnosis and treatment of cancer. Related candidate targeting molecules include monoclonal antibodies, antibody fragments, peptides, <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/sdab/">single-domain antibodies</a></strong></span>, etc. Among them, single-domain antibodies are the smallest naturally occurring antigen-binding fragments. They have a relative small molecular weight, high stability, a high clearance rate, and low immunogenicity. They have unique advantages in applications in the field of molecular imaging.</p> <p><img decoding="async" fetchpriority="high" class="aligncenter wp-image-165" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-scaled.jpg" alt="" width="364" height="205" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-scaled.jpg 2560w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-300x169.jpg 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-1024x576.jpg 1024w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-768x432.jpg 768w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-1536x864.jpg 1536w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-2048x1152.jpg 2048w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/12/1920452615-1600x900.jpg 1600w" sizes="(max-width: 364px) 100vw, 364px" /></p> <ol start="2"> <li><a href="/sdab/symbol-her2-6321.htm"><strong><span style="color: #0000ff;">HER2 single-domain antibodies</span></strong></a> under research</li> </ol> <p>According to literature reports, researchers used HER2 antigen, target DNA, or cells expressing HER2 antigen on their surfaces to immunize camels and screen for HER2-specific single-domain antibodies. Expression systems such as E. coli are then used to achieve large-scale preparation of single-domain antibodies. A number of HER2 single-domain antibodies have been developed to the clinical stage to evaluate their diagnostic potential against HER2-overexpressing tumors. Since HER2 overexpression accounts for the largest proportion of breast cancer, the research subjects mainly focus on the breast cancer population. Some of the HER2 single-domain antibodies under development are summarized as follows: 2Rs15d, NM-02, 5F7, MIRC208, MIRC213, 11A4, 18C3, 22G12, etc. Among them, the product candidate based on 2Rs15d and used for tumor imaging has been developed for Phase II clinical trials. The Phase 1 clinical trial of NM-02 for molecular imaging of breast cancer has been completed. 5F7 and MIRC213 are undergoing Phase 1 clinical trials, while 11A4, 18C3, 22G12, and others are in the preclinical research stage.</p> <p>2.1 HER2 single-domain antibody 2Rs15d</p> <p>2Rs15d, also known as anti-HER2-VHH1, has demonstrated good HER2 targeting in both preclinical and clinical trials. Based on 2Rs15d, a HER2-specific single-domain antibody, researchers have selected a variety of different imaging markers and developed a number of tumor molecular imaging candidate products, including 68Ga, 131I, 99mTc, 18F, and 89Zr. One of the 68Ga-labeled product candidates has entered Phase 2 clinical trials.</p> <p>2.2 HER2 single-domain antibody NM02</p> <p>NM-02 is another HER2 single-domain antibody. Based on this, the researchers combined the nuclide 99mTc to develop the candidate product 99mTc-NM-02. 99mTc-NM-02 as a tumor imaging drug for SPECT/CT imaging of HER2-positive tumors will be compared to the existing gold standard for HER2 expression detection, namely biopsy tissue immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) and 18F-FDG PET/CT imaging.</p> <p>2.3 HER2 single-domain antibody 5F7</p> <p>5F7 is a screened HER2 single-domain antibody. Various radionuclide-labeled 5F7s have shown tumor imaging potential in preclinical models.</p> <p>2.4 MIRC208 and MIRC213</p> <p>MIRC208 and MIRC213 are two HER-specific single-domain antibodies. The researchers used the nuclide 99mTc to label two single-domain antibodies and verified in a mouse model that the two tracers 99mTc-MIRC208 and 99mTc-MIRC213 could clearly visualize HER2-positive tumors, but could not visualize HER2-negative tumors.</p> <p> </p> <p>References</p> <p>Zhao L, Liu C, Xing Y, <em>et al</em>. Development of a 99mTc-Labeled Single-Domain Antibody for SPECT/CT Assessment of HER2 Expression in Breast Cancer. <em>Mol Pharm</em>. 2021 Sep 6;18(9):3616-3622. doi: 10.1021/acs.molpharmaceut.1c00569.</p> <p>D’Huyvetter M, De Vos J, Xavier C, <em>et al</em>. 131I-labeled Anti-HER2 Camelid sdAb as a Theranostic Tool in Cancer Treatment. <em>Clin Cancer Res</em>. 2017 Nov 1;23(21):6616-6628. doi: 10.1158/1078-0432.CCR-17-0310.</p> <p>Li L, Liu T, Shi L, <em>et al</em>. HER2-targeted dual radiotracer approach with clinical potential for noninvasive imaging of trastuzumab-resistance caused by epitope masking. <em>Theranostics</em>. 2022 Jul 18;12(12):5551-5563. doi: 10.7150/thno.74154.</p> ]]></content:encoded> </item> <item> <title>Research progress on the application of EGFR single domain antibodies in tumor imaging</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/research-progress-on-the-application-of-egfr-single-domain-antibodies-in-tumor-imaging/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Tue, 31 Oct 2023 09:29:33 +0000</pubDate> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[EGFR single domain antibodies]]></category> <category><![CDATA[sdAb]]></category> <category><![CDATA[sdAb conjugation]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/sdab/?p=159</guid> <description><![CDATA[Single domain antibodies (sdAbs) are the smallest units that retain antigen-binding sites. They have the advantages of high antigen affinity, high specificity, high solubility, high stability, high clearance, low immunogenicity, and can<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/research-progress-on-the-application-of-egfr-single-domain-antibodies-in-tumor-imaging/">Read More...</a>]]></description> <content:encoded><![CDATA[<p><span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/sdab/">Single domain antibodies</a> </strong></span>(sdAbs) are the smallest units that retain antigen-binding sites. They have the advantages of high antigen affinity, high specificity, high solubility, high stability, high clearance, low immunogenicity, and can be produced by microorganisms. Currently, two single-domain antibody therapeutic drugs have been approved for marketing. But in the field of tumor imaging, single-domain antibodies are still in the preclinical and clinical development stages.</p> <p><img decoding="async" class="aligncenter wp-image-160" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments.jpg" alt="" width="403" height="188" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments.jpg 1800w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments-300x140.jpg 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments-1024x478.jpg 1024w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments-768x359.jpg 768w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/Schematic-of-representative-antibody-and-antibody-fragments-1536x718.jpg 1536w" sizes="(max-width: 403px) 100vw, 403px" /></p> <p style="text-align: center;">Fig. 1 Schematic of representative antibody and antibody fragments. (Wei, 2020)</p> <ol> <li>Introduction to EGFR targets</li> </ol> <p>Human epidermal growth factor receptor (EGFR), also known as ERBB1 or HER1, is a transmembrane receptor tyrosine kinase (RTK) belonging to the ErbB receptor tyrosine kinase family. EGFR gene amplification, duplication, mutation, deletion, or whole-code mutation may lead to increased expression levels or functional activation of EGFR, which is associated with a poor prognosis of tumors. Multiple studies have shown that overexpression of EGFR has been detected in colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, head and neck cancer, and endometrial cancer. EGFR is a relatively mature target in the field of tumor treatment. It consists of three different domains, including an extracellular domain, a transmembrane domain, and an intracellular domain. Currently, a number of tyrosine kinase inhibitors (TKIs) targeting the intracellular domain of EGFR have been approved for marketing; in addition, a number of monoclonal antibodies targeting the extracellular domain of EGFR have been approved for marketing. In terms of tumor diagnosis, targeted drugs, such as specifically labeled EGFR antibodies, can detect cells overexpressing EGFR.</p> <ol start="2"> <li>EFGR single domain antibodies under research</li> </ol> <p>According to literature reports, researchers immunized camels with EGFR-overexpressing cells and obtained EGFR-specific single domain antibodies. Similar to monoclonal antibodies, single domain antibodies mainly target the extracellular domain (ECD) of EGFR. A number of <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/symbol-egfr-6238.htm">EGFR single domain antibodies</a></span></strong> have been developed for the diagnosis and treatment of EGFR overexpression tumors, including αEGFR-αEGFR-Alb, 8B6, 7D12, 7C12, EG2, 9G8, B39, EGa1, D10, etc. Generally speaking, most monovalent single domain antibodies are used for tumor imaging, and the commonly used expression system is <em>E. coli</em>. To perform tumor imaging, single domain antibodies also need to be linked to imaging labels.</p> <ol start="3"> <li>EFGR single domain antibody labeling strategy</li> </ol> <p>3.1 Radioactive metal element-labeled single domain antibodies</p> <p>Taking 99mTc as an example, some studies have reported the feasibility of EG2 as a molecular probe for detecting EGFR overexpressing tumor cells. The study labeled the EGFR single domain antibody EG2 with a tricarbonyl kit, and then tested the specificity of 99mTc-EG2 on A431 (EGFR overexpressing cells) and OCM-1 (EGFR low expressing cells) cell lysates. Its binding affinity is approximately 43.53 nmol/L. SPECT results showed that 99mTc-EG2 had a short half-life and a fast blood clearance rate, and EGFR overexpressing cells were observed one hour after injection. Another study reported detecting a reduction in tumor burden during EGFR TKI treatment using 99mTc-7C12 as a noninvasive tracer. 99mTc-D10 has also reported application potential for imaging small tumors or early-stage tumors, where tumors smaller than 100 mm in size can be visualized by SPECT imaging. This also means that 99mTc-D10 has the ability to evaluate EGFR expression levels during disease progression and may be a good tracer for non-invasive detection of tumors.</p> <p>68Ga and 89Zr are two other commonly used radiolabeled elements. Some researchers have prepared conjugates 68Ga-7D12 and 89Zr-7D12 based on the EGFR single domain antibody 7D12. The results showed that biodistribution and PET imaging experiments of 68Ga-7D12 and 89Zr-7D12 in mice with A431 transplants showed similar tumor uptake. Compared with radiolabeled monoclonal antibodies, 68Ga-7D12 showed stronger image contrast at early time points. The above results highlight the potential of 68Ga-7D12 for evaluating EGFR overexpressing tumors.