Recombinant antibodies

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Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. [1] They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of exploration and new production against specific targets. The most commonly used form is the single chain variable fragment (scFv), which has shown the most promising traits exploitable in human medicine and research. [2] In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity. [3] [4]

Contents

Structure and characterization

Formats

There are several known formats of recombinant antibodies which are commonly produced. These are the Fab recombinant antibodies, scFv and diabodies. [4] [5] [3] Each of the formats has a slightly different potential in applications and may be used in various fields of research as well as human and animal medicine. [6] Another researched possibility is the development of anti-idiotypic antibodies. Anti-idiotypic antibodies bind to a paratope of another specific antibody. Therefore, it can be used for measuring presence of antibodies and drug loads in patients' sera. [7] Based on their binding specificity 3 types of anti-idiotypic antibodies can be distinguished, which partially overlap with the previously mentioned formats: the classical ones, a group including Fab fragment antibodies, antibodies binding to idiotope outside of the drug binding site and antibodies, which only bind to the already assembled complex of drug bound to the target. [7] The most commonly used are the scFv, Fab fragments and bispecific antibodies.

Single chain variable fragment (scFv)

scFv is the smallest of the recombinant antibody formats, which is capable of antigen binding. [8] They have a molecular weight of approximately 27kDa. [9] They are formed by light and heavy chain of the variable region of an immunoglobulin. The two chains are linked by a flexible peptide linker. [2] The flexible peptide linker usually consists of short sequence repetition. The sequence is made up of four glycines and a serine [5] and it serves the purpose of stabilization of the fragment. [8] [10] The functionality may be enhanced by site-specific chemical modifications, adding a peptide-tag or by fusion with a gene to achieve production of bifunctional recombinant antibodies. [9] It is important to establish the binding activity in order to ensure good functionality of the product. To determine the binding activity, ELISA assay is routinely performed. [11]

Fab fragments

Structurally Fab fragments consist of two sets of variable and constant components, which create two polypetide chains. Together they form a stable structure. [5] As a member of the anti-idiotypic antibodies, Fab fragment recombinant antibodies bind directly to the paratope of the target antibody. That means that they compete with the drug for binding site and have an inhibitory function. Fab fragment antibodies can be used for detection of not bound drugs or free drugs in the serum. [7] Fab antibodies have also been used to avoid the adverse effects caused by unspecific binding of the Fc portion of the antibody, which is missing in the Fab fragment. [5] In case the IgG immunoglobulin was more suitable for the treatment or some other particular application, experiments have also been conducted, in which the recombinant Fab fragments were converted into recombinant IgG form. This possibility further broadens the pool of potential target structures. [12]

Bispecific recombinant antibodies

Along scFv and Fab fragments, diabodies or bispecific recombinant antibodies are the third major format. [5] Bispecific antibodies combine two different antigen binding specificities within one molecule. [10] The bispecific antibodies are used to crosslink the target molecules with two different cells and mediate direct cytotoxicity. [13] [14]

Production and development

Production of recombinant antibodies

The production of recombinant antibodies follows principally similar workflow. It consists of determining the sequence of the desired product followed by refinement of the codon, then gene synthesis and construct generation. Once the construct is delivered to the laboratory, expression constructs are produced, then they are transferred to a cell culture in the process called transfection and once the cell culture produces the desired recombinant antibody, it is regularly collected, purified and analyzed or used for further experimentation. For recombinant antibody production the stable cell lines such as CHO and HEK293 are used. [4] Optimizations of mammalian cell cultures have led to increase the yield of antibodies from HEK293 or CHO cell lines to over 12g/liter. [15] In the beginning phases of the recombinant antibody production it was important to achieve the assembly of a functional Fv fragment in Escherichia coli . The correct fold is essential for functionality of the antibody. [16] Second essential prerequisite for the modern day production of scFv was the successful assembly of recombinant antibodies from heavy and light chain of immunoglobulin. [17] These two experiments allowed for further development and refinement of the recombinant antibodies until modern day form. Today's in vitro production process eliminates the need for laboratory animals. Using a synthetic or human Ab library, as opposed to immunization of animals and the subsequent generation of stable hybridoma cell lines, requires fewer resources and produces less waste, making the entire process more sustainable. [18]

Hybridoma

Monoclonal antibodies are essential for many therapies applied today in human medicine. The first successful technology which was robust and led to a stable production of desired antibodies was hybridoma technology. The hybridoma cell lines, which produced large quantities of relatively pure and predictable antibodies was first introduced in 1975. [19] Since then, it has been used for various purposes scaling from diagnostic and therapeutic to research applications. Despite its indisputable role in scientific discoveries and numerous treatment strategies, the hybridoma technology presents researchers with some obstacles such as ethical issues, potential to lose expression of the target protein or lengthy production and most importantly the development of HAMA in patients as mentioned previously. [4] [20] Therefore, different methods need to complement or even partially replace the hybridoma. Hybridomas are an essential part of the recombinant antibody generation even today as they are still used to produce the monoclonal antibodies, from which the Fab fragments, scFv or somatically fused antibodies create a bispecific antibody. [5]

