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		<title>Common Engineering Techniques and Strategies in Antibody Discovery</title>
		<link>https://www.creative-biolabs.com/blog/index.php/engineering-techniques-antibody-discovery/</link>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Mon, 04 Jun 2018 05:36:29 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Biological Knowledge]]></category>
		<category><![CDATA[Drug Discovery Research]]></category>
		<category><![CDATA[adcc]]></category>
		<category><![CDATA[antibody fragment]]></category>
		<category><![CDATA[cdc]]></category>
		<category><![CDATA[chimerization antibody]]></category>
		<category><![CDATA[fcrn]]></category>
		<category><![CDATA[Humanized Antibody]]></category>
		<category><![CDATA[recombinant antibody]]></category>
		<category><![CDATA[ScFv]]></category>
		<category><![CDATA[Therapeutic Antibody]]></category>
		<guid isPermaLink="false">https://www.creative-biolabs.com/blog/?p=1259</guid>

					<description><![CDATA[In the process of discovering monoclonal antibodies, after screening antibody libraries to obtain excellent candidate molecules that can specifically bind to targets, it is necessary to enhance certain pharmacological and other functions<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/engineering-techniques-antibody-discovery/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p><span style="font-size:15px">In the process of discovering monoclonal antibodies, after screening antibody libraries to obtain excellent candidate molecules that can specifically bind to targets, it is necessary to enhance certain pharmacological and other functions through certain engineering techniques. Various strategies can improve the kinetic functions of antibody drugs, including affinity maturation, chimerization or humanization, and Fc modification. In order to improve the drug-formability and stability of antibodies, there is a need to improve the stability and aggregation of antibodies, as well as to reduce immunogenicity, some of which can also be achieved by computer tools.</span> </p>
<p><strong>1. Chimerization or humanization reduces immunogenicity</strong></p>
<p><span style="font-size:15px">In the early stage, when the mouse antibody enters the human body, it will respond to the human immune system and produce a human anti-mouse antibody (HAMA) response, inducing the hypersensitivity reaction or the effect of the mouse antibody to be neutralized by the anti-mouse antibody. The solution to this problem is to bring the antibodies closer to human antibodies by chimerization or humanization. A human-mouse chimeric antibody is a mouse antibody variable region gene fragment that is ligated to a human antibody constant region gene. <span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/antibody-humanization.html" target="_blank">Humanized antibodies</a></span> are transplanted into the CDR region of the mouse antibody to the corresponding site of human antibodies so that humanization can reach more than 90%.</span> </p>
<p><span style="font-size:15px">With the development of computer-assisted simulation technology, humanized antibody technology has entered the second generation. Its strategies include part of CDR transplantation, in which part of the CDRs necessary for antibody binding to antigens are grafted onto human antibody frameworks to obtain less immunogenic humanized antibodies;  Surface remodeling,  referring to the mosaic/remodeling of surface residues of mouse CDRs and FRs, similar to the profile of human antibody CDRs and the form of human FRs;  transplantation of specific decision regions, in which the SDR formed by the key amino acid in the antibody that closely interacts with the antigen is transplanted to the corresponding position of the human antibody. In addition, in order to ensure the affinity of antibodies, the researchers also proposed strategies for CDR compensation, positioning retention and template replacement.</span> </p>
<p><span style="font-size:15px">Although the humanization of non-human antibodies is still common today, the emergence of transgenic mice and, more importantly, the full human (synthetic) antibody library has reduced the need for these strategies. However, any therapeutic monoclonal antibody is immunogenic regardless of its source. Therefore, this question should always be considered when assessing the patient&#8217;s risk-return rate.</span> </p>
<p><strong>2. <em>In vitro</em></strong> <strong>affinity maturation</strong></p>
<p><span style="font-size:15px">For <span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creativebiolabs.net/Therapeutic-Antibodies.htm" target="_blank">therapeutic antibodies</a></span>, an increase in antibody affinity can help reduce dosage and toxic side effects. The <em>in vitro</em> affinity maturation strategy of antibodies is derived from the <em>in vivo</em> antibody maturation process (Figure 1). In this strategy, a small-capacity library of concentrated mutations and specific site mutation is constructed or the mutations are introduced into the variable regions of the original antibody, usually CDRs, and high-affinity antibodies are screened by surface display technology. Particular emphasis needs to be placed on the unique role of CDR H3 in antigen recognition and potential conformational changes. At the same time, the surrounding FR area should also be taken into account. In general, 10-fold improvement in affinity can be obtained.</span> </p>
<p><span style="font-size:15px">In order to mimic the adaptive immune system, an SHM <em>in vitro</em> mammalian system, mediated by recombinant AID, is developed with human variable sequence rearrangement, cell surface display and weak binding separation. Low-affinity monoclonal antibodies are preliminarily identified to produce a low pM affinity in SHM<em> in vitro</em>. In SHM, Ig gene insertion and deletion enhance<em> in vivo</em> and <em>in vitro</em> affinity maturation.</span> </p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/engineered-antibody-format.jpg"><img decoding="async" fetchpriority="high" class="size-full wp-image-1260 aligncenter" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/engineered-antibody-format.jpg" alt="engineered antibody format" width="600" height="350" /></a></p>
<p style="text-align: center;">Figure 1 Engineered technology enhances antibody pharmacology and stability</p>
<p><span style="font-size:15px">Computer tools also apply to the maturation of affinity. Early computer simulations focused on ionic interactions and single mutants to increase 10 to 100 times higher affinity of mAbs (such as cetuximab and bevacizumab). Another combination method based on CDR3 randomization and rational design increased the affinity of low μM scFv by 450 times.</span> </p>
<p><span style="font-size:15px">Not all therapeutic applications are eager for a high affinity for optimal efficacy. In the context of cancer, high-affinity binders are bound by &#8220;binding site barriers&#8221; and can only be located at the edge of the tumor. In another example, anti-transferrin receptor mAbs can only deliver their payload across the blood-brain barrier (BBB) in the blood with their affinity moderate. In both cases, mAbs with lower affinities are still able to bind to the target but are subsequently released, enhancing the ability to penetrate the tumor and BBB.</span> </p>
<p><strong>3. Fc modification enhances effector function and half-life</strong></p>
<p><span style="font-size:15px">Modification of the Fc region can have a profound effect on the pharmacological effects of a particular antibody. Importantly, the activity of therapeutic mAbs directed against tumors is dominated by activation and inhibition of the Fcγ receptor (FcγR), FcγRIII, or FcγRIIB. Studies have shown that cytotoxic mAbs will preferentially bind to FcγRIII, while reducing the involvement of FcγRIIB with greater clinical efficacy. In rituximab, only a small number of amino acids have been replaced, showing enhanced ADCC effects <em>in vitro</em> and <em>in vivo</em>. In addition, changes in glycosylation can also enhance FcR interactions. Using the GlycoMAb technology, the non-core fucose of the mAbs Fc region is expressed equally, and the anti-CD20 antibody GA101 has stronger ADCC effect, anti-tumor efficacy, and B cell depletion than <span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creativebiolabs.net/Anti-Human-CD20-Therapeutic-Antibody-rituximab-13492.htm" target="_blank">Rituximab</a></span>. This technology is combined with POTELLIGENT technology and the antibody drug <span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creativebiolabs.net/Mogamulizumab-32074.htm" target="_blank">Mogamulizumab</a></span> has been approved for the treatment of relapsed or refractory adult T cell leukemia or lymphoma.</span> </p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/fcrn-path.jpg"><img decoding="async" class="size-full wp-image-1261 aligncenter" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/fcrn-path.jpg" alt="fcrn path" width="450" height="500" /></a></p>