</p> <p>3.2 Near-infrared fluorescent dye-labeled single domain antibodies</p> <p>The use of near-infrared fluorescence technology for in situ imaging of tumor surgery has high application potential, allowing doctors to see the surgical steps instantly. Some studies have used the near-infrared fluorescent dye IRDye800CW to label 7D12 (EGFR single domain antibody) and EGFR monoclonal antibodies and compared the tumor imaging potential of the two. The results showed that 7D12-IR imaging of tumor cells was observed 30 minutes after injection, while the EGFR monoclonal antibody produced no signal. Tumor uptake was observed 2 hours after injection of 7D12-IR, and EGFR mAb accumulated in tumor cells after 24 hours. There are also studies comparing 7D12 labeled with different substances. The results show that 7D12 labeled with near-infrared fluorescent dyes has a higher tumor uptake efficiency than 7D12 labeled with radioactive elements, reflecting the excellent fast optical imaging performance of 7D12-IR.</p> <p>3.3 Crystal nanoparticle-labeled single domain antibodies</p> <p>Cobalt tetroxide (Co3O4) nanoparticles are crystalline nanoparticles based on their morphology and peroxidase-like (POD) activity. Some researchers have chosen to combine the EGFR single domain antibody B39 with Co3O4 nanopolyhedrons to detect EGFR overexpressing cells. The coupling strategy of B39 and crystal nanoparticles is to couple the nanoparticles to the C-terminus of the single-domain antibody B39, leaving the N-terminus containing the EGFR epitope completely free for detection of EGFR overexpressing cells. In addition, the researchers found that the conjugate had a low tendency to aggregate.</p> <p>3.4 Quantum dot-labeled single domain antibodies</p> <p>Quantum dots (QD) are a kind of semiconductor nanocrystal material. When excited by ultraviolet light, the quantum dots are in a high-energy state and release light of another wavelength. The wavelength depends on the size of the nanoparticles. QD-EG2 is the first imaging agent developed for tumor diagnosis. It is formed by coupling the EGFR single domain antibody EG2 and quantum dots. Other similar EGFR cell imaging reagents are QD-7D12, QD-EgA1 and QD-EgB. Quantum dots carrying the anti-cancer drug aminoflavonoids and coated with 7D12 have therapeutic, diagnostic and therapeutic properties. The single-domain antibody 7D12 enhances tumor uptake and delivers the drug to deep areas of the tumor.</p> <p>3.5 Contrast agent labeled single domain antibodies</p> <p>Contrast media is also an imaging marker. The iRGD (CRGDKGPDC) peptide is a type of CPP that can be localized on the cell membrane and enter the cell interior through the integrin receptor αVβ3. In order to improve the specificity of iRGD, the researchers designed the conjugate B39-iRGD of the EGFR single domain antibody and iRGD. B39-iRGD shows potential for tumor treatment and diagnostic applications. Compared with single domain antibodies alone, B39-iRGD combined with radiotherapy can enter deep areas of tumor cells and has stronger anti-tumor activity. In terms of imaging applications, the anti-EGFR-iRGD-DTPA-Gd obtained by coupling the single-domain antibody-iRGD with the contrast agent Magnevist can be combined with magnetic resonance imaging (MRI) technology to detect gastric cancer. (Learn more about our <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/single-domain-antibody-conjugation-services.html">sdAb conjugation</a></span></strong> services)</p> <p> </p> <p>References</p> <p>Piramoon M, Khodadust F, Hosseinimehr SJ. Radiolabeled nanobodies for tumor targeting: From bioengineering to imaging and therapy. <em>Biochim Biophys Acta Rev Cancer</em>. 2021 Apr;1875(2):188529. doi: 10.1016/j.bbcan.2021.188529.</p> <p>Wei W, Rosenkrans ZT, Liu J, Huang G, Luo QY, Cai W. ImmunoPET: Concept, Design, and Applications. <em>Chem Rev</em>. 2020 Apr 22;120(8):3787-3851. doi: 10.1021/acs.chemrev.9b00738.</p> <p>Tang H, Gao Y, Han J. Application Progress of the Single Domain Antibody in Medicine. <em>Int J Mol Sci</em>. 2023 Feb 20;24(4):4176. doi: 10.3390/ijms24044176.</p> <p>Yang E, Liu Q, Huang G, Liu J, Wei W. Engineering nanobodies for next-generation molecular imaging. <em>Drug Discov Today</em>. 2022 Jun;27(6):1622-1638. doi: 10.1016/j.drudis.2022.03.013.</p> <p>Brilhante-da-Silva N, de Oliveira Sousa RM, Arruda A, Dos Santos EL, Marinho ACM, Stabeli RG, Fernandes CFC, Pereira SDS. Camelid Single-Domain Antibodies for the Development of Potent Diagnosis Platforms. <em>Mol Diagn Ther</em>. 2021 Jul;25(4):439-456. doi: 10.1007/s40291-021-00533-7.</p> <p>Sharifi J, Khirehgesh MR, Safari F, Akbari B. EGFR and anti-EGFR nanobodies: review and update. <em>J Drug Target</em>. 2021 Apr;29(4):387-402. doi: 10.1080/1061186X.2020.1853756.</p> ]]></content:encoded> </item> <item> <title>Using single domain antibody targeted degradation technology to explore the subcellular functions of intracellular proteins</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/using-single-domain-antibody-targeted-degradation-technology-to-explore-the-subcellular-functions-of-intracellular-proteins/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Sat, 07 Oct 2023 07:12:55 +0000</pubDate> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[anti-membrane protein sdAb development]]></category> <category><![CDATA[single domain antibody library]]></category> <category><![CDATA[Single-domain Antibody]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/sdab/?p=144</guid> <description><![CDATA[Protein degradation is an important mechanism by which organisms regulate the amount and activity of proteins in cells. The ubiquitin-proteasome system is involved in regulating important physiological or pathological processes such as<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/using-single-domain-antibody-targeted-degradation-technology-to-explore-the-subcellular-functions-of-intracellular-proteins/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>Protein degradation is an important mechanism by which organisms regulate the amount and activity of proteins in cells. The ubiquitin-proteasome system is involved in regulating important physiological or pathological processes such as cell growth, proliferation, migration, differentiation, and death. The 2004 Nobel Prize in Chemistry was awarded to three Israeli scientists for their early discovery of the mechanism of ubiquitination-enzymatic intracellular protein degradation. Targeted protein degradation (TPD) is a novel drug development strategy that uses specifically recognized small molecules or macromolecules to form a targeting chimera (PROTAC) bifunctional molecule with a ubiquitin-proteasome. It can “hijack” the degradation process of cells and degrade key target proteins from cells to control key protein molecules in cells and explore their efficacy. Currently, large-scale screening is generally used to obtain chemical small molecule-mediated methods that specifically bind to target degradation proteins. However, its limitation lies in the need to find high-affinity small molecule ligands for the target protein, which is very difficult for some “untargetable” proteins. Scientists have achieved new results by using single-domain antibody targeted degradation technology to explore different subcellular functions of intracellular proteins. Relevant research results were recently published in the journal.</p> <p><img decoding="async" class="aligncenter wp-image-143" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/fx1.jpg" alt="" width="302" height="302" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/fx1.jpg 664w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/fx1-300x300.jpg 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/10/fx1-150x150.jpg 150w" sizes="(max-width: 302px) 100vw, 302px" /></p> <p style="text-align: center;">Fig. 1 Endogenous proteins degradation. (Dean Clift, 2017)</p> <p>Based on the previous construction of a camel-derived <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/sdab/custom-sdab-library-generation-services.htm">single domain antibody library</a></strong></span> and high-throughput screening, this study created a single domain antibody-based molecular toolkit-ubiquitin proteasome system – Nb4A-Fc-T2a-TRIM21. This system can target and degrade Survivin protein located in the cytoplasm and nucleus through the ubiquitination pathway, thereby exploring the potential functions of Survivin protein in different subcellular localizations. This reveals the different functions and activities in its different subcellular structures and provides a scientific basis for the further development of drugs that precisely target its subcellular structures.</p> <p>Most of the systems reported so far use chemical molecules to regulate protein degradation, with poor temporal and spatial resolution. Using antibodies instead of small molecule targeting will result in a larger molecular weight and poor membrane permeability, resulting in poor bioavailability and poor druggability. There are issues with the precision of regulation and possible off-target toxicity, as well as efficient cell production and cost when using whole antibodies. Single domain antibodies (sdAbs) have fully functional antigen-binding fragments that can target target proteins just like antibodies. They have the advantages of a small molecular weight, easy expression, high stability, and strong specificity. The Nb4A-Fc-TRIM21 system created in this study can rely on ubiquitination to target and degrade the Survivin protein located in the cytoplasm and nucleus and has the advantage of being fast and effective. Experimental results show that Survivin in the cytoplasm mainly exerts anti-apoptotic effects by directly or indirectly inhibiting the caspase pathway, while Survivin in the nucleus mainly promotes cell proliferation and participates in cell cycle regulation. This lays the theoretical foundation for an in-depth study of the mechanism of Survivin protein and the discovery of feasible and effective tumor drug targets. At the same time, this study also demonstrates the potential for wider application of single domain antibodies in basic research. (Learn more about our <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/sdab/one-stop-solution-for-anti-membrane-protein-sdab-development.htm">One-Stop Solution for Anti-Membrane Protein sdAb Development</a></strong></span>)</p> <p> </p> <p>Reference</p> <p>Wang, S., Xu, Y., Chan, H. F., Kim, H. W., Wang, Y., Leong, K. W., et al. (2016). Nanoparticle-mediated inhibition of survivin to overcome drug resistance in cancer therapy. <em>J. Control. Release</em> 240, 454–464.</p> <p>Fan, L., Sun, G., Ma, T., Zhong, F., and Wei, W. (2013). Melatonin overcomes apoptosis resistance in human hepatocellular carcinoma by targeting survivin and XIAP. <em>J. Pineal. Res.</em> 55, 174–183.</p> <p>Doudna, J. A., and Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. <em>Science</em> 346, 1258096.</p> <p>Clift, D., McEwan, W. A., Labzin, L. I., Konieczny, V., Mogessie, B., Jameset, L. C., et al. (2017). A method for the acute and rapid degradation of endogenous proteins. <em>Cell</em> 171, 1692–1706.e18.