Antibodies or immunoglobulins are a group of structurally & functionally similar glycoproteins that confer humoral immunity in humans and animals. In 1975, Köhler and Milstein developed mouse hybridoma technology to immortalize individual B cell clones producing a single (monoclonal) antibody. Since then, antibody structure and function have been studied extensively. The antibody backbone typically consists of two identical heavy chains and two identical light chains. Five antibody classes or isotypes (IgG, IgA, IgM, IgD, IgE) are recognized in mice and humans on the basis of different constant regions in the heavy chains. In the following sections, we will focus on the IgG subclasses (using human IgGs as examples), which are widely utilized for research, diagnostic and therapeutic applications.

Antibody Structure and Fucntion

Human IgG is a tetrameric protein comprising two identical 50-kDa heavy chains and two identical 25-kDa light chains. Each light chain is covalently linked to the N-terminal region of one heavy chain, while the two heavy chains associate covalently via disulfide bridges located in the hinge region, endowing an IgG with a characteristic Y-shaped structure. In addition intra-chain disulfide bonds are responsible for the formation of loops, leading to the compact, discrete folding of Ig domain-like structure of ~110 amino acids (see the domain structure of human IgG₁ below).

Each light chain or heavy contains a variable region (VL, VH) and one or three constant regions (CL, CH_1-3).  The amino acid sequences of these N-terminal regions are much more variable than the constant regions, which make up the rest of the IgG molecule. The variable regions and first constant region form the so-called fragment for antigen binding (Fab), while the remainder of the molecule constitutes the fragment crystalline (Fc), a region displaying little subclass variability. 

IgG is a truly “bifunctional” molecule, working through the coordinated actions of their two arms, Fab (or Fv) and Fc:

Antibody Variable Regions

The coding sequences for the variable regions are assembled from a number of mini-gene families (V and J for the light chain; V, D and J for the heavy chain), which are present in multiple variant copies in our genome. The DNA sequence of these mini-genes is modified further during the recombination and hypermutation events ("somatic hypermutation"), which occur during the development of antibody-producing B cells. The combination of heavy and light chain variable regions (VH+VL) at each N-terminal arm of a whole IgG is also known as Fv.

Within each variable region, there are three non-contiguous regions, which are exceptionally variable and thus referred to as "complementarity determining regions" (CDRs). CDRs provide the repertoire of complementary surfaces for recognizing different antigens or epitopes. The binding affinity is essentially mediated via the six CDRs, which fold as independent loops and together form the large surface patch making direct contact with the antigen. The variable region residues that are not part of the CDR's constitute the "framework" regions (FWRs) and generally do not interact with the antigen.  However certain residues in FWRS are key to positioning CDRs and therefore contribute to the binding affinity and specificity of an antibody.

Human IgG Subclasses & Properties

There are four subclasses of human IgG (IgG_1-4), which can be distinguished by their heavy chain’s constant regions. The primary sequences of these constant regions are >95% homologous. Major differences are found in the hinge region in terms of the numbers of residues and interchain disulfide bonds (see the table below). The hinge region is the most diverse structural feature of different IgGs. It connects two heavy chains through disulfide bonds in the middle. It also links the two Fab arms to the Fc portion and provides flexibilities to the IgG molecules. The flexibility is important for the Fab arm to interact with differently spaced epitopes and for Fc to adopt different conformations to induce immune effector functions. 

There are two types of light chains termed kappa (κ) and lambda (λ) chains.  The ratio of kappa and lambda light chain varies from species to species (e.g., 20:1 in mice vs. 2:1 in humans) and is also a characteristic of different IgG subclass (e.g., 2.4:1 for human IgG₁ vs. 8:1 for IgG₄). In addition, the point of light chain attachment to the heavy chain also differ among the IgG subclasses. For example, IgG₁ light chain and heavy chain are bound through a disulfide bond near the midpoint of heavy chain (i.e., near the end of CH₁).  In contrast, IgG₂, IgG₃ and IgG₄ are joined at one quarter the distance from the heavy chain amino termini (i.e., near the end of VH). 

Different human IgG subclasses have varying abilities to activate immune effector functions. For example, the Fc portion of human IgG₁ and IgG₃ (but nor IgG₂ or IgG₄) is capable of binding to C1q, leading to the activation of the classical complement (CDC) cascade. IgG1 and IgG3 can also bind Fcg receptors (FcgRs) on immune effector cells, such as natural killer (NK) cells and macrophages, and recruit them to induce strong ADCC.  Over the last decade, many technologies have emerged to improve antibody-directed immune effector functions. They include altering the glycosylation pattern or sequence of the Fc region with the aim to enhance its binding to C1q or FcgRs, and constructing bispecific antibodies or fragments (e.g., BiTE) that engage target cells and immune effector components simultaneously (see the text and graph below).

Mouse Antibody Classes and Subclasses

Ther are five Ig classes or isotypes (IgA, IgD, IgE, IgG, and IgM) from mice, same as humans. Each isotype has a different heavy chain. The mouse IgG subclasses include IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgG₃. For IgG_2a and IgG_2c, however, inbred mouse strains with the Igh1-b allele have IgG_2c instead of IgG_2a. The murine heavy chain locus has only one of these two subclass genes in addition to the others. Like human, mouse IgG subclasses are very important in immune effector function. For example, mouse IgGs display remarkable differences in anti-bacterial responses (IgG₃ >> IgG_2b > IgG_2a >> IgG₁) and opsonophagocytic activities (IgG₃ > IgG_2b = IgG_2a >> IgG₁).