<p style="text-align: center;">Figure 2 Interaction of Fc fragments of IgG in blood and endosomes with neonatal receptor FcRn affected by pH</p>
<p><span style="font-size:15px">Fc also leads to increased blood circulation time. Many studies have investigated the modification of Fc to extend the half-life of the antibody. Due to pH-dependent FcRn interactions, mutagenesis studies focus on replacing Fc amino acids that bind directly to FcRn to increase the affinity at pH=6 rather than pH=7.4. Although Fc modifications to enhanced circulating time may affect Fc&#8217;s effector function, studies have shown that mutations in different AAs of bevacizumab and cetuximab lead to more effective antitumor activity in mouse cancer models, thereby validating the strategy of increasing the half-life of therapeutic antibodies. Since then, a large number of favorable mutations have been reported. Unfortunately, the results of animal models are not always related to humans. However, 5-10 times increase in affinity with FcRn usually results in 2-4 times increase in half-life.</span> </p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/The-effect-principle-of-Antibody-ADCC-and-CDC.jpg"><img decoding="async" class="size-full wp-image-1262 aligncenter" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/The-effect-principle-of-Antibody-ADCC-and-CDC.jpg" alt="The effect principle of Antibody ADCC and CDC" width="500" height="500" /></a></p>
<p style="text-align: center;">Fig. 3 The effect principle of Antibody ADCC and CDC</p>
<p><strong>4. Improve stability and reduce polymerization</strong></p>
<p><span style="font-size:15px">Many therapeutic mAbs have limited thermal and colloidal stability when expressed under non-physiological conditions, making their manufacture and shelf life frustrating. Therefore, some engineering strategies aim to improve the physical characteristics of mAbs to increase their overall efficacy or productivity. The research for reducing polymerization has focused on changing the antibody framework, the antigen-binding loop, and the binding domain-binding interface. More specifically, stabilization strategies include: (a) adjustment of the dosage form; (b) specific point mutations (framework and/or CDRs); (c) addition of additional intradomain disulfide bonds in the CH2-CH3 domain; (d) change the disulfide structure of the Fab region; (e) and/or the charged fusion tag. Similarly, in the development of computer algorithms, rational design and in vitro selection methods can be used to counteract aggregation and predict high viscosity, chemical degradation, and rapid plasma clearance.</span> </p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/Non-natural-polymerization-of-antibodies.jpg"><img decoding="async" loading="lazy" class="size-full wp-image-1263 aligncenter" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/06/Non-natural-polymerization-of-antibodies.jpg" alt="Non-natural polymerization of antibodies" width="500" height="350" /></a></p>
<p style="text-align: center;">Figure 4 Non-natural polymerization of antibodies</p>
<p><span style="font-size:15px">The above engineering strategies are mainly focused on improving purified mAbs. In addition, there are other bioconjugation strategies to improve the overall mAb treatment index, each with its own advantages and disadvantages. For example, the coupling of an approved monoclonal antibody to a small molecule drug or a combination of radioisotopes aims to concentrate the vector&#8217;s power to the target cell/tissue. Another alternative is a bispecific antibody and CAR-T cell therapy that aims to relocate T cells to cancer for cancer treatment, while nanotechnology uses antibodies as targeting ligands or as diagnostics and/or drug delivery load.</span> </p>
<p>&nbsp;</p>
<pre>References: Kennedy P J, Oliveira C, Granja P L, et al. Monoclonal antibodies: technologies for early discovery and engineering [J]. Critical reviews in biotechnology, 2018, 38(3): 394-408.</pre>
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		<item>
		<title>What You Should Know about Antibody Monomer</title>
		<link>https://www.creative-biolabs.com/blog/index.php/what-you-should-know-about-antibody-monomer/</link>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Fri, 30 Mar 2018 07:18:49 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Biological Knowledge]]></category>
		<category><![CDATA[News]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[fab]]></category>
		<category><![CDATA[monoclonal antibody]]></category>
		<category><![CDATA[recombinant antibody]]></category>
		<category><![CDATA[ScFv]]></category>
		<category><![CDATA[Therapeutic Antibody]]></category>
		<guid isPermaLink="false">https://www.creative-biolabs.com/blog/?p=1245</guid>

					<description><![CDATA[Most proteins have a tendency to form polymers, which are accompanied by slow polymer formation when they are produced, stored, transported, and injected into a patient. There are two reasons for why<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/what-you-should-know-about-antibody-monomer/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p><span style="font-size:15px">Most proteins have a tendency to form polymers, which are accompanied by slow polymer formation when they are produced, stored, transported, and injected into a patient. There are two reasons for why to control the content of monoclonal antibody polymers. On the one hand, it can reduce impurities and increase purity. On the other hand, the presence of polymers in the treatment process will not only reduce the effect of the drug, but also lead to the immune response. In the experiments on animal <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/in-silico-Immunogenicity-Assessment.html">immunogenicity</a></span></strong>, it was found that the proportion of natural structures in the polymer and the size of the polymer determine the immunogenicity of the polymer. Therefore, some scholars concluded that the immunogenicity of the polymer is determined by repetitiveness of the surface antigenic epitopes.</span></p>
<p><span style="font-size:15px">The production of polymer is complex. In simple terms, monomeric protein polymer is a stable complex composed of two or more proteins. The formation process includes the destruction of the natural structure of the protein and the formation of a reversible self-polymerization, the reversible self-polymerization becomes irreversible due to the conformational change, the addition of other monomers makes the polymer larger and the polymers interact with each other to form soluble polymers with the size increases once again, and the soluble polymers are separated from each other, resulting in the formation of insoluble polymers.</span></p>
<p><span style="font-size:15px">Polymers are usually linked by strong non-covalent binding forces and require a certain degree of conformational changes (misfolded or unfolded) to exhibit key amino acid sequences (also known as &#8220;hot spots&#8221;, which have strong ability to form irreversible polymers, and are the core of the polymers), thereby aggregating different monomers, which for sure does not mean that no polymer is formed when properly folded. Monoclonal antibodies can form different forms of polymers, as shown in Figure 1. The red area indicates &#8220;hot spots&#8221;, the blue double-headed arrow indicates a reversible process, the red one-way arrow indicates an irreversible process. Misfolding, unfolding, and correct folding of the <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="https://www.creative-biolabs.com/recombinant-igG-production.html">Fab and Fc segments of the monoclonal antibody</a></strong></span> can all form polymers, and they do not dissolve when the soluble polymer splits. However, there is neither definite method nor specific measure to determine in which way does a protein form a polymer, but the content of the polymer is not impossible to be reduced.</span></p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/What-You-Should-Know-About-Antibody-Monomer.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-1246" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/What-You-Should-Know-About-Antibody-Monomer.jpg" alt="Pathway of Monoclonal Antibody Forming polymers" width="526" height="311" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/What-You-Should-Know-About-Antibody-Monomer.jpg 526w, https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/What-You-Should-Know-About-Antibody-Monomer-300x177.jpg 300w" sizes="(max-width: 526px) 100vw, 526px" /></a></p>