</p> <p>Miao H, Liu C, Ouyang H, Zhang P, Liu Y, Zhang C, Deng C, Fu Y, Niu J, Zheng W, You F, Yang Y, Ma X. A nanobody-based molecular toolkit for ubiquitin-proteasome system explores the main role of survivin subcellular localization. <em>Front Bioeng Biotechnol</em>. 2023 Jan 20;10:952237. doi: 10.3389/fbioe.2022.952237. PMID: 36743654; PMCID: PMC9895104.</p> ]]></content:encoded> </item> <item> <title>Single Domain Antibody-Drug Conjugates</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibody-drug-conjugates/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Mon, 28 Aug 2023 02:04:02 +0000</pubDate> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[antibody-drug conjugate]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[Therapeutic sdAb]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=130</guid> <description><![CDATA[The development concept of single domain antibody-drug conjugates (sdAb-drug conjugates) is similar to that of antibody-drug conjugates (ADCs). That is, the antibody in the ADC is replaced with a single domain antibody<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibody-drug-conjugates/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>The development concept of single domain antibody-drug conjugates (<span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/novel-sdab-development.htm">sdAb-drug conjugates</a></span>) is similar to that of antibody-drug conjugates (ADCs). That is, the antibody in the ADC is replaced with a single domain antibody with a higher affinity. Compared with ADCs, sdAb-drug conjugates have the advantages of a fast clearance rate, strong stability, and strong tumor penetration ability.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-131" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/1.png" alt="" width="409" height="270" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/1.png 985w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/1-300x198.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/1-768x508.png 768w" sizes="(max-width: 409px) 100vw, 409px" /></p> <p style="text-align: center;">Fig. 1 Number of publications related to single domain antibodies in chemotherapy from 2000 to 2020 (Panikar SS, 2021)</p> <p><strong>Potential advantages of sdAb-drug conjugates compared to mAbs in drug development</strong></p> <p><strong>Tissue penetration</strong>: The larger size and presence of the Fc region of mAbs can improve pharmacokinetic properties, but also affect their tissue penetration ability. Smaller volumes of sdAb-drug conjugates enable higher tissue penetration and enhanced cell killing <em>in vivo</em>.</p> <p><strong>Antigen recognition ability</strong>: Among all complementarity determining regions, CDR3 accounts for 60–80% of antigen recognition specificity. The CDR3 loop is long and extended, which endows VHH with better antigen recognition specificity and affinity, which can enhance the recognition of hidden tumor antigen epitopes.</p> <p><strong>Strong stability</strong>: Compared with mAbs, sdAbs have significantly higher thermal stability, Tm values, and reversible thermal denaturation. In addition, sdAbs are resistant to the denaturing effects of proteases, extreme pH, and hydrophobic agents.</p> <p><strong>Tandem into a multispecific or <a href="/sdab/multivalent-sdab-development.htm"><span style="color: #0000ff;">multivalent configuration</span></a></strong>: Compared with mAbs, sdAbs are small in size, which is conducive to tandem with a variety of antibody domains with different functions and can be used for the delivery of multifunctional drugs. It can also avoid rapid renal clearance and increase the drug ratio while enhancing affinity.</p> <p>Taking these characteristics together, compared with mAbs or their fragment counterparts, sdAbs are ideal candidates for the development of highly stable and specific drug-conjugated carriers.</p> <p><strong>Key factors to consider in the development of sdAb-drug conjugates</strong></p> <p>In order to improve the efficiency of sdAb-drug conjugates, several factors can be considered—enhancing the specificity and affinity of sdAb-drug conjugates, the selection of conjugated drugs, site-specific attachment strategies, drug sites, and the ratio of drugs to sdAb-drug conjugates.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-132" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/2.png" alt="" width="353" height="258" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/2.png 1031w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/2-300x219.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/2-768x561.png 768w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/2-1024x748.png 1024w" sizes="(max-width: 353px) 100vw, 353px" /></p> <p style="text-align: center;">Fig. 2 Factors to consider when developing sdAb-drug Conjugates (Panikar SS, 2021)</p> <p><strong>Drug type</strong>: The drug conjugates used to prepare sdAb-drug conjugates are mainly divided into DNA damage agents and microtubule inhibitors.</p> <p><strong>Linker</strong>: Linker molecular design is a key factor affecting pharmacokinetics and pharmacodynamics. In order to maximize the efficiency of sdAb-drug conjugates, the ideal linker should meet the following conditions:</p> <ol> <li>High stability in human plasma</li> <li>It can break itself under specific tumor environments</li> <li>Use a hydrophilic linker to reduce the aggregation of sdAb-drug conjugates.</li> </ol> <p><strong>Ratio of drug to antibody</strong>: Although theoretically sdAbs do not show drug-loading advantages over mAbs, the effective accumulation and stability of sdAb-drug conjugates at the tumor site are superior to those of ADCs. The robust properties of sdAb-drug conjugates (high antigen specificity, stability, solubility, and lower immunogenicity) will allow them to enter target cells more efficiently than mAbs.</p> <p><strong>Site-specific conjugation strategy</strong>: Compared with ADCs, the simple chemical conjugation strategy of sdAb-drug conjugates and drugs has wider applicability, even surpassing ADCs in terms of low production cost and long-term stability. For sdAb-drug conjugates, lysine, cysteine, aspartic acid, and glutamic acid are usually selected as chemical conjugation sites to achieve a higher ratio of drug to sdAbs. However, this may also result in a heterogeneous mixture of sdAb-drug conjugates, affecting overall potency. In recent years, advances in protein chemistry have enabled the use of new tools to achieve homogeneous conjugation of drugs to sdAbs, including lysine amide coupling, insertion of cysteine residues, insertion of unnatural amino acids, and enzymatic conjugation, among others.</p> <p>In clinical practice, although therapeutic antibody drugs have strong targeting, their therapeutic effect on solid tumors is limited due to their large molecular weight. Although small-molecule chemical drugs have a high killing effect on cancer cells, their selectivity is poor, and they often accidentally injure normal cells, causing serious side effects. As sdAb-drug conjugates are receiving more and more attention in diagnosis and therapeutic applications, chemotherapy drugs based on sdAb-drug conjugates are expected to achieve better efficacy in tumor treatment.</p> <p> </p> <p>Reference</p> <p>Panikar SS, <em>et al</em>. Nanobodies as efficient drug-carriers: Progress and trends in chemotherapy. <em>J Control Release</em>. 2021 Jun 10;334:389-412. doi: 10.1016/j.jconrel.2021.05.004. Epub 2021 May 6. PMID: 33964364.</p> ]]></content:encoded> </item> <item> <title>Single domain antibodies with flexible half-life</title> <link>https://www.creative-biolabs.com/blog/sdab/biological-knowledge/single-domain-antibodies-with-flexible-half-life/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Tue, 01 Aug 2023 01:58:51 +0000</pubDate> <category><![CDATA[Biological Knowledge]]></category> <category><![CDATA[Half-life]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[Therapeutic sdAb]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=125</guid> <description><![CDATA[As a type of antibody with good application prospects, single domain antibodies have advantages in the diagnosis and treatment of many diseases. However, its short half-life in plasma limits its application in<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/biological-knowledge/single-domain-antibodies-with-flexible-half-life/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>As a type of antibody with good application prospects, single domain antibodies have advantages in the diagnosis and treatment of many diseases. However, its short half-life in plasma limits its application in the therapeutic field. The half-life refers to the time required for the drug concentration in plasma to drop to half after the injection is completed. This article will discuss how to regulate the half-life of single domain antibodies to improve their application value in the field of therapy.</p> <p><strong>Why do single domain antibodies have a shorter half-life than traditional antibodies?</strong></p> <p>There are two main reasons for the short <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/sdab/extending-half-life-of-sdab-for-drug-development.htm">half-life of single domain antibodies</a></strong></span>:</p> <ol> <li>Glomerular filtration results in rapid clearance of single domain antibodies from the body, and the limit of glomerular filtration protein drugs is generally around 60 kDa. That is to say, small molecular weight antibody fragments or derivatives, such as single domain antibody (15 kDa) and scFv (28 kDa) or even Fab (50 kDa), will be cleared from the body through glomerular filtration, resulting in a short half-life. Among them, single domain antibodies have the smallest molecular weight among all antibody types, and their half-life is often only tens of minutes.</li> <li>Single domain antibodies lack the ability to specifically bind to FcRn. Traditional antibodies usually have a longer half-life because of their larger molecular weight and the ability to specifically bind to FcRn (neonatal Fc receptor). FcRn can protect the antibody from being degraded by lysosomes, and then the antibody is transported to the surface of the cell membrane with FcRn, loses its combination with FcRn, and is re-released into the plasma, thereby prolonging the half-life of the antibody.</li> </ol> <p><strong>Strategies to regulate the half-life of single domain antibodies</strong></p> <ol> <li>Increase the molecular weight: Increase the molecular weight of single domain antibodies through fusion proteins, such as HSA (human serum albumin) or multimer structures, and reduce the renal clearance rate.</li> <li>Combining with Fc fragments: Combining single domain antibodies with Fc fragments to extend its half-life by borrowing the function of FcRn.</li> <li>PEGylation: Modification of single domain antibodies with polyethylene glycol to prolong their circulation in the body.</li> <li>Liposome encapsulation: Encapsulate the single domain antibody in liposomes through nanotechnology to improve its stability and prolong its half-life.