Antibody Applications

Conventional antibodies have been utilized in research for protein detection through Western blot, immunohistochemistry (IHC) and enzyme-linked immunosorbent assays (ELISA) for decades. Antibodies have also been developed for diagnostic applications such as pregnancy tests and detection of the viruses in the blood, such as an ELISA that detects HIV. Moreover, antibodies are used commonly in therapeutic applications. For example, Infliximab (Humira) is a human antibody that recognizes tumor necrosis factor alpha (TNFα) and is used in the treatment of Crohn's disease and rheumatoid arthritis. Trastuzumab, or Herceptin, is an antibody used in the treatment of metastatic breast cancer that binds to the epidermal growth factor receptor 2 (EGFR2 or Her2).

Typical antibody applications include:

• Therapeutic use (cancers, infectious diseases & inflammation)

Recombinant Antibody Production

Antibodies are unique in their high affinity and specificity for recognizing a target antigen, a quality that has made them one of the most useful macromolecules in Life Sciences, Biotechnology and Biomedical applications. Modern biotechnology has facilitated the large-scale production of recombinant antibodies. To date, almost all therapeutic antibodies in the clinic and on the market are expressed recombinantly in mammalian cells.

Recombinant antibodies have the highest standards of quality and purity in terms of the composition and specificity of antigen-binding. They are able to target specific epitopes, recruit the immune system where appropriate, maintain long serum half-lives, and deliver clinical benefits in patients. The generation of antibody-producing stable cell lines is an important component of the therapeutic antibody development process. CHO has become the industry “workhorse” for the production of therapeutic antibodies. However, the industry relies on several proprietary expression systems and selection methods (e.g., DHFR and GS) for antibody cell line generation, which is a time and resource consuming process (typically 6 to 18 months).

Recombinant Antibodies for Research Use

Antibodies are highly sensitive and specific for particular epitopes, which makes them ideal reagents for research, in particular in antigen detection and quantification. Currently, most research antibodies are produced in animals as monoclonal (with homogenous isotype and antigen specificity) and polyclonal (heterogeneous isotype and antigen specificity) antibodies. A polyclonal antibody supply is dependent on the source animal, and thus no two batches against a particular antigen will be identical. In contrast, monoclonal antibodies are grown from hybridomas, which can produce a continuous supply of homogenous antibody and are the current standard for research antibody production.

There are growing interests in using recombinant antibodies for research due to the homogeneity and reproducibility for a recombinant product with defined sequence and composition. It also allows a continuous supply of homogenous antibody (homogeneous composition and antigen specificity) and will probably replace hybridoma to become the future standard for research antibody production. In addition genetic engineering enable the quick switching of isotype, species, and/or subclass of a specific antibody, thus making it possible to generate a complete set of monoclonal antibody from all classes and subclasses with an identical antigen-binding specificity. This is particularly useful for the applications that classes/subclasses matter, e..g., immunostaining and flow cytometry analysis involving the use of secondary antibody as well as in vivo functional studies.

Antibody Fragments and Derivatives

Antibody fragments can be produced through chemical or genetic mechanisms. Chemical fragmentation utilizes reducing agents to break the disulfide bonds and digests the antibody with proteases such as pepsin and papain. For example, chemical and protease digestion of full size antibodies yield antigen binding fragments (Fab) from the variable regions of IgGor IgM. Although biochemical methods are able to generate antibody fragments, it is quite laborious and requires a large quantity of purified antibody starting material. In contrast, genetic engineering and construction of fragments offers the ability to create a multitude of fragment containing molecules, each with unique binding and functional characteristics.

The abbreviations in the graph above are as follows:

Genetic engineering allows the production of a single chain variable fragment (scFv) , which is Fv fragment (VH and VL) linked by a flexible peptide. Manipulation of the orientation of V-domains and the linker length creates different forms of Fv molecules. For example, when the linker is at least 12 amino acids long, the scFv fragment is primarily a monomer. Linkers of 3-11 amino acid long yield a dimeric scFv, which thus creates a bivalent “diabody”. If the linker length is less than three amino acids, scFv molecules associate into “triabody” or “tetrabody”, a multivalent form of scFv with greater binding avidity to the target antigen than a monmeric form. scFv fragments can be generated with two different variable domains, yielding a bispecific molecule to bind to two different epitopes. “Minibodies” are scFv-CH3 fusion proteins that assemble into bivalent dimers. Genetic engineering can also be used to create bispecific (Fab’)2 and trifunctional antibody (see the graph above). 

Disadvantages of full size antibodies include their inability to penetrate into certain tissues due to their relatively large size. The Fc region will frequently elicit an immune response, which may be detrimental in certain patients. For research, the Fc domain often causes nonspecific binding, which may impair detection specificity. Fragments offer advantages over a full size antibody for some applications. For example, antibody fragments are small enough to infiltrate into some tissues that full size antibodies are unable, which may help in both therapeutic and immunostaining procedures. However, these fragments lacking Fc are degraded in the body much more rapidly than the full length antibodies.

G&P Biosciences Antibody Production Capability

G&P Biosciences has developed unique mammalian expression systems for recombinant antibody production in a high throughput and time effective manner, especially suitable for research laboratory needs. We have exploited a large panel of expression vectors and selection methods for stable antibody cell line generation. Our expression vectors are designed to allow high throughput cloning of immunoglobulin genes and subsequent expression as whole antibodies or fragments. We can express a variety of different class and subclasses of IgG (e.g., human IgG1-4 including some allotypic variants) from many species, including human, mouse and rabbit. We can also produce many Fv and Fab fragment-derived molecules, such as Fab, Fab’, F(ab)’2, “minibody”, scFv-Fc and bispecific antibodies (visit our "Antidoy Products" and “Antibody Services” to learn more and order).

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