<p style="text-align: center;">Pathway of Monoclonal Antibody Forming polymers (Source: reference )</p>
<p><span style="font-size:15px">An amino acid sequence or residue with the tendency to form a polymer, i.e. a &#8220;hot spot&#8221;, exists not only at a certain position of the protein. Under normal circumstances, the amino acid sequence or residue is highly hydrophobic, low-charged, and easy to form β-sheets. That is, the force that normally promotes the folding of the protein also results in the formation of a polymer. If the “hot spots” exposed in the amino acid sequence can be accurately located and modified to cause repulsion between the proteins, the inherent tendency of the polymers will be affected. However, this genetic approach does not necessarily work because there is no accurate prediction and determination of whether “hot spots” are actually exposed on the surface of proteins and whether they are involved in the formation of polymers.</span></p>
<p><span style="font-size:15px">For monoclonal antibodies, modification of the complement regions (CDRs) of their variable regions, such as facilitating the amino acid mutation at the CDRs edge, and adding glycosides near the CDRs can all increase the resistance to formation of the polymer. In addition, the change in the protein charge is also a measure of the formation of resistance polymers, such as amino acid residues exposed by mutation and the addition of a charged polypeptide at the end of the antibody. Studies have pointed out that the existence of strong negatively charged amino acids (such as peptides) can enhance the electrostatic repulsive force between polymers, so that the formation of a polymer can be suppressed. Oxidation of methionine residues has an important influence on the propensity of many proteins to form polymers. To prevent the oxidation of methionine residues, freezing is usually the choice of antibody storage and transport, which can reduce the formation of polymers.</span></p>
<p><span style="font-size:15px">In the process of antibody production, the upstream usually determines the rate of formation of a polymer, and its environment such as temperature, protein concentration, pH, and ionic strength all influence the number of polymers. In a study on polymer, the SEC method was used to determine the content of the polymer. The experimental results are shown in Figure 2 as below. During D3-D13, the polymer content increased linearly with the increase of antibody concentration. At D13, the cells were removed from the medium and the harvested antibody-containing culture fluid was incubated at 37°C for two weeks. It was found that the content was still increasing slowly, even if the antibody concentration did not change. The longer the incubation time was, the higher the polymer content. This study shows that the polymer content can be reduced by reducing the culture time.</span></p>
<dl id="attachment_1247" class="wp-caption aligncenter" style="width: 248px;">
<dt class="wp-caption-dt"><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Effect-of-antibody-charge-on-polymer-growth.png"><img decoding="async" loading="lazy" class="size-full wp-image-1247" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Effect-of-antibody-charge-on-polymer-growth.png" alt="Changes in polymer content during cell culture" width="500" height="300" /></a></dt>
</dl>
<p style="text-align: center;">Figure 2 Changes in polymer content during cell culture (Image from reference 2)</p>
<p><span style="font-size:15px">The solution environment of the antibody, adsorption with the container, and chemical degradation can all affect the concentration of the intermediates and their interactions. The rate of formation and content of the polymer can be reduced by changing the dissolution environment. For example, the adjustment of pH (adjusting pH to keep it away from the isoelectric point pI) and ionic strength can increase the difficulty of polymer growth, as shown in Figure 3. The adjustment of pH and ionic strength can change the charge that the antibody carries, so that the electrostatic repulsive force between the monomers is increased to counteract the strong hydrophobic interaction force between the &#8220;hot spots&#8221; and inhibit the formation of the polymer. It is generally believed that the effect of negative charge increase is more significant than positive charge. This study has been applied to a variety of polymer systems, such as monoclonal antibodies, cytokines, and globulins.</span></p>
<p><a href="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Therapeutic-Protein-AggregationMechanisms-Design-and-Control.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-1248" src="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Therapeutic-Protein-AggregationMechanisms-Design-and-Control.jpg" alt="Therapeutic Protein AggregationMechanisms, Design, and Control" width="388" height="374" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Therapeutic-Protein-AggregationMechanisms-Design-and-Control.jpg 388w, https://www.creative-biolabs.com/blog/wp-content/uploads/2018/03/Therapeutic-Protein-AggregationMechanisms-Design-and-Control-300x289.jpg 300w" sizes="(max-width: 388px) 100vw, 388px" /></a></p>
<p style="text-align: center;">Fig 3 Effect of antibody charge on polymer growth (Source: reference 2)</p>
<p><span style="font-size:15px">In addition, disulfide bonds are also considered to be a factor for the formation of polymers (disulfide bonds can increase β-sheets), and the normal attachment of disulfide bonds is one of the conditions for the normal folding of proteins. The thiol group is an important source of disulfide bonds. The free thiol group on the cysteine that is not bound will result in a decrease in the disulfide bond content, which in turn will cause the protein to misfold. The formation of disulfide bonds requires a certain oxidizing environment, so the addition of oxidant Gu2+ promotes the formation of disulfide bonds. In addition, free thiol groups will affect the long-term stability of the antibody, and the removal of free thiol groups can increase the stability of the antibody.</span></p>
<p><span style="font-size:15px">For the upstream uncontrollable polymer, the downstream will take the removal role of the polymer. Variety of buffers being used in the purification process is an important factor affecting the polymer content. The acid buffer used to elute the antibody from protein A will cause the formation of polymers. Studies have found that the addition of arginine to the buffer in this step will reduce the amount of the solution compared to citrate, glycine and histidine. Multi-screen SEC column analysis can remove the polymer based on the size of the monomer and the polymer, but sacrifices some of the recovery. The mechanism of AEX and CEX to reduce the rate of the polymer is mainly that it can decompose the dimer or the multimer into monomers, and the content of the polymer under optimal conditions can be reduced to less than 0.5%. Frequent filtration and buffer exchange in the UF/DF stage, the mechanical pressure may cause the increase of protein polymers. However, as with the SEC principle, ultrafiltration can also be used in polymer removal.</span></p>
<p><span style="font-size:15px">The control of antibody polymers can be achieved not only during production and storage, but also during initial drug development. For example, a. experiments performed under high temperature conditions screen for proteins that are least likely to be misfolded and polymer; b. looking for proteins of different origins that are similar to the target protein (naturally can inhibit the formation of polymers), and compare the structure and sequence of the two proteins, and screen out proteins that are not easily aggregated.</span></p>
<p>References:<br />
1.Therapeutic Protein Aggregation: Mechanisms, Design, and Control<br />
2.Protein Aggregation and Bioprocessing<br />
3.When GoodGoes Awry: The Aggregation of Protein Therapeutics</p>
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		<item>
		<title>Definition and Production of Recombinant Antibody</title>
		<link>https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/</link>
					<comments>https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/#comments</comments>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Fri, 28 Apr 2017 08:47:01 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[antibody fragment]]></category>
		<category><![CDATA[antibody isotypes]]></category>
		<category><![CDATA[antibody production]]></category>
		<category><![CDATA[IgG]]></category>
		<category><![CDATA[phage display]]></category>