</li> </ol> <p>At present, single domain antibody drugs that have entered the clinic or have been marketed mainly use the method of fusing Fc, HSA, or HSA single domain antibodies to prolong the half-life.</p> <p><strong>Example</strong></p> <p><strong>Example 1</strong><strong>—</strong><strong>Half-life extension by fusion of anti-HSA single domain antibodies</strong></p> <p>HSA is the most abundant and most stable protein in human plasma, containing 585 amino acids and a molecular weight of about 66.5 kDa. The X-ray crystal structure of HSA shows that it is a heart-shaped molecule consisting mainly of α-helices and lacking β-sheets. It contains three homologous domains, DI, DII, and DIII, each of which is subdivided into A and B subdomains (DIA, DIB, DIIA, DIIB, DIIIA, and DIIIB). These domains are connected by long and flexible loops. Plasma concentrations of HSA are approximately 45 mg/ml (0.6 mM), with a circulating half-life of up to 20 days. When single domain antibodies are combined with HSA, their hydrodynamic radius and molecular weight will increase, glomerular filtration will be reduced, and their residence time <em>in vivo</em> will be greatly extended.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-126" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/X-ray-crystal-structure-of-HSA.png" alt="" width="302" height="223" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/X-ray-crystal-structure-of-HSA.png 561w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/X-ray-crystal-structure-of-HSA-300x221.png 300w" sizes="(max-width: 302px) 100vw, 302px" /></p> <p style="text-align: center;">Figure 1. X-ray crystal structure of HSA (PDB ID: 1uor)</p> <p><strong>Example 2</strong><strong>—</strong><strong>Extending the half-life of single domain antibodies by binding to Fc fragments</strong></p> <p>Antibody Fc fragments are constant regions of antibodies in which the amino acid sequence remains unchanged in different types of antibodies. The antibody type is determined by the specific structure of the FC fragment. Unlike monoclonal antibodies, single domain antibodies cannot achieve antibody-dependent cell-mediated cytotoxicity (ADCC) effects and serum stability due to the lack of Fc fragments, which require the participation of Fc receptors. The Fc receptor FcγRIIIa (CD16a) plays a key role in natural killer cells (NK)-mediated ADCC. By conjugating a single domain antibody to a recombinant humanized heavy-chain antibody form of the Fc fragment, its Fc-mediated effector function and blood residence time can be increased, but its diffusion advantage can also be reduced. IgG1 Fc was chosen as the fusion fragment of the single domain antibody because of the affinity of IgG1 and the Fc receptor protein.</p> <p><strong>Example 3</strong><strong>—</strong><strong>Multivalent Single Domain Antibodies</strong></p> <p><strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/multivalent-sdab-development.htm">Multivalent single domain antibodies</a></span></strong> have a larger molecular weight and are effective against glomerular filtration.</p> <p><strong>Example 4</strong><strong>—</strong><strong>Extending the half-life of single domain antibodies by PEGylation</strong></p> <p>Polyethylene glycol (PEG for short) is formed by the polymerization of ethylene glycol and belongs to the class of polyether substances. It has good water solubility, extremely low immunogenicity and toxicity. PEGylation refers to the covalent combination of drugs with PEG to improve the pharmacokinetics, pharmacodynamics, and immunological properties of drugs and improve their therapeutic effects. PEGylation can change the physical and chemical properties of drugs, enhance the retention time of drugs in vivo, prolong the half-life, and improve the binding affinity of cell receptors, thereby improving tumor targeting and reducing the incidence of adverse events. PEG can also improve the solubility and stability of proteins, which is helpful for the production and storage of drugs. Therefore, PEG is widely used in drug delivery and drug modification technologies and can be directly coupled with drugs or attached to the surface of drugs and encapsulated in nanomaterials.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-127" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/Main-advantages-of-PEGylated-protein.png" alt="" width="460" height="233" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/Main-advantages-of-PEGylated-protein.png 1320w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/Main-advantages-of-PEGylated-protein-300x152.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/Main-advantages-of-PEGylated-protein-768x389.png 768w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/08/Main-advantages-of-PEGylated-protein-1024x519.png 1024w" sizes="(max-width: 460px) 100vw, 460px" /></p> <p style="text-align: center;">Figure 2. Main advantages of PEGylated protein (Francesco M. Veronese, 2005)</p> <p>Due to the small molecular weight and short half-life of single domain antibodies, their performance in clinical applications is limited. Therefore, regulating the half-life of single domain antibodies is of great significance for expanding their application in the therapeutic field. At present, there are many methods that can effectively increase the half-life of single domain antibodies, such as increasing the molecular weight, combining with Fc fragments, fusing carrier proteins or anti-HSA single domain antibodies, pegylation, and liposome encapsulation. Some methods have entered the clinical trial stage or have been approved for marketing. We expect more single domain antibody drugs to be promoted to the clinic and marketed to promote the development of human health.</p> <p> </p> <p><strong>Reference</strong></p> <p>Chaudhury C, <em>et al</em>. The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. <em>J Exp Med</em>. 2003 Feb 3;197(3):315-22. doi: 10.1084/jem.20021829.</p> <p>Coppieters K, <em>et al</em>. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. <em>Arthritis Rheum</em>. 2006 Jun;54(6):1856-66. doi: 10.1002/art.21827.</p> <p>Hua, B. <em>et al</em>. A novel single domain antibody targeting TIGIT for cancer use in combination therapies. <em>Cancer Research</em> 81, 2451-2451 (2021).</p> ]]></content:encoded> </item> <item> <title>Advantages of Applying Single Domain Antibodies in the Field of Cell Therapy</title> <link>https://www.creative-biolabs.com/blog/sdab/uncategorized/advantages-of-applying-single-domain-antibodies-in-the-field-of-cell-therapy/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Thu, 01 Jun 2023 03:06:43 +0000</pubDate> <category><![CDATA[Uncategorized]]></category> <category><![CDATA[CAR-T]]></category> <category><![CDATA[single domain antibody-based CAR-T]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[Therapeutic sdAb]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=117</guid> <description><![CDATA[Single domain antibodies have been widely used as antigen-binding domains in CAR-T due to their small size, excellent stability, high affinity, and strong manufacturing feasibility. Single domain antibody-based CAR constructs have shown<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/uncategorized/advantages-of-applying-single-domain-antibodies-in-the-field-of-cell-therapy/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>Single domain antibodies have been widely used as antigen-binding domains in CAR-T due to their small size, excellent stability, high affinity, and strong manufacturing feasibility. Single domain antibody-based CAR constructs have shown promising functions on more than a dozen different tumor-specific targets. The resulting <a href="http://E:\sdab\novel-sdab-development.htm"><span style="color: #0000ff;"><strong>single domain antibody-based CAR-T</strong></span></a> or CAR-NK cells have shown antitumor effects both<em> in vivo</em> and <em>in vitro</em>. The application of single domain antibodies in CAR-T therapy has been well-documented from the laboratory to the bedside and is showing great potential in forming more challenging advanced CAR-T.</p> <p><strong>A Brief History of CARs </strong></p> <p>Chimeric receptors were first proposed by Zelig Eshhar and Gideon Gross in 1989. They replaced the Vα and Vβ regions of the αβ-T cell receptor (TCR) with the VH and VL of the antibody, respectively, to construct an artificial chimeric TCR in the form of VH-Cα/VL-Cβ or VL-Cα/VH-Cβ. This chimeric TCR can bind target cells in an MHC-independent manner and activate cells through the TCR mechanism.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-120" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-1.jpg" alt="" width="547" height="239" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-1.jpg 554w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-1-300x131.jpg 300w" sizes="(max-width: 547px) 100vw, 547px" /></p> <p style="text-align: center;">Figure 1. Schematic diagram of TCR, chimeric TCR, and different generations of scFv as CAR domains</p> <p>The currently used CARs are composed of four components: an extracellular antigen recognition domain (including a single-chain antibody fragment scFv or single domain antibody VHH); structural components, such as hinge and transmembrane domains; co-stimulatory signaling domains that provide maintenance of CAR-T cell effector functions; and CD3ζ activation domains.</p> <p>The first-generation CARs contained only the CD3ζ chain signaling domain. Although the first generation of CAR-T cells can specifically kill targeted tumor cells <em>in vitro</em> and in mouse models. However, tumor cells rarely express co-stimulatory receptor ligands such as B7, and the first-generation CARs did not have co-stimulatory receptor domains. As a result, they showed low cell killing activity and short persistence <em>in vivo</em>, and did not have clinical effectiveness.</p> <p>The second-generation CARs were inserted with co-stimulatory domains, such as CD28, 4-1BB, or OX40, between the transmembrane sequence and the ITAM domain of CD3ξ. The second-generation CAR-Ts exhibited optimized T cell activation, increased antigen-dependent proliferation <em>in vitro</em>, enhanced persistence <em>in vivo</em>, and more potent antitumor activity. The second-generation CARs with CD28 or 4-1BB co-stimulatory domains are currently well established, and three FDA-approved anti-CD19 CAR-T cells are designed based on this structure.</p> <p>The third-generation CARs were added with additional co-stimulatory domains to further enhance T cell activation. Typical intracellular domains consisted of CD28/4-1BB/CD3ξ or CD28/OX40/CD3ξ. Clinical trials showed that compared with the second-generation CARs, CAR-T cells carrying the third-generation CARs exhibited stronger expansion and longer survival time, especially in patients with mild disease and lower levels of normal B cells .