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First draught of the entry about recombinant antibodies, including sections on structure, forms, advantages and production. There are still missing aspects, which I will add tomorrow.
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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. Recombinant antibodies have many advantages in both medical and research application, which make them a popular subject for further 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 for further research.[2] In contrast to monoclonal antibodies produced by hybridoma cell lines, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which effect its functionality.[3][4]

Structure and characterization

Formats

There are several know formats of recombinant antibodies which are commonly produced. These are the Fab recombinant antibodies, scFv and diabodies.[4][5][6] Each of the formats has a slightly different potential in applications and may be used in various fields of research and both human and animal medicine.[7] 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.[8] 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.[9]

Single chain variable fragment (scFv)

scFv is the smallest of the recombinant antibody formats.[2] They are formed by light and heavy chain of the variable region. The two chains are linked by a flexible peptide linker. [2]

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.[9] Fab antibodies have also been used to avoid the adverse side affects, which may be caused by unspecific binding of the Fc portion of the antibody.[5]

Advantages of using recombinant antibodies

Recombinant antibodies bring many advantages into 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. Thanks to their size, which is smaller than complete antibody and particularly than 2000 nm[10], yet not smaller than 8 nm[11] they are cleared from the organism with ease and in a timely manner, through the renal pathway, which is the desirable clearance.[10][11] Another great advantage is their monovalency, which means that they are highly specific and bind to a single antigen. The research is so far that the antibodies carry no other activity than the antigen binding.[12] 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.[13] 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][14]

These were the top advantages for the use in patients. However, the use of recombinant antibodies is also advantegeous compared to traditional antibodies derived from hybridoma cell lines during their production as well. Even though the yields of the final product are not as high, the production is much faster and we have better control over the process of production than in hybridoma cell lines. 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 structure 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]

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 generated, 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.[4]

Monoclonal antibodies are essential for many therapies applied today in human medicine. The first successful technology which was robust and led to 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. [15] Since then, it has been used for various purposes scaling from diagnostic to therapeutic and research applications. Despite its indisputable role in scientific discoveries and numerous treatment strategies, the hybridoma technology presents 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.[4] Therefore, different methods need to complement or even partially replace the hybridoma.

The most commonly applied technique to produce recombinant antibodies in the laboratory settings today is the phage display.[2][12][14][16][17][18] 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.[16]

Medical and research applications

The potential of recombinant antibodies in human and animal medicine is immense as shown 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]

  1. ^ a b Creative Biolabs (2017-04-28), Introduction of Recombinant Antibody, retrieved 2017-08-18
  2. ^ a b c d Ahmad, Zuhaida Asra; Yeap, Swee Keong; Ali, Abdul Manaf; Ho, Wan Yong; Alitheen, Noorjahan Banu Mohamed; Hamid, Muhajir (2012). "scFv Antibody: Principles and Clinical Application". Clinical and Developmental Immunology. 2012: 1–15. doi:10.1155/2012/980250. ISSN 1740-2522.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  3. ^ Kunert, Renate; Reinhart, David (2016-04-01). "Advances in recombinant antibody manufacturing". Applied Microbiology and Biotechnology. 100 (8): 3451–3461. doi:10.1007/s00253-016-7388-9. ISSN 0175-7598.
  4. ^ a b c d e f g h Miltenyi Biotec (2017-03-22), Webinar: Recombinant Antibodies for Improved Flow Cytometry, retrieved 2017-08-20
  5. ^ a b c Kriangkum, Jitra; Xu, Biwen; Nagata, Les P.; Fulton, R.Elaine; Suresh, Mavanur R. "Bispecific and bifunctional single chain recombinant antibodies". Biomolecular Engineering. 18 (2): 31–40. doi:10.1016/s1389-0344(01)00083-1.
  6. ^ Kunert, Renate; Reinhart, David (2016-04-01). "Advances in recombinant antibody manufacturing". Applied Microbiology and Biotechnology. 100 (8): 3451–3461. doi:10.1007/s00253-016-7388-9. ISSN 0175-7598.
  7. ^ Ma, Julian K.-C.; Hikmat, Ban Y.; Wycoff, Keith; Vine, Nicholas D.; Chargelegue, Daniel; Yu, Lloyd; Hein, Mich B.; Lehner, Thomas (May 1998). "Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans". Nature Medicine. 4 (5): 601–606. doi:10.1038/nm0598-601.
  8. ^ Bio-Rad Laboratories (2013-12-03), Developing Recombinant Anti Idiotypic Antibodies for PK/PD and Immunogenicity Assays, retrieved 2017-08-20
  9. ^ a b Bio-Rad Laboratories (2013-12-03), Developing Recombinant Anti Idiotypic Antibodies for PK/PD and Immunogenicity Assays, retrieved 2017-08-20
  10. ^ a b Blanco, Elvin; Shen, Haifa; Ferrari, Mauro. "Principles of nanoparticle design for overcoming biological barriers to drug delivery". Nature Biotechnology. 33 (9): 941–951. doi:10.1038/nbt.3330.
  11. ^ a b "Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats". Nanomedicine. 3 (5): 703–717. 2008-09-25. doi:10.2217/17435889.3.5.703. ISSN 1743-5889.
  12. ^ a b Neri, D.; Petrul, H.; Roncucci, G. (August 1995). "Engineering recombinant antibodies for immunotherapy". Cell Biophysics. 27 (1): 47–61. ISSN 0163-4992. PMID 7493398.
  13. ^ 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.
  14. ^ a b "Therapeutic targeting in nanomedicine: the future lies in recombinant antibodies". Nanomedicine. 12 (15): 1873–1889. 2017-07-13. doi:10.2217/nnm-2017-0043. ISSN 1743-5889.
  15. ^ Köhler, G.; Milstein, C. (1975-08-07). "Continuous cultures of fused cells secreting antibody of predefined specificity". Nature. 256 (5517): 495–497. doi:10.1038/256495a0.
  16. ^ a b Frenzel, André; Hust, Michael; Schirrmann, Thomas (2013). "Expression of Recombinant Antibodies". Frontiers in Immunology. 4. doi:10.3389/fimmu.2013.00217. ISSN 1664-3224.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  17. ^ Jørgensen, Mathias Lindh; Friis, Niels Anton; Just, Jesper; Madsen, Peder; Petersen, Steen Vang; Kristensen, Peter (2014-01-15). "Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae". Microbial Cell Factories. 13: 9. doi:10.1186/1475-2859-13-9. ISSN 1475-2859.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  18. ^ Bagheri, Vahid; Nejatollahi, Foroogh; Esmaeili, Seyed Alireza; Momtazi, Amir Abbas; Motamedifar, Mohamad; Sahebkar, Amirhossein. "Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library". Life Sciences. 169: 1–5. doi:10.1016/j.lfs.2016.11.018.
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