		<category><![CDATA[recombinant antibody]]></category>
		<category><![CDATA[ScFv]]></category>
		<category><![CDATA[shark antibody]]></category>
		<category><![CDATA[single-chain antibody]]></category>
		<guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=808</guid>

					<description><![CDATA[What is antibody (conventional antibody)? Before we begin to introduce recombinant antibody, we should learn about antibody. Antibody is known as immunoglobulins which are generally Y-shaped. They are peptide molecule secreted by<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<h2>What is antibody (conventional antibody)?</h2>
<p>Before we begin to introduce <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creativebiolabs.net/Hi-Affi-TM-Recombinant-Antibodies.htm" target="_blank">recombinant antibody</a></span>, we should learn about antibody. Antibody is known as immunoglobulins which are generally Y-shaped. They are peptide molecule secreted by b cells, mostly by differentiated B cells called plasma cells. Basically the function of antibodies is that control and stop pathogens and to assist in an immune response. They can protect us against infection and intoxication by mechanism of action for antibody functionality, such as antagonism, agonism, or cell killing via ADCC, ADCP, CDC, apoptosis PCD, or lysosomal-related PCD.</p>
<p>Much has been learned about the antibody structure (Fig. 1). IgG is a heterotetrameric protein assembled from two identical heavy and light chains (H, L), assembled by disulfide bonds. Each chain has two regions, the constant region (C) and the variable region (V). There are two parts on the right chain. The top is the variable region of the light chain (VL) and the bottom is the constant region of light chain (VH). Similarly, the heavy chain is divided into four parts. The top one-fourth of it is the variable region of the heavy chain and the other three parts are the constant regions (CH1, CH2, CH3). The “arms” of the “Y” bind antigens. The tail of the “Y” is responsible for biological activity, such as C’ activity or binding to cells. The antigen binding region also called paratope is a small part of variable region which recognizes and binds to an antigen. Each arm of the Y shape of an antibody monomer is tipped with a paratope, which is a set of complementarity determining regions that short for CDR.<br />
<a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-809" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg" alt="Structure of Antibody (Immunoglobulins)" width="876" height="382" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg 876w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins-300x131.jpg 300w" sizes="(max-width: 876px) 100vw, 876px" /></a></p>
<p style="text-align: center;">Fig. 1 Structure of Antibody (Immunoglobulins)</p>
<h2>Antibody Isotypes</h2>
<p>The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The sequence of the heavy chain defines the class of Ig, such that α, δ, ε, γ and μ heavy chains define the immunoglobulins A (IgA), immunoglobulins D (IgD), immunoglobulins E (IgE), immunoglobulins G (IgG), and immunoglobulins M (IgM) classes, respectively, each with a distinct role in the human adaptive immune system (Fig. 2). The light chains are either kappa (κ) or lambda (λ) isoforms for all classes.</p>
<p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg"><img decoding="async" loading="lazy" class="aligncenter  wp-image-810" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg" alt="Fig. 2 Antibody Isotypes" width="404" height="505" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg 567w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes-240x300.jpg 240w" sizes="(max-width: 404px) 100vw, 404px" /></a></p>
<p>&nbsp;</p>
<p style="text-align: center;">Fig. 2 Antibody Isotypes</p>
<p>Human IgA constitutes only 13% (2.1 mg/ml) of the antibody in human serum, but it is the predominant class of antibody in extravascular secretions. They are monmeric form in blood. The J chain is a 15-kD polypeptide consisting of 129 amino acid residues and one carbohydrate group. IgA1 is the most prevalent form in serum, but IgA2 is slightly more prevalent in secretions. Human IgE (190 kD) makes up less than 0.003% (0.4mg/ml) of the antibody in serum. IgE binds mast cells or basophils through its Fc part. IgE protects against parasites by releasing mediators that attract eosinophils. IgE is made up of about 13% carbohydrate. IgG is the most thoroughly studied in all five isotypes and recombinant antibody engineering is based on it. IgG constitutes about 80% (12.5 mg/ml) of the antibody in serum. Human IgG consists of four subclasses (isotypes), which are numbered in order of their serum concentrations (IgG1, IgG2, IgG3, and IgG4). The chief distinguishing characteristic among the four IgG subclasses is the pattern of inter chain linkages in the hinge region. IgM, primarily induced by polysaccharide antigens, is a 950-kD pentamer that makes up about 8% (1.25mg/ml) of the antibody in the serum. The five monomeric IgM molecules are arranged radially, the fab fragments pointing outward and the Fc fragments pointing to the center of the circle. IgM can quickly clump antigen and efficiently activate complement. IgM acts as one of the main receptors on the surface of mature B cells, along with IgD. When IgM is a surface receptor, it is in its monomeric form.</p>
<h2>Recombinant Antibody (rAb) Definition</h2>
<p>What are recombinant antibodies? Recombinant antibodies are antibody fragments generated by using recombinant antibody coding genes as a source and display technology, delivering high reproducibility, specificity and scalability. Unlike monoclonal antibodies (mAbs) which are produced using traditional hybridoma technologies, rAbs do not need hybridomas and animals in the production process if you only use synthetic genes.</p>
<h2><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/recombinant-igG-production.html">Production of Recombinant Antibody</a></span></h2>
<p>How is recombinant antibody produced? Let me show you the process of recombinant antibody production (Fig.3). As we know, antibody is composed of two heavy chains (VH) and two light chains (VL).</p>
<p>Step 1: Variable genes of heavy chain and light chain antibody should be cloned by PCR and designed primers. About the detailed strategy of PCR, <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/scfv-library-construction-protocol.html">scfv library construction protocol</a> </span>is introduced in resource of Creative Biolabs. By the recombinant DNA technology, link purified genes of VH and VL with prokaryotic expression vector which requires select in advance based on your target recombinant antibody fragments. If you need scfv fragment antibody, linker must be designed in your PCR strategy.</p>
<p>Step 2: Transformation: Electroporate ligation product of VH and VL received in previous step into cloned expression host cells. In this step, it is very important that highly competent of commercial source be recommended to obtain high transformation efficiency.</p>
<p>Step 3: Choose appropriate antibody display technology, such as phage display (which can screening small antibody fragment to obtain large antibody library); ribosome display (which can get a library of a capacity of 10<sup>14 </sup>without limitation of transformation efficiency and acquire mutant antibody library); yeast display (which make human antibody expression more superior because that yeast expression system is similar with mammalian cells). According to these above procedures, some recombinant antibodies can be constructed successfully, such as scfv antibody fragments, fab antibody fragments, and single domain antibodies.</p>
<p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-811" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg" alt="Production of Recombinant Antibody" width="1174" height="464" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg 1174w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody-300x119.jpg 300w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody-1024x405.jpg 1024w" sizes="(max-width: 1174px) 100vw, 1174px" /></a></p>
<p>&nbsp;</p>
<p style="text-align: center;">Fig. 3 Production of Recombinant Antibody</p>
<h2>Common Types of Recombinant Antibody</h2>
<p>There are 3 common types of recombinant antibody fragments listed: Fab, scfv, single domain antibody (sdab) (Fig. 4).</p>