</p> <p>The fourth-generation CARs were added with the ability to secrete cytokines or antibodies, as well as enhanced anti-tumor activity by regulating the tumor immune microenvironment. The fourth-generation CAR-T cells combined direct anti-tumor function with the immunomodulatory ability to release cytokines at the tumor site, avoiding the adverse effects of cytokines caused by systemic administration, and they were expected to be used in the targeted therapy of solid tumors.</p> <p>The fifth-generation CAR is designed based on the second-generation CAR, adding cytokine receptors to its intracellular domain.</p> <p><strong>Advantages of Replacing scFv with Single Domain Antibody As CAR Targeting Domain </strong></p> <p><strong>No Aggregation Risk</strong></p> <p>ScFv, as a CAR, is easy to accumulate on the cell surface to trigger the activation of effector cells and a cytotoxic signaling cascade, leading to T cell exhaustion. The main reasons for the high self-aggregation tendency of scFvs are exposed free hydrophobic residues on the weight variable domains, and unstable folding of the VH/VL regions. In contrast, VHH-based CAR-T cells do not have this risk.</p> <p style="text-align: center;"><img decoding="async" loading="lazy" class="aligncenter wp-image-118" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-2.png" alt="" width="317" height="358" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-2.png 554w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-2-265x300.png 265w" sizes="(max-width: 317px) 100vw, 317px" /></p> <p style="text-align: center;">Figure 2. Schematic diagram of scFv aggregation on the surface of CAR-T</p> <p><strong>Low immunogenicity</strong></p> <p>The linker peptide sequence linking VH and VL in the scFv structure, as well as the backbone sequence of the murine antibody, can lead to immunogenicity risks, leading to the generation of anti-drug antibodies (ADA) <em>in vivo</em>. The ADA effect may neutralize the function of CAR-T cells, causing severe side effects, the loss of CAR-T cells, and even the failure of CAR-T therapy. In contrast, VHH does not require linker peptides, and the sequence similarity of VHH to human VH (VH3 gene family) is as high as 75-90%, so the risk of immunogenicity is unlikely to occur.</p> <p><strong>Bispecific CAR</strong></p> <p>The structure of scFv limits the potential to construct more complex CARs. When a bispecific CAR is constructed, the cross-pairing of VH and VL in two independent scFv molecules may lead to reduced affinity, and the size of multiple scFv inserted genes affects the viral packaging efficiency. VHH is more favorable than scFv, which is prone to be mismatched, to be constructed as the targeting domain of bispecific CAR, and the efficiency of transfection and virus packaging is also better than that of scFv.</p> <p>In addition to the above advantages, VHH also has a more favorable structure in terms of antigen epitope binding, solubility, and stability. Due to these properties, VHHs have a unique potential for developing various forms of CAR-T.</p> <p><strong>Research Progress of Single Domain Antibodies in the Field of CAR-T Therapy</strong></p> <p><strong>Single Domain Antibody-Based CAR</strong></p> <p style="text-align: center;"><img decoding="async" loading="lazy" class="aligncenter wp-image-119" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-3.png" alt="" width="348" height="411" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-3.png 554w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-3-254x300.png 254w" sizes="(max-width: 348px) 100vw, 348px" /></p> <p style="text-align: center;">Figure 3. Schematic diagram of CAR-T design with single domain antibody as targeting domain</p> <p>BCMA is a transmembrane activator and calcium regulator that plays an important role in the maturation and differentiation of B cells into plasma cells. Because malignant plasma cells express high levels of BCMA, they are an important target for immunotherapy of various cancers. Cilta-cel was approved by the FDA in 2022 for the treatment of adult relapsed/refractory multiple myeloma (MM). It is the first FDA-approved CAR-T in China and the world that uses a single domain antibody as the targeting domain. Cilta-cel adopts a unique <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://E:\sdab\multivalent-sdab-development.htm">bivalent single domain antibody</a></span></strong> design, and its approval for marketing shows that it is feasible to use single domain antibodies to develop and manufacture bispecific/bivalent CAR-T cells.</p> <p>In addition to the approved Cilta-cel using a single domain antibody as the CAR-T targeting domain, current research on the CAR-T targeting domain of single domain antibodies has involved many popular targets, including VEGFR2, HER2, PSMA, TAG-72, GPC2, CD38, CD33, CD7, MUC1, EGFR, CD20, CD105, PD-L1, and EIIIB.</p> <p><strong>Single Domain Antibody-Based Targeting Modules</strong></p> <p>UniCAR T cells are an improved T cell therapy based on CAR technology. Unlike traditional CAR T cells, UniCAR T cells do not directly bind to tumor cells, but are redirected to specific cell surface antigens by binding to targeting modules. Due to the small size of single domain antibody molecules, they can penetrate tumor tissues more easily, and their preparation and synthesis are more flexible, so they are a good choice for targeting modules. Research data also demonstrated that bivalent VHH-based targeting modules can redirect UniCAR-expressing T cells to cancer cells expressing low levels of EGFR antigen.</p> <p style="text-align: center;"><img decoding="async" loading="lazy" class="aligncenter wp-image-121" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-4.png" alt="" width="307" height="405" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-4.png 554w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/06/fig.-4-227x300.png 227w" sizes="(max-width: 307px) 100vw, 307px" /></p> <p style="text-align: center;">Figure 4. Redirection of UniCAR-expressing T cells to target specific antigens of interest using scFv, monovalent VHH, and bivalent VHH-based targeting modules</p> <p><strong>CAR-T with Autocrine Single Domain Antibody</strong></p> <p>At present, a number of CAR-T drugs have been approved for marketing in the world, but they are all targeting hematological tumors. While there are more patients with solid tumors, and the local microenvironment of tumors is more complex. The fourth-generation CAR-T is expected to regulate the tumor immune microenvironment of solid tumors through autocrine antibodies or cytokines to enhance its anti-tumor activity. Among them, the fastest progress is the autocrine PD1 single domain antibody MSLN-CAR T cell product (BZD1901). The drug is administered through intravenous infusion, and the reinfused CAR-T cells are activated after recognizing tumor antigens, killing tumor cells through cytotoxicity, and releasing PD1 single domain antibodies at the same time, which bind to PD1 on the surface of T cells to block PD- 1/PD-L1 signaling, change the inhibitory immune microenvironment, and exert a synergistic effect to kill tumor cells.</p> <p><strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://E:\sdab\">Single domain antibodies</a></span></strong> are an emerging and malleable tool in the field of cell therapy. Compared with traditional single-chain antibodies, single domain antibodies can more easily penetrate tumor cells, better recognize tumor targets, and produce anti-tumor effects. Single domain antibodies can also be used to construct multifunctional CAR-T cells, which can improve the efficacy and safety of treatment by targeting multiple targets. In addition, single domain antibodies can also be used as the targeting module of UniCAR-T to dynamically regulate and monitor CAR-T cells. With the deepening of its research, it is believed that the application of single domain antibodies in cell therapy will become more and more extensive.</p> <p> </p> <p><strong>Reference</strong></p> <p>Safarzadeh Kozani, Pouya, <em>et al</em>. “Nanobody-based CAR-T cells for cancer immunotherapy.” <em>Biomarker Research</em> 10.1 (2022): 24.</p> <p>Bao, C., <em>et al</em>. “The Application of Nanobody in CAR-T Therapy. <em>Biomolecules</em> 2021, 11, 238.” (2021).</p> <p>Ramos, Carlos A., <em>et al</em>. “In vivo fate and activity of second-versus third-generation CD19-specific CAR-T cells in B cell non-Hodgkin’s lymphomas.” <em>Molecular Therapy</em> 26.12 (2018): 2727-2737.</p> <p>Albert, Susann, <em>et al</em>. “From mono-to bivalent: improving theranostic properties of target modules for redirection of UniCAR T cells against EGFR-expressing tumor cells in vitro and in vivo.” <em>Oncotarget</em> 9.39 (2018): 25597.</p> ]]></content:encoded> </item> <item> <title>Most Popular Antibody Fragment: Single Domain Antibody vs. ScFv</title> <link>https://www.creative-biolabs.com/blog/sdab/biological-knowledge/most-popular-antibody-fragment-single-domain-antibody-vs-scfv/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Fri, 14 Apr 2023 07:25:18 +0000</pubDate> <category><![CDATA[Biological Knowledge]]></category> <category><![CDATA[scFv]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[VHH]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=111</guid> <description><![CDATA[With the advent of recombinant DNA technology, many antibody fragments have been developed. Among them, camelid heavy chain variable domains (VHHs) and single chain variable domains (ScFv) are the most favored variable<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/biological-knowledge/most-popular-antibody-fragment-single-domain-antibody-vs-scfv/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>With the advent of recombinant DNA technology, many antibody fragments have been developed. Among them, camelid heavy chain variable domains (VHHs) and single chain variable domains (ScFv) are the most favored variable domains. Camelid <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/">single domain antibodies</a></span></strong> (sdAbs) are being extensively investigated as an alternative due to their superior chemical and physical properties such as higher solubility, stability, smaller size, and lower production cost.</p> <p><strong>Structures of Single Domain Antibodies and ScFv</strong></p> <p>ScFv is an engineered form of Fv in which instead of a disulfide bond, two variable domains are linked together by a flexible linker. The length and amino acid composition of this linker play an important role in the correct folding of the protein. It is usually 10-25 amino acids in length, in which the Glu-Lys stretch increases solubility, and the Gly-Ser stretch increases flexibility. Each of the two variable domains in ScFv has three complementarity determining regions (CDRs) linked to the framework regions (FRs). CDRs are responsible for antigen binding, and their structure is complementary to the epitope, while the framework regions (FRs) act as scaffolds without much variability compared with CDRs. The contribution of each CDR in antigen binding is different. For example, HCDR3 in the heavy chain contributes 29% to binding specificity, whereas LCDR2 only contributes 4%.