Phage display

The most commonly applied technology to produce recombinant antibodies in the laboratory settings today is the phage display. [2] [9] [10] [11] [21] [22] Phage display is a method, in which the target recombinant antibody is produced on the surface of a bacteriophage. This allows for a fast recombinant antibody production and easy manipulation in the laboratory conditions. Both scFv and Fab fragment recombinant antibodies are routinely produced using the antibody phage display. [10] From all the possible phage display systems, the most common is the Escherichia coli, due to its rapid growth and division rate and cheap set up and maintenance. [20]

Engineering and development

Two main strategies have been described to engineer the scFv fragments. The first one is the so-called non-colinear approach. It works on the principle of heterodimerization of two chains. Non-colinear approach leads to production of diabodies and recombinant antibodies, which combine two specificities. The second approach is called colinear and it described the process of fusion of two different scFv with a biologically active protein. [5]

Medical and research applications

Recombinant antibodies fulfill a large spectrum of functions spanning from research to diagnosis and treatment therapies for various diseases. Their specificity and low immunogenicity make them a great alternative to traditional forms of treatment, increasing the accuracy of targeting specific molecules and avoiding adverse side effects.

Recombinant antibodies have been explored as a treatment for cancer, [23] HIV, [24] herpes simplex virus (HSV) [22] and more. ScFv have been a part of the highly promising therapeutic approach of universal chimeric antigen receptors (uniCAR) technology, which shows promising results. The scFv are part of the technology in the form of target modules, which direct the immune response to specific cancer cells, expressing the target antigen. [23] [25] [26] In case of research into HIV treatment, the recombinant antibodies are rather used for their neutralizing quality. [24] The same goes for HSV infection. Specific recombinant antibodies are designed to bind with to surface heparin sulphate proteoglycan (HSP), which complicates or even disables the entry of the HSV into the host cell. This is a method which significantly decreases the severity of HSV infection. [22]

As was mentioned in the beginning of this section, recombinant antibodies can also be used in diagnosis, an example of such diagnostic application is the detection of rabies virus. [3] [20] [27] Since the current diagnostic antibodies are not as accurate as would be desired, the recombinant antibodies offer a promising alternative. In case of rabies infection, which is only treatable shortly after exposure, accurate and precise diagnosis is vital for survival of the patient. In comparison to commercially produced and commonly available antibodies, the recombinant antibodies are cheaper to produce and more accurate in determining the infection. Another advantage of the recombinant antibody is the potential application as a neutralizing antibody as part of the subsequent treatment. [20]

The potential of recombinant antibodies in human and animal medicine is immense as shown even by the few selected examples. As mentioned previously the recombinant antibodies and especially those, which have been developed in phage display are highly specific, have great pharmacokinetics and could be used in wide range of treatments. However, it is important to realize that it is not expected or desired for the recombinant antibodies created in phage display to completely replace the hybridoma antibody production but rather to complement it. [4]

Advantages of using recombinant antibodies

Recombinant antibodies bring many advantages with their application in human medicine and research. The first one is the complete elimination of ethical issues because there is no need for animal immunization. The cultivation of CHO cells for recombinant antibody expression is a popular strategy for antibody producers since the cell structure is similar to that of the human body. Thanks to their size, which is smaller than complete antibody and particularly than 2000 nm, [28] yet not smaller than 8 nm [29] they are cleared from the organism with ease and in a timely manner, through the renal pathway, which is the desirable clearance. [28] [29] Another great advantage is their monovalency, which means that they are highly specific and bind to a single antigen. Researchers have managed to produce antibodies carrying no other activity than the antigen binding. [9] Since the recombinant antibodies are sequence defined they are more reliable as well as reproducible. [4] In combination with their small size the great specificity can be exploited to deliver highly specific drug to a specific site precisely because the small size predisposes the recombinant antibodies to penetrate tissues more easily. It has been reported that the recombinant antibodies penetrate tumor tissue better than the full-length IgG immunoglobulins. [30] The small size also adds to better biodistribution in the patient. [1] In comparison to antibodies derived from hybridoma cell lines the recombinant antibodies do not cause immunogenicity, the infamous human anti-mouse antibody (HAMA). [4] [21] Further advantages show afucosylated recombinant antibodies which are used successfully in the fight against cancer. [31]