<p>Fab fragment was originally defined as one of the cleavage products after treatment of rabbit IgG with papain, which cleaves the core hinge, resulting in two identical fab fragments and the intact Fc as products. The molecular weight of Fab fragment is ~50 kDa. The nonspecific protease pepsin cuts below the first disulfide bond in the hinge region giving rise to a F(ab’)<sub>2</sub> fragment. The molecular weight of F(ab’)<sub>2</sub> fragment is ~100 kDa. The Fab contains four domains: the heavy chain variable domain (VH) linked to constant domain 1 (CH1), and the light chain variable domain (VL) linked to a constant domain (CL). Due to the hinge region which allows for flexibility of fabs in relation to the Fc, intact IgGs have proven difficult to crystallize in forms suitable for diffraction studies, and thus, to this time, only a few structures for intact IgGs have been determined.</p>
<p>Single-chain fragment variable (scfv) molecules combine the coding sequence of the variable heavy (VH) and sequence of the variable light chain (VL) domains of an antibody in a single-gene encoded format. The resulting polypeptides, with the variable light (VH ) and heavy chain (VH ) domains connected by a flexible peptide linker, were assemble into functional antigen-binding sites. The linker technology is a key step of success of constructing scfv antibody library. Scfvs have been developed as possible drug candidates in their own right, as well as components or domains of drug candidates.</p>
<p>Single domain antibody (sdAb) is discovered in in camelids and nurse sharks that consisted of a lone VH domain, lacking a paired VL, attached to a constant region. The primary advantages of domain antibodies as compared with scFvs are generally better folding and stability characteristics, the absence of the linker, and size.</p>
<p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-812" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg" alt="Common Types of Recombinant Antibody" width="847" height="451" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg 847w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody-300x160.jpg 300w" sizes="(max-width: 847px) 100vw, 847px" /></a></p>
<p style="text-align: center;">Fig. 4 Common Types of Recombinant Antibody</p>
<p>Compared with conventional antibody, recombinant antibody (rAb) possesses some advantages, such as smaller size, monovalency, ease of engineering and manufacture, improved tissue penetration, no animal immunization and broader biodistribution, as well as lack of potentially deleterious Fc effector function. However, there are some drawbacks in recombinant antibodies, such as lower antibody yield, highly training and experienced technician. Most of scientists need to get them from outsourced companies because of complexity and intensive high technology of recombinant antibody production.</p>
<p>Application of Recombinant Antibody (rAb):</p>
<ul>
<li>desire or requirement for a short circulating half-life in serum;</li>
<li>a smaller biologic that would have broader tissue distribution or the ability to penetrate tumors;</li>
<li>a molecule that can be manufactured in either yeast or <em>E. coli </em>to potentially reduce cost of goods or increase scale of manufacturing;</li>
<li>a molecule lacking an Fc effector functionality to eliminate both cellular responses against the target and potential for dimerization of receptors due to bivalency;</li>
<li>a <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creativebiolabs.net/bispecific-antibody-production.htm" target="_blank">bispecific antibody </a></span>fragment, such as has been demonstrated by BiTEs (bispecific T cell engagers; diabodies and most recently, DARTs;</li>
<li>a molecule lacking an Fc effector functionality to eliminate both cellular responses against the target and potential for dimerization of receptors due to bivalency; a molecule that can be manufactured in either yeast or <em>E. coli </em>to potentially reduce cost of goods or increase scale of manufacturing.</li>
</ul>
<p>Creative Biolabs is a professional custom service provider with extensive experience in antibody engineering. With over ten years of efforts, we have extended our services to over 26 countries. We provide recombinant antibody product, such as scfv antibody fragments, fab antibody fragments, single domain antibody, rabbit monoclonal antibodies, as well as recombinant antibody construction and expression services. If you are interested in learning more about how we can help with your projects, please don’t hesitate to contact us.</p>
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		<title>Combinatorial Antibody Libraries Overview</title>
		<link>https://www.creative-biolabs.com/blog/index.php/antibody-libraries/</link>
					<comments>https://www.creative-biolabs.com/blog/index.php/antibody-libraries/#comments</comments>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Mon, 27 Feb 2017 09:41:37 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[antibody libraries]]></category>
		<category><![CDATA[phage display]]></category>
		<category><![CDATA[ScFv]]></category>
		<guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=786</guid>

					<description><![CDATA[Phage display has proven to be a powerful technique for the interrogation of libraries containing millions or even billions of different peptides or proteins. Phage display libraries permit the selection of peptides<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/antibody-libraries/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p><strong><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Phage-Display-Platform.html">Phage display</a></strong> has proven to be a powerful technique for the interrogation of libraries containing millions or even billions of different peptides or proteins.<strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/phage-display-library-construction.html"> Phage display libraries</a></span></strong> permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. Antibody phage display libraries obviate the need for immunization and the concomitant laborious hybridoma protocols for obtaining mAbs, directly afford the cloned antibody genes in single-chain Fv (scFv) or Fab format for convenient manipulation, and, importantly, can be derived from the human antibody repertoire. For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and isolate a wide variety of antibodies. Combinatorial antibody library technology represents a powerful tool for discovering and designing antibodies that bind targets with high affinity and specificity. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Using this technique, antibody genes have been cloned from multiple species or expressed directly from large man-made repertoires of antibody-encoding genes. Recent studies demonstrate that the technique allows for the in vitro evolution of antibodies to create molecules whose affinity for antigen exceeds that observed in nature.</p>
<h2>The type of combinatorial antibody library</h2>
<p>There are four types of antibody library in combinatorial antibody libraries: <strong>immune libraries,</strong> <strong>naïve</strong>, <strong>semisynthetic</strong> and <strong>synthetic libraries</strong>, the latter three have been included as “single-pot” libraries, as they are designed to isolate antibody fragments binding to every possible antigen in theory. We discriminate ‘naïve’ and ‘synthetic’ antibody libraries, depending on the source of immunoglobulin genes. For most applications, the availability of large pre-made collections of non-immune repertoires has superseded the use of immune repertoires.</p>
<p><strong>Immune libraries</strong> from human or animals are constructed from immunized donors or natural infections and typically used in biopharmaceutical research to obtain an antibody against a particular target antigen. From immune libraries, V genes of these libraries contain hypermutations and are affinity matured, antibody fragments with monovalent dissociation constants in the nanomolar range can be isolated. Immune libraries are typically created and used in medical research to isolate an antibody fragment against one particular antigen, and therefore would not be the source of choice for the selection of a large number of different specificities.</p>
<p><strong>The naïve libraries</strong> are construct from natural unimmunized human rearranged V genes, synthetic human V genes, or shuffled V genes. These universal (non-immunized) libraries differ from libraries derived from immune antibodies, in that the former are antigen-independent (i.e. a single library can be panned for specificity against virtually any antigen). Examples of naïve library type are the naïve human Fab library constructed by de Haard and colleagues, the McCafferty libraries, or the HAL scFv libraries. The affinity of Abs selected from a naïve library is proportional to the size of the library, ranging from 10<sup>6–7</sup> M<sup>–1</sup> for a small library of 3&#215;10<sup>7</sup> clones to 10<sup>8–10</sup> M<sup>–1</sup> for a large repertoire of 10<sup>10</sup> clones made by brute-force cloning.</p>