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-113" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-1-The-differences-between-scFv-and-single-domain-antibody-in-structure.png" alt="" width="485" height="335" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-1-The-differences-between-scFv-and-single-domain-antibody-in-structure.png 554w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-1-The-differences-between-scFv-and-single-domain-antibody-in-structure-300x207.png 300w" sizes="(max-width: 485px) 100vw, 485px" /></p> <p style="text-align: center;">Fig. 1 The differences between scFv and single domain antibody in structure. (Asaadi, Y., <em>et al</em>., 2021)</p> <p>A hypothesis whether a single heavy chain could retain the binding affinity of the parental Ab was raised due to the higher contribution of VH in antigen binding. After some research, it was found that some of their unfavorable properties, such as low affinity, poor solubility, and higher production cost hindered their development.</p> <p>The discovery of heavy chain-only antibodies (HCAbs) in camels and immunoglobulin neoantigen receptors (IgNARs) in cartilaginous fish represents a new starting point for the development of single domain antibodies. The antigen-binding domain (VHH and V-NAR) of these specific immunoglobulins is a high-affinity V-type domain that has evolved without the drawbacks of the conventional single heavy chain segment of the past. These excellent properties originate from major adjustments in sequence and structure. VHH fragments are used more frequently due to easier handling, stronger antibody responses, and higher recombinant expression yields than shark V-NARs for camelid single domain antibodies.</p> <p>Similar to VH, camelid VHH consists of nine β-bands that form a typical IgV fold, but deletion of VL results in significant differences between these two fragments, especially in FR2 and hypervariable loops. In a traditional VH region, FR2 consists of four highly conserved hydrophobic amino acids (Val37, Gly44, Leu45, and Trp47), which together with Gln39, Gly44, Tyr91, and Trp103 form a conserved hydrophobic interface to facilitate VL connection. In single domain antibodies absent VL, these four hydrophobic residues were replaced by more hydrophilic amino acids (Phe37, Glu44, Arg45, and Gly47) to avoid exposure of such a large hydrophobic region to solvent. In addition to this substitution, residues near this interface rotated their side chains without deforming the C-α backbone, thereby increasing the hydrophilicity of the VHH surface. Furthermore, the CDR3 domain of VHH folds over this interface to protect amino acids previously covered by VL. These changes illustrate the higher solubility of VHHs than ScFv and conventional single heavy chain VHs.</p> <p>In VHH, the expansion of loops in the hypervariable region compensates for the loss of diversity in the 3 VL CDRs and VH-VL combinations. The extension of CDR1 and CDR3 provides a 600-800A2 antigen interaction surface provided by six loops from the VH-VL domain. Furthermore, elongated CDR1 imprinted with mutational hotspots in the VHH germline compensates for VL partner variability. While the extended CDR3 can reach epitopes that are barely accessible to conventional antibodies, the enlarged loop implies greater flexibility, hindering the entropic binding of antigens. To solve this problem, VHHs have evolved an additional disulfide bond towards CDR1, CDR2, or FR2. All of these structural features increase epitope diversity and allow for a wide variety of geometric loop structures that deviate radically from the canonical loop structures defined by conventional antibodies and contribute to the orientation of the CDR3 toward the antigen.</p> <p><strong>Physicochemical Properties of Single Domain Antibody and ScFv</strong></p> <p>Due to significant structural differences, ScFvs and single-domain antibodies exhibit different properties <em>in vitro</em> and <em>in vivo</em>.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-114" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-2-A-comparison-between-scFv-and-single-domain-antibody-in-properties.png" alt="" width="584" height="299" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-2-A-comparison-between-scFv-and-single-domain-antibody-in-properties.png 831w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-2-A-comparison-between-scFv-and-single-domain-antibody-in-properties-300x153.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2023/04/Fig.-2-A-comparison-between-scFv-and-single-domain-antibody-in-properties-768x393.png 768w" sizes="(max-width: 584px) 100vw, 584px" /></p> <p style="text-align: center;">Fig. 2 A comparison between scFv and single domain antibody insdAb properties. (Asaadi, Y., <em>et al</em>., 2021)</p> <p><strong>Size</strong></p> <p>First and foremost, the two fragments differ significantly in size. ScFvs are almost twice the size of single domain antibodies and weigh around 30 kDa. The smaller size facilitates the genetic manipulation of VHH, and the presence of only three antigen-binding loops can easily enhance its inherent antigenic propensity. The smaller size of VHHs also results in their shorter half-life in the blood due to filtration and degradation by the kidneys. This feature may be helpful as it leads to high tissue permeability. But disadvantageously, because their molecular weight is lower than the glomerular filtration cut-off size (65 KDa), there may be problems in some clinical treatments that require prolonged antibody circulation. This limitation has led to the development of half-life extension strategies that combine VHHs with additional molecules. One of the most popular of these involves the addition of stabilizing groups, such as polyethylene glycol (PEG) molecules, which can slow blood clearance. A fusion of long-circulating serum proteins such as albumin or specific binders of albumin can effectively <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/extending-half-life-of-sdab-for-drug-development.htm">increase the half-life of VHH</a></span></strong> in blood. Fc fusions can stabilize them in the blood and meanwhile stimulate the immune system to reach the target site. In addition, Fc or albumin fusions make antibody fragments larger and enable FcRn-mediated circulation to increase the half-life in the blood.</p> <p><strong>Solubility and Stability</strong></p> <p>As mentioned earlier, in VHH, four highly conserved hydrophobic amino acids were replaced by more hydrophilic residues, resulting in a marked difference in properties between single domain antibodies and ScFv. In ScFv, these four residues (V37, G44, L45, and W47) in FR2 form a hydrophobic interface that facilitates VH-VL linkage. However, on the downside, this hydrophobic region reduces the solubility of ScFvs, causing them to have a high tendency to aggregate. Substitution of polar and minor amino acid residues at this position (F37 or Y37, E44, R45, and G47) makes them more hydrophilic and therefore more soluble than ScFv. Furthermore, this nonpolar-to-polar transition results in the molecular and thermodynamic stability of VHHs compared to ScFvs. Therefore, single domain antibodies are more resistant to chemical denaturants and proteases, and have higher stability under harsh pH or ionic strength. This <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/sdab-stability-improvement-services.htm">higher conformational stability of VHH</a></span></strong> also stems from the presence of additional disulfide bonds, which reduce the possibility of heat-induced aggregation and limit the flexibility of VHHs. Due to the higher stability, they show high refolding efficiency, which means that increasing or decreasing the sample temperature does not affect the single domain antibody conformation, or disengagement and binding of the target without any aggregation or denaturation. This structural rigidity is a clinically preferred attribute, as non-native protein aggregation is a common drawback of antibody therapy which in severe cases can increase immune response.</p> <p><strong>Immunogenicity</strong></p> <p>A major disadvantage of scFvs is that they are mostly derived from rodents, since hybridoma technology is only well developed in mice and rats. The sequence homology of mouse VL and VH to the human corresponding regions is only 53% and 51%, respectively. However, single domain antibodies show high sequence similarity to human VH (VH3 gene family), and about 75-90% homology is associated with lower immunogenicity in clinical applications. Therefore, VHH is more humanized. Even after the humanization of the murine scFv, the variable region of the scFv can still elicit an anti-idiotypic response. In addition, engineering scFv to reduce human anti-mouse antibody (HAMA) responses may inactivate injected scFv and reduce its clinical effectiveness, and hypersensitivity reactions may occur upon repeated administration. Furthermore, humanization reduced the binding affinity of these fragments, and CDR grafting may represent novel immunogenic epitopes. In general, the humanization of murine scFv can overcome some but not all these immunogenicity problems.</p> <p><strong>Affinity</strong></p> <p>Although ScFvs and single domain antibodies share similar affinities, they display a clear preference for epitopes. Single domain antibodies are more likely to access grooves and clefts on the surface of antigens such as ion channels, viral glycoproteins, or immune synapses, while ScFvs prefer flat and linear epitopes. These differences are due to the longer CDR3 loop in single domain antibodies that allows a highly convex shape to access concave epitopes. Single domain antibodies also exhibit good affinity for planar epitopes, suggesting that these fragments can form various interfacial complexes. Furthermore, single domain antibodies show a lower nonspecific background binding than ScFv.</p> ]]></content:encoded> </item> <item> <title>Molecular Therapy: Development of New-Generation of Fully Human Single-Domain Antibody-Drug Conjugates Targeting Solid Tumors</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/molecular-therapy-development-of-new-generation-of-fully-human-single-domain-antibody-drug-conjugates-targeting-solid-tumors/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Wed, 07 Sep 2022 06:25:08 +0000</pubDate> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[antibody-drug conjugate]]></category> <category><![CDATA[Single-domain Antibody]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=99</guid> <description><![CDATA[An antibody-drug conjugate (ADC) is formed by linking monoclonal antibodies and small molecule cytotoxic drugs through linkers. It has both the tumor targeting of antibody drugs and the strong tumor-killing activity of<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/molecular-therapy-development-of-new-generation-of-fully-human-single-domain-antibody-drug-conjugates-targeting-solid-tumors/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>An antibody-drug conjugate (ADC) is formed by linking monoclonal antibodies and small molecule cytotoxic drugs through linkers. It has both the tumor targeting of antibody drugs and the strong tumor-killing activity of chemotherapeutic drugs, becoming one of the hottest research and development directions in the field of biotechnology drugs. However, antibody-drug conjugates are limited by issues including the large molecular weight of full-length antibodies, poor tumor penetration, and low intratumoral diffusion efficiency, and their efficacy in solid tumors still needs to be broken through.