![]() Both of these domains contain long CDR loops, often larger than those observed in conventional murine and human antibodies. Discovery of two types of organisms, the camelids and cartilaginous fish, that have evolved high affinity single variable domains (called the VhH domain in camelids (11) and the V-NAR domain in sharks (12)) mounted on an Fc equivalent constant domain framework resparked interest in this area. However, they rarely retain the affinity of their parent antibody and are poorly soluble and prone to aggregation (10). When mouse single VH domains were shown to be functional (10) it was proposed that because of their small size they could potentially target cryptic epitopes that have evolved in many pathogenic viruses to contain narrow cavities that bind to target receptors but are poorly accessible to intact antibodies. For this reason scFvs have become one of the main building blocks of antibody fragments. ScFvs naturally multimerize (7) and this can be controlled by linker length creating dimeric, trimer or tetrameric forms (8, 9). However, Fv domains tend to have limited stability due to the dissociation of the domains and thus a peptide linker was introduced between the VH and VL domains to create the scFv (5, 6). The Fv portion of an IgG, consisting of the VH and VL domains, is the smallest fragment that maintains the full binding capacity of the intact antibody. These are mainly based on either Fab fragments or the single chain Fv (scFv) as building blocks. The image above shows a selection of the antibody fragments that have been engineered. Over the past three decades antibodies have been dissected into smaller antigen binding fragments, initially by proteolysis and later by genetic engineering to produce mono or multivalent fragments. This approach and others will be described in more detail in the bispecifics section.įigure. Antibody fragments that have been engineered to be multimeric are of use when targeting multiple disease associated antigens. Fragments are also useful in imaging and cancer therapy, where a long serum half-life mediated by Fc interaction with the FcRn receptor results in poor contrast and in the case of radiolabelled antibodies fast clearance from the circulation via the kidney is also advantageous to reduce prolonged exposure (4). The use of smaller fragments enables deeper penetration with the affinity of the antibody also being critical and if it is too high this will restrict its ability to penetrate a tumour (3). Solid tumours have substantial physical barriers often preventing the penetration of antibodies to the centre and resulting in reduced therapeutic effects (2). This also helps to reduce the other main failure of therapeutic antibodies, namely the lack of delivery, which is especially true for anti-cancer antibodies. A common solution for applications where the antibody is only being used to block a signalling molecule or receptor is the use of antibody fragments that lack the Fc domain (1). There are a range of applications in which Fc mediated effects are not required and are even undesirable. ![]() Click here to read about our engineering services, including converting any antibody into a fragment. FleXpress™ High-Throughput Antibody Production.Immunoglobulin Fc domains (Ig Fc domains).Anti-Non-Human Primate (NHP) Immunoglobulin Antibodies. ![]() VivopureX™ Antibodies for In Vivo Research.Recombinant Antibodies from Human Patients.Separation of antibody fragments on non-reducing SDS-PAGE. The procedure from digestion to collection of fragments using FabRICATOR Fab2 Kit takes about one hour.įigure 2. The F(ab’)2 fragments were purified on the CaptureSelect ™ spin column and the bound Fc fragments eluted in a single step by lowering the pH followed by immediate adjustment back to neutral pH. First, the antibody was digested on the FabRICATOR Immobilized spin column at RT for 15 minutes followed by a short centrifugation step. To generate and purify intact F(ab’)2 and Fc/2 fragments, trastuzumab was processed using the FabRICATOR Fab2 Kit and analyzed by SDS-page (Fig. ![]() The FabRICATOR Immobilized contains immobilized FabRICATOR (Ides) to prepare F(ab’)2 and Fc fragments from several species of IgG (for rabbit IgG, please inquire), while mouse IgG2a and IgG3 can be processed using the FabRICATOR Z Fab2 Kit containing FabRICATOR Z Immobilized. Our IgG specific proteases are therefore also available in immobilized formats for fast and easy generation of homogenous F(ab’)2 and Fc/2 fragments. Some application such as binding studies are highly sensitive and therefore require pure antibody fragments without residual enzyme in the final preparation. ![]()
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