<p><strong>Semi-synthetic libraries </strong>is constructed<strong> </strong>wherein diversity is controlled by the oligonucleotide synthesis. Semi-synthetic libraries are derived from unrearranged V-genes from pre-B cells or a single antibody framework with at least one complementary determining region (CDR) region genetically randomized. The combinations create new diversity. The character of the naïve repertoire will be influenced or edited by the animal and will be extremely susceptible to contamination from materials derived from activated B cells or plasma cells, which would limit the diversity of the library. Semi-synthetic libraries can overcome this diversity problem.</p>
<p><strong>Synthetic libraries </strong>contain a framework with randomly integrated codons into CDRs. Antibodies are built artificially, by <em>in vitro</em> assembly of V-gene segments and D:J segments. V genes may be assembled by introducing a predetermined level of randomization of CDR regions into germline V-gene segments, or rearranged V-genes. Through synthetic library construction, new diversity can be obtained. Synthetic libraries are antigen-independent and particularly useful for unbiased selection of antibodies against any target antigen. These kinds of libraries were optimized in the past years by mimicking the <em>in vivo</em> amino acid distribution in the CDRs. Some of these libraries have yielded antibodies against many different antigens, including haptens, proteins, and cell-surface markers, but their affinities are typically in the micromolar range.</p>
<h2>The construction of phage antibody Libraries</h2>
<p><strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/phage-display-library-construction.html">Library construction</a></span></strong> is facilitated by the ready availability of phagemid vectors, which allow for construction and display of libraries of all of these types of antibody fragments using a single rare cutting restriction enzyme. Selection of interesting members from the library based on the displayed antibodies’ binding specificity and affinity is generally performed over several rounds of selection and amplification in a process known as panning.</p>
<p>For library construction, large repertoires of single-chain Fv antibody fragment (scFv) or Fab antibody genes are cloned into engineered phage or phagemid vectors in a way that these antibody fragments can be displayed on the bacteriophage surface. One of the most successful applications of phage display has been the isolation of monoclonal antibodies from large random combinatorial phage antibody libraries (Fig. 1). Such libraries have been built in scFv and fab format by researchers. Typically, the antibody fragments are displayed on the surface of phage as either fab fragments, single-chain variable region fragments (scFvs), or dimeric scFvs, also known as diabodies, which differ from scFvs in the reduced length of the linker peptide used and their preference to associate as dimers.</p>
<p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/02/combinatorial-library.png"><img decoding="async" class=" aligncenter" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/02/combinatorial-library.png" alt="Construction of a human Ab library displayed on phage" /></a></p>
<p><strong>Figure 1</strong> Construction of a human Ab library displayed on phage. cDNA encoding for the heavy and the light variable regions of Abs (V<sub>H</sub>, V<sub>L</sub>) are amplified from human B cells by PCR and assembled. The assembled genes are inserted into a phagemid vector in frame with the gene encoding the CP pIII. The vector is introduced into E. coli. After rescue with helper phage, the random combinatorial library of Abs is displayed on phage and selection can be performed(<em>Hennie R. Hoogenboom.etc</em>).</p>
<p>The standard procedure of constructing antibody library is that:<br />
1. Construction of Phage-Display Vector (choose suitable vector based on antibody format ).<br />
2. Preparation of the cDNA Template: After the isolation of mRNA from the desired cell type and the preparation of cDNA, the construction of immune libraries is usually done by a two-step cloning or assembly PCR (choose appropriate clone methods based on library type).<br />
3. Amplification of Antibody Variable Region Genes (choose proper PCR strategy and optimizing PCR reaction based on targeting sequence size and primer design requirement).<br />
4. Construction of the scFv/fab Library (after target genes (scfv/fab genes) and optimized vectors are digested by same restriction enzymes, ligate their purified segments).<br />
5. Rescue of scFv-Phage.<br />
6. Panning of scFv/fab-Phage: The library was subjected to three or four rounds of panning. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning.<br />
7. ELISA and purification of scFv/fab-Phage Binding: choose proper antigen and expression system is the key factor of success.<br />
To gain a high-quality library, firstly, the individual steps were optimized to increase the antibody variable region gene diversity and the efficiency of scFv/fab gene assembly and cloning. The primer design was optimized for amplification of variable region gene pools to maintain maximum diversity. Second, about scFv, the efficiency of scFv gene assembly was increased by exploiting the presence of the DNA encoding the (G<sub>4</sub>S)<sub>3</sub> peptide linker included at the end of the 3’-primer of the V<sub>H</sub> gene and 5’-primer of the V<sub>L</sub> gene. This design allowed us to assemble scFv genes from only two DNA fragments. Several parameters about human universal libraries are given in Table 1.</p>
<p style="text-align: center;"><strong>Table 1</strong> Human “single-pot”/universal phage display libraries.</p>
<table class="table text-align-cent table-bordered">
<tbody>
<tr>
<td style="text-align: center;">Library vector(library name)</td>
<td style="text-align: center;">Library type</td>
<td style="text-align: center;">Antibody type</td>
<td style="text-align: center;">Library cloning strategy</td>
<td style="text-align: center;">Library size</td>
</tr>
<tr>
<td style="text-align: center;">fdDOG-2lox,pUC19-2lox</td>
<td style="text-align: center;">Semisynthetic</td>
<td style="text-align: center;">Fab</td>
<td style="text-align: center;">PCR with random CDR3 primer, Cre-lox</td>
<td style="text-align: center;">6.5 x 10<sup>10</sup></td>
</tr>
<tr>
<td style="text-align: center;">fdTet</td>
<td style="text-align: center;">Naïve</td>
<td style="text-align: center;">scFv</td>
<td style="text-align: center;">Recloning of a naïve library</td>
<td style="text-align: center;">5 x 10<sup>8</sup></td>
</tr>
<tr>
<td style="text-align: center;">pComb3</td>
<td style="text-align: center;">Semisynthetic antitetanus Ab framework</td>
<td style="text-align: center;">Fab</td>
<td style="text-align: center;">PCR with random CDR H3 primer</td>
<td style="text-align: center;">&gt;10<sup>8</sup></td>
</tr>
<tr>
<td style="text-align: center;">pMorph series (HuCAL library)</td>
<td style="text-align: center;">Synthetic</td>
<td style="text-align: center;">scFv</td>
<td style="text-align: center;">PCR with random CDR H3 primer</td>
<td style="text-align: center;">2 x 10<sup>9</sup></td>
</tr>
<tr>
<td style="text-align: center;">pR2 (DAb library)</td>
<td style="text-align: center;">Semisynthetic</td>
<td style="text-align: center;">V<sub>H</sub> dAb</td>
<td style="text-align: center;">Assembly PCR random CDR V<sub>H</sub></td>
<td style="text-align: center;">3 x 10<sup>9</sup></td>
</tr>
<tr>
<td style="text-align: center;">pMorph series (HuCAL GOLD library)</td>
<td style="text-align: center;">Synthetic</td>
<td style="text-align: center;">Fab</td>
<td style="text-align: center;">Two-step cloning, all CDR replacement</td>
<td style="text-align: center;">1.7 x 10<sup>7</sup></td>
</tr>
<tr>
<td style="text-align: center;">pSEX81</td>
<td style="text-align: center;">Naïve</td>
<td style="text-align: center;">cFvs (with N-terminus of CH1 and CL)</td>
<td style="text-align: center;">Two-step cloning</td>
<td style="text-align: center;">4 x 10<sup>9</sup></td>
</tr>
</tbody>
</table>
<p>In summary, antibodies with subnanomolar affinities can be selected from either type of library, naïve or synthetic. If the assembly by cloning or PCR and preservation of molecular complexity is carefully controlled at every step of its construction, high-quality libraries of more than 10<sup>10</sup> independent clones can be generated, with the potential to yield hundreds of different antibody fragments to any given antigen. The library yielded antibodies with in some cases nanomolar affinity against numerous different antigens. ScFv phage library proved to be difficult to re-propagate without significant loss of diversity. However, a more stable, large (1.2 x 10<sup>9</sup>) clone scFv phagemid library made by standard cloning methods and using the synthetic V-genes, was recently shown to be equally effective.</p>