</p> <p>Recently, a study developed a fully human <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/novel-sdab-development.htm">single-domain antibody-drug conjugate</a></span></strong> (UdADC) with high stability, rapid distribution, and strong tumor penetration, and confirmed that this new drug has significant advantages over traditional antibody-coupled drugs in tumor organs, tumor microspheres, and tumor models in mice.</p> <p>In the preliminary research, the team integrated the design concept of synthetic biology with the theoretical technology of antibody engineering, and established an innovative fully human single-domain antibody research and development system. In this study, the team further developed a high-affinity fully human single-domain antibody n501 against carcinoembryonic antigen 5T4.</p> <p><img decoding="async" loading="lazy" class="aligncenter wp-image-100" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/09/1-s2.0-S1525001622002428-fx1_lrg.jpg" alt="" width="435" height="435" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/09/1-s2.0-S1525001622002428-fx1_lrg.jpg 996w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/09/1-s2.0-S1525001622002428-fx1_lrg-150x150.jpg 150w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/09/1-s2.0-S1525001622002428-fx1_lrg-300x300.jpg 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/09/1-s2.0-S1525001622002428-fx1_lrg-768x768.jpg 768w" sizes="(max-width: 435px) 100vw, 435px" /></p> <p style="text-align: center;">A fully human single-domain antibody (UdAb) targeting the oncofetal antigen 5T4. (Wu, 2022)</p> <p>The team found that compared with the traditional camel-derived single-domain antibody, the fully human single-domain antibody n501 has better stability and can still maintain antigen-binding activity under severe storage conditions such as high temperature. The team analyzed the high-resolution three-dimensional structure of the complex of n501 and 5T4 by means of structural biology, and the result showed that the fully human single-domain antibody has a spatial structure very similar to the traditional camel-derived single-domain antibody. Moreover, n501 can bind tightly to eight leucine-rich repeats (LRRs) of the 5T4 antigen at the same time, revealing the molecular mechanism of n501’s high stability and high antigen affinity.</p> <p>Further, the researchers mutated the serine at position 85 in the fully human single-domain antibody n501 to cysteine to realize the site-specific conjugation of the small molecule drug SN38. This new type of drug is named fully human single-domain antibody-drug conjugate (UdADC). In order to make a systematic comparison with traditional mAb-based ADC drugs, the team developed a high-affinity fully human single-domain antibody m603 targeting the same target 5T4 using the fully human <a href="/sdab/one-stop-solution-for-sdab-development.htm"><span style="color: #0000ff;"><strong>single-domain antibody R&D platform</strong></span></a> established earlier. And then they used traditional coupling technology to couple SN38 to m603 to prepare the ADC. In <em>in vitro</em> anti-tumor cell experiments, both UdADC (n501-SN38) and ADC (m603-SN38) can effectively and specifically kill cell lines with high expression of 5T4. Because the fully human single domain antibody (13 kDa) has a smaller molecular weight than the traditional IgG mAb (150 kDa), the team found that UdADC has a far more superior tumor tissue penetration ability far superior than ADC.</p> <p>In the pancreatic cancer organ model and three-dimensional tumor microsphere model, the penetration depth of UdADC is much better than that of ADC based on traditional monoclonal antibodies, and can accumulate more effectively in the tumor, while ADC mostly stays on the superficial surface of the tumor. In addition, in the pancreatic cancer tumor-bearing mouse model, UdADC can quickly locate the tumor site and accumulate efficiently within half an hour after injection, so it can more effectively inhibit the growth of the tumor in mice and show more efficient anti-tumor activity.</p> <p>In summary, fully human single-domain antibody-drug conjugates have excellent characteristics of high stability, rapid distribution to tumor sites, deep tumor penetration, high tumor uptake, etc. It can be used as a research and development platform for a new generation of antibody conjugated drugs, in order to develop efficient anti-tumor targeted drugs for clinical treatment of solid tumors.</p> ]]></content:encoded> </item> <item> <title>Single-domain Antibodies and Undruggable Targets</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibodies-and-undruggable-targets/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Tue, 02 Aug 2022 02:46:33 +0000</pubDate> <category><![CDATA[sdAb News]]></category> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[Single-domain Antibody]]></category> <category><![CDATA[Therapeutic sdAb]]></category> <category><![CDATA[Tumor Targets]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=95</guid> <description><![CDATA[The single-domain antibody has similar antigen binding characteristics to the traditional antibody. But, because single-domain antibodies use a single Ig variable domain for antigen recognition, they can access epitopes that cannot be<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibodies-and-undruggable-targets/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>The <a href="/sdab/"><span style="color: #0000ff;">single-domain antibody</span></a> has similar antigen binding characteristics to the traditional antibody. But, because single-domain antibodies use a single Ig variable domain for antigen recognition, they can access epitopes that cannot be reached by conventional antibodies or antibody derivatives (e.g., scFv), such as penetrating protein surfaces or gaps in domain-domain media. In some cases, single-domain antibodies can cross-react with homologous targets from other species, which may contribute to the transition from preclinical application to clinical application. For example, anti-EGFR single-domain antibodies 8B6, anti-HER2 single-domain antibodies 2Rs15d, and single-domain antibodies against fibronectin EIIIB splicing variants cross-react with human and mouse antigens.</p> <p style="text-align: center;"><img decoding="async" loading="lazy" class="aligncenter wp-image-96" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/08/fig-1.png" alt="" width="596" height="428" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/08/fig-1.png 1080w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/08/fig-1-300x216.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/08/fig-1-768x552.png 768w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/08/fig-1-1024x736.png 1024w" sizes="(max-width: 596px) 100vw, 596px" /></p> <p style="text-align: center;">Fig.1 Single-domain antibodies and their targets associated with the tumor microenvironment</p> <ol> <li>The EGFR Family</li> </ol> <p>Members of the <span style="color: #0000ff;"><a style="color: #0000ff;" href="/sdab/symbol-egfr-6238.htm">epidermal growth factor receptor</a></span> (EGFR) family are usually overexpressed on the surface of epithelial-derived tumor cells and play a role in their proliferation, survival, and angiogenesis. Antibodies targeting EGF receptors have been proved to be successful in cancer treatment. For example, the commercial product cetuximab is a human-mouse chimeric monoclonal antibody that specifically binds to EGFR. Therefore, members of the EGFR family have become one of the first tumor markers targeted by single-domain antibodies. The single-domain antibodies targeting EGFR1 were identified by phage display, and specific conjugates were identified by competitive elution with ligand EGF. Using the same EGFR phage single-domain antibody library and selecting EGFR extracellular domain, the researchers identified single-domain antibodies 7C12, 7D12, and 9G8, in which 7C12 and 7D12 competed with cetuximab.</p> <p>Multivalent single-domain antibody molecules can be constructed by fusing single-domain antibody gene fragments or by chemical coupling. EGFR-specific single-domain antibodies form bivalent molecules in different combinations, all of which inhibit the proliferation of tumor cells in an epidermoid carcinoma model <em>in vitro</em>. Specifically, the combination of 7D12-9G8 anti-EGFR single-domain antibody performed best in inhibiting EGFR signal transduction and reducing the growth of human epidermoid carcinoma A431 cells. When bound to serum albumin binding single-domain antibody Alb1, this structure is called CONAN-1, which strongly inhibits EGF-induced signal transduction and leads to tumor regression in A431 xenografted mice.</p> <p>Using similar methods, other researchers have obtained anti-EGFR single-domain antibodies 8B6 and OA-cb6. In addition, single-domain antibodies that recognize HER2, another member of the EGFR family, have been shown to specifically target tumors derived from HER2+SKOV3 ovarian cancer cells. Single-domain antibodies 11A4 and 5F7GGC targeting HER2 have been used in various clinical applications.</p> <ol start="2"> <li>VEGFR2 and VEGF</li> </ol> <p>Vascular epithelial growth factor receptor 2 (VEGFR2) is a part of the human VEGFR receptor family, which exists in vascular endothelial cells. Its ligand VEGF is secreted by cell types such as macrophages and tumor cells, inducing downstream signaling pathways to participate in cell proliferation, angiogenesis, and metastasis, which makes VEGF and VEGFR2 attractive targets for single-domain antibody-based therapies. For example, VEGF (VEGFR) target antibodies can specifically bind to VEGF (VEGFR) to inhibit downstream signal pathways and angiogenesis.</p> <p>Anti-VEGFR2 single-domain antibody 3VGR19 was obtained by phage display on the recombinant extracellular domain of the VEGFR2 receptor. It can inhibit VEGFR2 signal transduction and thus inhibit the formation of capillary-like structure, which has been confirmed in an <em>in vitro</em> study of human umbilical vein endothelial cells (HUVEC).