These were the top advantages for use in patients. However, the use of recombinant antibodies is also advantageous compared to traditional monoclonal antibodies derived from hybridoma cell lines during their production as well. The production is much faster and we have better control over the process than in hybridoma technology. Moreover, the recombinant antibodies may be designed virtually against any antigen, of the proper size and shape, but they are not solely limited to the peptide nature of an antigen. The recombinant antibodies may also be used in fused form with drugs and/or toxins, which may be further exploited in the medical applications. Last but not least of their advantages during production is the possibility to optimize and genetically engineer the recombinant antibodies based on the current demand of the patient or researcher. [4] An experienced technician is required to perform the phage display and third it is almost inevitable to include outsource companies in the process for the gene synthesis and construct generation. [1] [4] However, in a systematic comparison of animal derived antibodies verus phage display derived recombinant antibodies used for research and diagnostic applications, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) released a recommendation in favor of on non-animal derived antibodies in May 2020, [32] mainly based on the fact that in contrast to animal derived antibodies, recombinant antibodies are always sequence defined protein reagents, allowing to eliminate some of the quality issues attributed to current research antibodies when made in animals. [33] [34]

Related Research Articles

<span class="mw-page-title-main">Antibody</span> Protein(s) forming a major part of an organisms immune system

An antibody (Ab) is the secreted form of a B cell receptor; the term immunoglobulin (Ig) can refer to either the membrane-bound form or the secreted form of the B cell receptor, but they are, broadly speaking, the same protein, and so the terms are often treated as synonymous. Antibodies are large, Y-shaped proteins belonging to the immunoglobulin superfamily which are used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that cause disease. Antibodies can recognize virtually any size antigen with diverse chemical compositions from molecules. Each antibody recognizes one or more specific antigens. Antigen literally means "antibody generator", as it is the presence of an antigen that drives the formation of an antigen-specific antibody. Each tip of the "Y" of an antibody contains a paratope that specifically binds to one particular epitope on an antigen, allowing the two molecules to bind together with precision. Using this mechanism, antibodies can effectively "tag" a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

<span class="mw-page-title-main">Monoclonal antibody</span> Antibodies from clones of the same blood cell

A monoclonal antibody is an antibody produced from a cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.

<span class="mw-page-title-main">Phage display</span> Biological technique to evolve proteins using bacteriophages

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. The proteins that the phages are displaying can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those of other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

<span class="mw-page-title-main">Single-chain variable fragment</span> Fragment

A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The image to the right shows how this modification usually leaves the specificity unaltered.

<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients. The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab.

<span class="mw-page-title-main">Protein A</span> Surface protein in bacteria cell walls

Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family. Through these interactions in serum, where IgG molecules are bound in the wrong orientation, the bacteria disrupts opsonization and phagocytosis.

<span class="mw-page-title-main">Monoclonal antibody therapy</span> Form of immunotherapy

Monoclonal antibodies (mAbs) have varied therapeutic uses. It is possible to create a mAb that binds specifically to almost any extracellular target, such as cell surface proteins and cytokines. They can be used to render their target ineffective, to induce a specific cell signal, to cause the immune system to attack specific cells, or to bring a drug to a specific cell type.

<span class="mw-page-title-main">Fragment antigen-binding region</span> Part of an antibody that binds to antigens

The fragment antigen-binding region is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope, comprising a set of complementarity-determining regions, at the amino terminal end of the monomer. Each arm of the Y thus binds an epitope on the antigen.

<span class="mw-page-title-main">Fragment crystallizable region</span> Tail region of an antibody

The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This region allows antibodies to activate the immune system, for example, through binding to Fc receptors. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains in each polypeptide chain. The Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and α-2,6 linked sialic acid residues.

<span class="mw-page-title-main">Protein L</span>

Protein L was first isolated from the surface of bacterial species Peptostreptococcus magnus and was found to bind immunoglobulins through L chain interaction, from which the name was suggested. It consists of 719 amino acid residues. The molecular weight of protein L purified from the cell walls of Peptostreptoccus magnus was first estimated as 95kD by SDS-PAGE in the presence of reducing agent 2-mercaptoethanol, while the molecular weight was determined to 76kD by gel chromatography in the presence of 6 M guanidine HCl. Protein L does not contain any interchain disulfide loops, nor does it consist of disulfide-linked subunits. It is an acidic molecule with a pI of 4.0. Unlike protein A and protein G, which bind to the Fc region of immunoglobulins (antibodies), protein L binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than protein A or G. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (scFv) and Fab fragments also bind to protein L.