<p>Methodologies for the creation and selection of combinatorial antibody libraries on the surface of phage continue to be improved and extended. Now new library methodologies offer many alternative and at least as powerful routes, by combining the generation of billions of components with a fast screening or selection procedure to identify the most interesting lead candidates. Antibodies have been selected to molecules both large and small to study protein recognition, immunity, and problems in basic and applied sciences. In conclusion, antibody phage display will continue to be a main method for the generation of human therapeutic antibodies in the near future.</p>
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		<title>Fundamental Technologies for Antibody Production- Phage Display</title>
		<link>https://www.creative-biolabs.com/blog/index.php/fundamental-technologies-for-antibody-production-phage-display/</link>
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		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Thu, 22 Dec 2016 10:28:02 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[antibody fragment]]></category>
		<category><![CDATA[fab]]></category>
		<category><![CDATA[phage display]]></category>
		<category><![CDATA[ScFv]]></category>
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					<description><![CDATA[The phage display technology, first described over a decade ago, has opened new windows in the production of recombinant antibody fragments with the promise of trapping the naive immune repertoire in vitro.<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/fundamental-technologies-for-antibody-production-phage-display/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p>The <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Phage-Display-Platform.html">phage display technology</a></span></strong>, first described over a decade ago, has opened new windows in the production of <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Recombinant-Antibodies.html">recombinant antibody fragments</a></span></strong> with the promise of trapping the naive immune repertoire <em>in vitro</em>. Phage display is a term describing display of foreign (poly) peptides on the surface of phage particle. The technique differs from conventional expression systems, however, in that the foreign gene sequence is spliced into the gene for one of the phage coat proteins, so that the foreign amino acid sequence is genetically fused to the endogenous ones of the coat protein to make a hybrid “fusion” protein. Using recombinant DNA technology, collections of billions of certain ligands (e.g., peptides or proteins, antibody fragments), cDNA-encoded or gene fragment-proteins presented on phage (so called phage display libraries) can be constructed and surveyed for specific affinity or activity (Fig. 1).</p>
<p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2016/12/phage-display.png"><img decoding="async" loading="lazy" class=" size-full wp-image-751 aligncenter" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2016/12/phage-display.png" alt="phage display" width="564" height="388" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2016/12/phage-display.png 564w, https://www.creative-biolabs.com/blog/wp-content/uploads/2016/12/phage-display-300x206.png 300w" sizes="(max-width: 564px) 100vw, 564px" /></a></p>
<p>Fig. 1. Phage display cycle. DNA encoding for millions of variants of certain ligands (e.g., fab,peptides, proteins, or fragments thereof) is batch-cloned into the phage genome as part of one of the phage coat proteins (pIII, pVI, or pVIII). Large libraries containing millions of different ligands can be obtained by force-cloning in <em>E. coli</em>. From these repertoires, phage carrying specific-binding ligands can be isolated by a series of recursive cycles of selection on Ag(agar plate), each of which involves binding, washing, elution, and amplification.</p>
<p>Antibody was the first protein to be displayed successfully on the surface of phage. For therapeutic monoclonal antibodies, phage display system is typically used for three applications: identification and isolation of target specific antibodies either from non-immune, naïve, libraries or from libraries derived from genetic engineering of antibodies or immunized animals to optimize them for increased higher affinity or improved biophysical characteristics.</p>
<p>The main features of phage display technology in antibody production includes: sequences cloning of antibody library; selection of phage vector that can make large antibody libraries; linkage of the gene sequence with the displayed antibody; a screening (or selection) of identifying antibody variants that uniquely bind the target of choice; amplification of the nucleic acid sequence; either without or with introduction of diversity; and production of soluble antibody for functional characterization. The widespread use of antibody phage display comes from its relative simplicity, durability, stability of the phage particles and the ability to generate large libraries suitable for both <em>de novo</em> discovery and antibody optimization.</p>
<p>The phage display process for discovering antibodies or proteins that bind specifically to a given target will be detailed in the following so will only be highlighted here. Selection of phage Antibody involves the sequential enrichment of specific binding phage from an excess of nonbinding clones, which is achieved by multiple rounds of phage binding to the target, washing to remove unbound phage, and elution to retrieve specific binding phage. The target protein is tagged with biotin; target ones can be bound with very high affinity to streptavidin-coated plates or beads. Typically the target protein is either immobilized on a surface or beads that can be washed to remove unbound phage.</p>
<p>Effective display formats for Antibodies(Abs) are scFv, Fabs, immunoglobulin variable fragments (Fvs) with an engineered intermolecular disulphide bond to stabilize the VH–VL pair and diabody fragments. Antibody libraries are constructed by reverse-transcribing and PCR amplifying genes encoding antigen-binding domains of heavy (VH) and light (VL) chains from lymphocyte total RNA. By combining different VH and VL, a process termed chain shuffling, unique antibody fragments are formed. The smaller size of the scFv format makes their libraries genetically more stable than Fab libraries. To display Fabs on phage, either the light or heavy chain is fused via its C-terminus to vector, the partner chain is expressed and secreted into the periplasmic space where chain association forms an intact Fabs. Over the past decade, several fully human antibody phage display libraries have been constructed using either Fab or scFv formats. There are two general formats used for phage display of antibodies based on where the antibody (ab) genes are inserted. In one, the display protein-ab fusion is encoded on a separate plasmid, a phagemid that is subsequently encapsulated into phage particles upon infection with helper phage. In this case, the number of pIII-fusion proteins in the mature phage is either zero or one (i.e. monovalent display), allowing high affinity binders to be more easily isolated due to the absence of avidity effects. In the second format, the ab and display protein gene sequences are fused and incorporated directly into the phage genome so that five copies of a pIII-antibody fusion protein are produced. This format works reasonably well for libraries and general screenings but falls short when seeking high affinity binders as these are difficult to distinguish from lower affinity binders that have apparent higher affinity resulting from the avidity effect created by the binding of five copies to the target.</p>
<p>To date, there are at least 25 therapeutic antibodies that are derived from <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/phage-display-library-construction.html">phage display libraries</a></span></strong> approved for clinical use or in various stages of clinical trials. One advantage of phage display is that fully human antibody can be isolated. Furthermore, phage display offers greater control over the selection process than using animal immunization. Finally, compared to other<em> in vitro </em>selection methods such as cell display, phage display is more readily accessible as it does not require expensive equipment like fluorescence-activated cell sorter (FACS). One shortcoming of phage display is that selection of antibody fragments on phage is based on expression. Some antibodies can be toxic to <em>E. coli</em> and accumulate stop codons throughout their coding sequence during the phage display process and be eliminated. Because of phage volume and size, as well as the maximum efficiency of <em>E. coli</em> transformation, phage display libraries have been limited to about 10<sup>11</sup> unique members. Hybridoma-based antibodies, on the other hand, are subject to <em>in vivo</em> “editing” which tends to reduce problematic antibody properties such as aggregation. One difference from hybridoma technology is that the output of most display systems include antibody fragments such as scFvs or Fabs that must be further manipulated genetically to yield complete IgGs which are often required for initial functional evaluation.</p>