</p> <p>Some researchers isolated anti-angiogenic VEGFR2-D3 specific single-domain antibody NTV1 from HuSdl<img src="https://s.w.org/images/core/emoji/14.0.0/72x72/2122.png" alt="™" class="wp-smiley" style="height: 1em; max-height: 1em;" />. HuSdl<img src="https://s.w.org/images/core/emoji/14.0.0/72x72/2122.png" alt="™" class="wp-smiley" style="height: 1em; max-height: 1em;" /> is a human single-domain antibody library of “camelid” human antibodies. In a similar manner, VEGF single-domain antibodies were also obtained. These single-domain antibodies inhibited endothelial cell proliferation<em> in vitro</em> angiogenesis experiments using HUVEC. One of the humanized versions of Nb42 has also been produced. In addition, the single-domain antibody VA12, which specifically targets the VEGF-A binding domain, has shown the potential for anti-angiogenesis in chorioallantoic membrane assay.</p> <ol start="3"> <li>C-Met and HGF</li> </ol> <p><a href="/sdab/symbol-hgf-sf-6325.htm"><span style="color: #0000ff;">Hepatocyte growth factor</span></a> (HGF) binding to c-Met receptors can activate pathways that lead to cancer progression, angiogenesis, and metastasis. The overexpression of HGF and c-Met receptors is associated with a poor prognosis of several different epithelial and non-epithelial cancers. Currently, single-domain antibodies targeting c-Met and HGF have been produced. Schmidt Sl ø rdahl <em>et al.</em> used a bispecific single-domain antibody, one of which targets c-Met and the other that binds to human serum albumin to prolong the half-life. This bispecific anti-c-Met single-domain antibody inhibits the interaction between c-Met and HGF and leads to a decrease of cell migration and adhesion in multiple myeloma cells. This bispecific single-domain antibody is more effective than the traditional bivalent anti-c-Met monoclonal antibody in inhibiting tumor growth.</p> <p>Jo Vercammen <em>et al.</em> reported that two α HGF- single-domain antibodies called 1E2 and 6E10 showed a dose-dependent inhibition of HGF-induced proliferation of Bx-PC3 human pancreatic cancer cells. Nude mice bearing human glioma U-87MG xenograft were treated with an anti-HGF single-domain antibody. Compared with the control group, the anti-HGF single-domain antibody significantly inhibited tumor growth. Both of the above single-domain antibodies show potential for the treatment of multiple myeloma and other HGF-c-Met-driven cancers.</p> <ol start="4"> <li>Other Targets</li> </ol> <p>In addition to the above molecules, many other tumor-associated antigens have also been used as targets for the development of single-domain antibodies. Chemokine receptors, belonging to G protein-coupled receptors (GPCR), are overexpressed in many kinds of malignant tumors. Chemokines and their receptors drive the migration and activation of various cell types associated with innate and adaptive immune responses. If the goal is to interfere with cell migration, given the excellent tissue permeability of single-domain antibodies, these molecules will be ideal targets. Single-domain antibodies targeting GPCR and its ligands include reagents against human CXCR2, CXCR4, CXCR7, CXCL11, and CXCL12, as well as viral GPCRUS28.</p> <p>In addition, single-domain antibodies targeting human tumor-associated transmembrane/membrane proteins have been identified, such as carcinoembryonic antigen (CEA), prostate-specific membrane antigen (PSMA), and human and mouse macrophage mannose receptor (MMR).</p> <p>Immune cell markers are also important targets being investigated, such as human CD7, human and mouse CTLA-4, human and mouse PDL-1, mouse CD8, mouse CD11b, human CD20, human CD38, mouse CD45, mouse Ly-6C/Ly-6G, and human and mouse MHC-II. Other targets include fibronectin, TUFM, CapG, CAIX, CD33, human and mouse CD47, mouse ARTC2, and TNF α.</p> ]]></content:encoded> </item> <item> <title>Single Domain Antibodies and Specific Membrane Receptors (Blood-brain Barrier)</title> <link>https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibodies-and-specific-membrane-receptors-blood-brain-barrier/</link> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Tue, 28 Jun 2022 05:42:16 +0000</pubDate> <category><![CDATA[sdAb News]]></category> <category><![CDATA[sdAb Research]]></category> <category><![CDATA[Blood-brain Barrier]]></category> <category><![CDATA[Single-domain Antibody]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/sdab/?p=73</guid> <description><![CDATA[Single domain antibodies are derived from llama heavy chain-only antibodies (HCAbs). They represent a new generation of biologics with unique properties. Single domain antibodies have excellent tissue distribution, high temperature and pH<a class="moretag" href="https://www.creative-biolabs.com/blog/sdab/sdab-research/single-domain-antibodies-and-specific-membrane-receptors-blood-brain-barrier/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>Single domain antibodies are derived from llama heavy chain-only antibodies (HCAbs). They represent a new generation of biologics with unique properties. <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="https://www.creative-biolabs.com/sdab/">Single domain antibodies</a></strong></span> have excellent tissue distribution, high temperature and pH stability, are easy to produce recombinantly, and can be easily converted into different formats. In addition, single domain antibodies have the unique ability to bind molecular clefts, such as the active site of an enzyme, thereby interfering with the function of the target protein. Over the past decade, a number of single domain antibodies against inflammation-related proteins have been developed with the aim of modulating their immune function. Here, we outline recently developed single domain antibodies targeting immune pathways associated with neuroinflammation. Furthermore, we highlight strategies to modify single domain antibodies, which enable them to overcome the blood-brain barrier and serve as highly specific therapies for acute inflammatory brain injury.</p> <p><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-65" src="http://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/06/sdab-BBB.png" alt="" width="850" height="401" srcset="https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/06/sdab-BBB.png 850w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/06/sdab-BBB-300x142.png 300w, https://www.creative-biolabs.com/blog/sdab/wp-content/uploads/sites/8/2022/06/sdab-BBB-768x362.png 768w" sizes="(max-width: 850px) 100vw, 850px" /></p> <p style="text-align: center;">Single domain antibodies are derived from llama heavy chain-only antibodies (HCAbs)</p> <p>To combat infectious diseases, many single domain antibodies against bacterial and viral antigens have been produced to prevent or improve pathogenicity. More recently, key players in immune pathways have become targets of single domain antibodies to modulate immune responses. This has generated single domain antibodies against Fc receptors (FcRs), chemokine receptors, chemokines, cytokines and extracellular enzymes. These single domain antibodies generally exhibit high target specificity and can modulate the function of their targets in an agonistic or antagonistic manner.</p> <p>In 2010, Martine Smit’s group reported the generation of two single domain antibodies that specifically target the chemokine receptor CXCR4. Single domain antibodies 238D2 and 238D4 showed potent competitive inhibition of CXCL12 binding to CXCR4. When injected into monkeys, anti-CXCR4 single domain antibodies induced the mobilization of hematopoietic stem cells by disrupting the CXCR4/CXCL12 axis that contributes to hematopoietic stem cell residency in the bone marrow. In 2013, the same group reported the generation of antagonistic single domain antibodies against CXCR7. When injected into mice, these single domain antibodies showed beneficial effects in an in vivo xenograft model of head and neck cancer. Meanwhile, the same group published a set of single domain antibodies specifically targeting CCL2, CCL5, CXCL11 and CXCL12. The binding of single domain antibodies to CXCL11 and CXCL12 inhibited chemokine receptor binding, thereby preventing in vitro cell migration induced by chemokine receptor activation. Since a variety of chemokines and their receptors are known to facilitate the migration of immune cells to the brain following brain injury, the single domain antibodies described above may be promising therapeutic alternatives for the treatment of acute brain injury. Muruganadam et al. in 2002 described the selection of a single domain antibody (FC5) that crossed human blood-brain barrier endothelial cells in vitro. Later, the same group proposed that FC5 binds to the putative α(2,3)-sialoglycoprotein receptor and is transcytosed via clathrin vesicles. The results demonstrate that FC5 conjugated to the opioid peptide Dal can be used as an in vivo drug delivery shuttle to induce significant analgesic responses compared to the unconjugated Dal peptide. Other approaches utilize receptor-mediated transcytosis for brain targeting. In addition, transferrin receptors and insulin receptors are also used for receptor-mediated endocytosis of small-molecule drugs and therapeutic proteins. These studies suggest that single domain antibodies that bind to these receptors and trigger transcytosis may be a promising alternative for ligand-based drug delivery to the brain. A study by Pierre Lafaye’s group reports that single domain antibodies with a high electrical point (pI) can spontaneously cross the blood-brain barrier. Such single domain antibodies can not only enter the brain, but can even penetrate cells and bind to intracellular proteins.</p> <p>After acute brain injury, such as ischemic stroke or trauma, danger-associated molecular patterns (DAMPs) released by necrotic cells activate resident microglia, leading to the production of pro-inflammatory mediators such as cytokines and chemokines that attract other immune cells. Preventing local inflammation may be a means of preventing further loss of brain tissue. High mobility group box 1 (HMGB1) and nucleotide DAMPs such as adenosine triphosphate (ATP) are released during cerebral ischemia. Antibody-mediated neutralization of HMGB1 or its receptor antagonism significantly reduced infarct size in middle cerebral artery occlusion in a mouse model of ischemia/reperfusion. The ATP receptor P2X7, which mediates inflammasome formation and cell death, was antagonized by small-molecule inhibitors in a mouse model of transient focal ischemia, again resulting in reduced infarct size. Both the HMGB1/RAGE and ATP/P2X7 pathways show promising targets for single domain antibody-mediated antagonism.</p> <p>Single domain antibodies have been shown to be versatile and effective biologics suitable for the treatment of inflammatory diseases. Due to their unique structure, single domain antibodies have the potential to modulate the function of cell surface and secreted proteins in an agonistic or antagonistic manner. They can be genetically engineered to extend their half-life in the body, and in proof-of-concept studies, they have been shown to act as shuttles for the delivery of therapeutic agents through the BBB. Future studies will have to demonstrate whether this strategy can also be used to deliver therapeutic single domain antibodies to the brain to improve neuroinflammation outcomes.</p> ]]></content:encoded> </item> </channel> </rss>