Small modular immunopharmaceuticals, or SMIPs for short, are artificial proteins that are intended for use as pharmaceutical drugs. They are largely built from parts of antibodies (immunoglobulins), and like them have a binding site for antigens that could be used for monoclonal antibody therapy. SMIPs have similar biological half-life and, being smaller than antibodies, are reasoned to have better tissue penetration properties. They were invented by Trubion and are now being developed by Emergent BioSolutions, which acquired Trubion in 2010.

A bispecific monoclonal antibody is an artificial protein that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen. Naturally occurring antibodies typically only target one antigen. BsAbs can be manufactured in several structural formats. BsAbs can be designed to recruit and activate immune cells, to interfere with receptor signaling and inactivate signaling ligands, and to force association of protein complexes. BsAbs have been explored for cancer immunotherapy, drug delivery, and Alzheimer's disease.

<span class="mw-page-title-main">Trifunctional antibody</span> Monoclonal antibody

A trifunctional antibody is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen, making it a type of bispecific monoclonal antibody. In addition, its intact Fc-part can bind to an Fc receptor on accessory cells like conventional monospecific antibodies. The net effect is that this type of drug links T cells and monocytes/macrophages, natural killer cells, dendritic cells or other Fc receptor expressing cells to the tumor cells, leading to their destruction.

<span class="mw-page-title-main">Chemically linked Fab</span>

Two chemically linked fragments antigen-binding form an artificial antibody that binds to two different antigens, making it a type of bispecific antibody. They are fragments antigen-binding of two different monoclonal antibodies and are linked by chemical means like a thioether. Typically, one of the Fabs binds to a tumour antigen and the other to a protein on the surface of an immune cell, for example an Fc receptor on a macrophage. In this way, tumour cells are attached to immune cells, which destroy them.

A rabbit hybridoma is a hybrid cell line formed by the fusion of an antibody producing rabbit B cell with a cancerous B-cell (myeloma).

<span class="mw-page-title-main">ImmTAC</span>

ImmTACs are a class of bispecific biological drug being investigated for the treatment of cancer and viral infections which combines engineered cancer-recognizing TCRs with immune activating complexes. ImmTACs target cancerous or virally infected cells through binding human leukocyte antigen (HLA) presented peptide antigens and redirect the host's cytotoxic T cells to recognise and kill them.

Synthetic antibodies are affinity reagents generated entirely in vitro, thus completely eliminating animals from the production process. Synthetic antibodies include recombinant antibodies, nucleic acid aptamers and non-immunoglobulin protein scaffolds. As a consequence of their in vitro manufacturing method the antigen recognition site of synthetic antibodies can be engineered to any desired target and may extend beyond the typical immune repertoire offered by natural antibodies. Synthetic antibodies are being developed for use in research, diagnostic and therapeutic applications. Synthetic antibodies can be used in all applications where traditional monoclonal or polyclonal antibodies are used and offer many inherent advantages over animal-derived antibodies, including comparatively low production costs, reagent reproducibility and increased affinity, specificity and stability across a range of experimental conditions.

Creative Biolabs, Inc. is a life-science company which produces and supplies biotech products and services for early drug discovery and development, including various phage display libraries such as pre-made libraries, phage display services, antibody sequencing, and antibody humanization. Customers include pharmaceutical companies, academic institutions, government agencies, clinical research organizations and biotechnology companies.

<span class="mw-page-title-main">Pretargeting (imaging)</span>

Pretargeting (imaging) is a tool for nuclear medicine and radiotherapy. Imaging studies require a high contrast of target to background. This can be provided by using a biomarker which has a high affinity and specificity for its target.

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  28. 1 2 Blanco E, Shen H, Ferrari M (September 2015). "Principles of nanoparticle design for overcoming biological barriers to drug delivery". Nat. Biotechnol. 33 (9): 941–51. doi:10.1038/nbt.3330. PMC   4978509 . PMID   26348965.
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  30. ↑ Yokota, T.; Milenic, D. E.; Whitlow, M.; Wood, J. F.; Hubert, S. L.; Schlom, J. (1993-08-15). "Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other immunoglobulin forms". Cancer Research. 53 (16): 3776–3783. ISSN   0008-5472. PMID   8339291.
  31. ↑ "What's the difference between afucosylated antibodies and recombinant antibodies? - evitria". 2021-03-29. Retrieved 2021-04-28.
  32. ↑ EURL ECVAM Recommendation on Non-Animal-Derived Antibodies. Publications Office of the European Union. 2020. ISBN   978-92-76-18346-4.
  33. ↑ Baker, Monya (May 2015). "Reproducibility crisis: Blame it on the antibodies". Nature. 521 (7552): 274–276. Bibcode:2015Natur.521..274B. doi: 10.1038/521274a . PMID   25993940.
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