<p>Creative Biolabs provides comprehensive antibody <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/scfv-fab-production.html">Fab/scFv production</a></span></strong> service by phage display to meet your biopharmaceutical and biotechnology goal. We have long-term devoted to the development and application of phage display technology.</p>
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		<title>The Latest Research Progress in ScFv</title>
		<link>https://www.creative-biolabs.com/blog/index.php/the-latest-research-progress-in-scfv/</link>
					<comments>https://www.creative-biolabs.com/blog/index.php/the-latest-research-progress-in-scfv/#comments</comments>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Tue, 29 Sep 2015 01:21:10 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[ScFv]]></category>
		<guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=566</guid>

					<description><![CDATA[Recently, some scientists have raised a idea that scFv shouldn’t focus on the traditional antibody because modern new drugs require more efficient antibodies. The latest research progress in scFv has indicated there<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/the-latest-research-progress-in-scfv/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p style="text-align: justify;">Recently, some scientists have raised a idea that scFv shouldn’t focus on the traditional antibody because modern new drugs require more efficient antibodies. The latest research progress in scFv has indicated there are two new types, namely the single-chain antibody multimer and bispecific single-chain antibody.</p>
<p style="text-align: justify;"><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2015/09/244x300.jpg"><img decoding="async" loading="lazy" class="alignleft size-full wp-image-567" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2015/09/244x300.jpg" alt="244x300" width="244" height="300" /></a>As for single-chain antibody multimer, it is different from typical antibody that merely owns one light chain and one heavy chain, which is slightly inferior to the parent antibody in antigen gene. Therefore, people are trying to use polypeptide gene that can easily form spiral and make it co-express with VH gene and VL gene in expression system, for the purpose of producing bivalent single-chain antibody. In fact, some relevant research progresses have shown the length of linker (connect VH and VL) is connected with the single-chain antibody multimer.</p>
<p style="text-align: justify;">For the bispecific single-chain antibody, it can be completed utilizing the VH and VL gene of two different single-chain antibody. Generally, it can be recombined during the expression process. Such antibody can specifically link two antigens with dual specificity. Compared with conventional bispecific antibodies, bispecific single-chain antibody has a variety of advantages. Firstly, the absence of Fc fragments reduces the possibility of non-specific binding in FcR positive cells. Secondly, it will minimize the murine protein, which could reduce the possibility of immune hypersensitivity. Finally, its small molecules is beneficial in penetrating the tumor tissue.</p>
<p style="text-align: justify;">While on the other hand, <strong><a href="http://www.creative-biolabs.com/scfv-fab-production.html">scFv</a></strong> still has some deficiencies in practical applications even if it has achieved massive progresses through 20 years of development, such as low affinity, poor stability, short half-life, etc.. Taking the affinity as an example, scFv’s affinity is ten to one thousand times lower than that of parent single-chain antibody. More specifically, scFv has small molecules. Its in vivo clearance speed is fast so that sometimes it cannot reach enough time in treatment and diagnosis. Given the discussed details above, increasing scFv’s antigen-binding ability might be a choice for improving its disadvantages. Moreover, with rapid advance in molecular biology, molecular immunology, single-chain antibody display and in-depth technical study of expression, better scFv that is accessible to a variety of purposes is just around the corner.</p>
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		<title>Modern Research in ScFv</title>
		<link>https://www.creative-biolabs.com/blog/index.php/modern-research-in-scfv/</link>
					<comments>https://www.creative-biolabs.com/blog/index.php/modern-research-in-scfv/#comments</comments>
		
		<dc:creator><![CDATA[biolabs]]></dc:creator>
		<pubDate>Mon, 28 Sep 2015 01:18:27 +0000</pubDate>
				<category><![CDATA[Antibody Engineering Research]]></category>
		<category><![CDATA[Antibody Discovery]]></category>
		<category><![CDATA[ScFv]]></category>
		<guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=562</guid>

					<description><![CDATA[In recent years, an increasing number of researchers have payed attention to the modern advance of scFv, such as its clinical applications, practical functions, and so on. As for such issue, people<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/modern-research-in-scfv/">Read More...</a>]]></description>
										<content:encoded><![CDATA[<p style="text-align: justify;">In recent years, an increasing number of researchers have payed attention to the modern advance of scFv, such as its clinical applications, practical functions, and so on. As for such issue, people hold various ideas. Some think scFv is significant in pharmaceuticals while others who against the idea by saying that it might has some serious side effects. For me, the latest research progresses in scFv have been meaningful to people’s life.</p>
<p style="text-align: justify;"><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2015/09/260px-Heavy_chain_and_common_antibody.svg_.png"><img decoding="async" loading="lazy" class="alignleft size-full wp-image-564" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2015/09/260px-Heavy_chain_and_common_antibody.svg_.png" alt="260px-Heavy_chain_and_common_antibody.svg" width="260" height="175" /></a>Generally speaking, scFv owns some special characteristics in a wide range of areas. In fact, scFv can be regarded as a kind of new recombinant protein, because it can combine the heavy chain and light chain in antibody’s variable region together through the elastic short peptide that is composed of 15 to 25 amino acid residues. Specifically, scFv is the smallest structure unit, with a parent antibody. Briefly, it has various advantages, such as small molecular, strong penetration on tumor, short bloodstream period, low immunogen, and so on.</p>
<p style="text-align: justify;">Actually, in recent years, <strong><a href="http://www.creative-biolabs.com/scfv-fab-production.html">scFv</a></strong> has stepped into a new era because of its modern research progress. Taking monovalent single-chain antibody as an example, some scientists have found scFv in E.coli can be spontaneously folded into a native conformation, owing a extremely similar antigen affinity as the intact antibody, which can be obtained through cell expression. Specifically, using oligo nucleotides with encoded elastic short peptides to connect some crucial elements together and then insert the combined substance into prokaryotic cell expression.</p>
<p style="text-align: justify;">In practice, Challdhary includes several other scientists successfully prepared the single-chain antibody in pseudomonas exotoxin in 1990; the expressed immunotoxins can recognize and then kill those cells that carry OVB3 antigen. Since then, more and more scientists have stressed such research and have made great achievements as well. For example, some researchers begin to take further steps in trying to use yeast or plant cells to conduct the practical express. They have realize great success in the subsequent ten years. However, even if massive achievements have been realized, we should keep the advance, because only in this way can the biotechnology benefits people’s modern life to a larger degree.</p>
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