MOJ ISSN: 2373-4442MOJI

Immunology
Mini Review
Volume 3 Issue 4 - 2016
Advances in Monoclonal Antibodies Production and Cancer Therapy
Abdullah Farhan ul Haque Saeed1* and Saima Ashraf Awan2
1Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, Fujian Agriculture and Forestry University, China
2Fujian Agriculture and Forestry University, China
Received: June 22, 2016 | Published: July 15, 2016
*Corresponding author: Abdullah Farhan ul Haque Saeed, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou-350002, China, Email:
Citation: Saeed AFUH, Awan SA (2016) Advances in Monoclonal Antibodies Production and Cancer Therapy. MOJ Immunol 3(4): 00099. DOI: 10.15406/moji.2016.03.00099

Abstract

Monoclonal antibodies are highly specific class of antibodies which are produced by identical hybridoma cells. These antibodies are developed by various advanced and powerful technologies being used in the areas of cell biology, biochemistry, biotechnology, immunology and medical sciences. The techniques involved hybridoma technology, phage display technology, single B-cell amplification by polymerase chain reaction (PCR) and many other modified methods. The advancement in antibody therapeutics is an essential area of growth in the pharmaceutical manufacturing and more than 30 monoclonal antibodies have been approved on the US and Europe markets. Regardless of these advancements, certain significant target antigens remain intractable to therapeutic antibodies due to complexity of the target molecules. The present review describes recent advances in monoclonal antibody production and cancer therapy for better understanding the therapeutic efficacy.

Keywords: Monoclonal antibody; Hybridoma; Phage display; B-Cell amplification; Therapeutics

Abbreviations

PCR: Polymerase Chain Reaction; mAbs: Monoclonal Antibodies; IP: Intellectual Property; HAMA: Human Anti-Mouse Antibodies; PEG: Polyethylene Glycol; CDRs: Complementarity-Determining Regions; FDA: Food and Drug Administration; VH: Variable Domains; Ig: Immunoglobulins

Introduction

Monoclonal antibodies (mAbs) are the dominant group of recombinant proteins used in human therapy. These proteins were first successfully developed by Köhler and Milstein [1] in mid-1970s and published in Nature in 1975 with free of intellectual property (IP) rights, and produced a method for B-cell immortalization and the production of monoclonal antibodies. The mAbs have various applications in the fields of cell biology, immunology, biotechnology and medicines. They are also being used in vivo imaging techniques of different kinds of diseases [1-4].

Antibodies are also called immunoglobulins (Ig), they play a vital role in internal defenses or immunity against a vast number of invading microorganisms and other disease causing agents or antigens. They are a major group of glycoproteins which are a part of neutralizing immune response present in blood or other body fluids of vertebrates. These antibodies have five common isotypes (IgG, IgE, IgA, IgM and IgD), among them, IgGs are permeable to extravascular spaces, have a longer half-life as they bind to neonatal Fc receptor (FcRn) as compared to other isotypes and have the most therapeutic potential. FcRn binding affinity, cross-reactivity, and the elimination pathways are comparable in nonhuman primates and humans. Therapeutic mAbs usually bind to nonhuman primate antigens than rodent antigens because of the greater sequence homology [5-18].

Köhler and Milstein [1] developed mAbs by the isolation of primary B-cells which were later differentiated and matured in vivo and further fused with immortalized myeloma cells or lymphoblastoid cells. These myeloma cell lines were already being cultured since the 1960s and were used for differentiation of fused and unfused cells in hybridoma technology. Subsequently, the hybridoma technology destined for immortalized differentiated cells was soon recognized as the potential technique for the production of mAbs on a large scale in pharma industry as future blockbusters [1,4].

 Till now, the mAbs are being developed by a number of pharmaceutical industries on a large scale. It has been projected that more than 30 mAbs and their derivatives (fusion proteins and antibody fragments) have been developed; furthermore, more than 350 mAbs are in various phases of drug trials and patents. Significantly, antibodies are reflected to be individual products and can be patented to produce market exceptionality [19-21].

Production, genetic manipulation and progress in molecular biology techniques enabled development of recombinant hybridoma cell lines instead of less-defined and undifferentiated cells. The next generation of mAb development cell lines was determined by the glitches of hybridoma instability. The production of chimeric antibodies with human Fc and mouse Fv regions helped the prohibition of human anti-mouse antibodies (HAMA) responses in human patients [22-24]. The chemistry, and advances in production technologies and therapeutic uses of mAbs have been discussed in the current study.

Chemistry of mAbs

The mAbs exhibit biological effector functionality by interacting with complementary and Fc domains. A typical IgG has total molecular weight of approximately 150 kDa. It is a symmetrical Y-shaped globular molecule comprising of two identical heavy chains (~50 kDa each) and two identical light chains (~25 kDa each) respectively. The heavy chains are further consisted of one variable domain and three constant domains, and the light chains consist of one variable and one constant domain. The variable domain is moreover consisted of three small sections of peptide which is called complementarity-determining regions (CDRs) (Figure 1). CDRs are the hypervariable regions that determine the specific antibody binding [25-28].

Figure 1: The structure of monoclonal antibody and its components.

Advances in mAbs production technologies

The development and recognizing mAb by immortalized hybridoma technology has long been in used since 1977. Since then the rodent mAb technology has been rapidly and successfully applied for the development of new and improved diagnostics and therapeutics to enhance the understanding of human biology [29].

Hybridoma technology

The mAbs have been developed by using mouse hybridoma technology for therapeutic applications. This technique uses innate functionality of B-lymphocytes and myeloma or immortalized cancerous cells for the effective production of immortal hybridoma cells secreting mAbs specific for antigen of interest. The B-cells are fused with myeloma cells in the presence of polyethylene glycol (PEG) as a fusing agent (Figure 2). The antibodies produced by this technique are specific in nature to the target antigen and has various application in therapeutics. However, immune responses are often weak in mice, resulting in low affinity and/or non-specific mAbs. Animals other than rodents have not been usually used to produce mAb due to the problems associated in the establishment of immortalized antibody-producing cell lines by hybridoma [1,30,31].

Phage display

Phage display technology was developed by G. Smith in 1985 to display the peptides on the surface of lysogenic filamentous bacteriophages. Since then, this technology has become one of the leading method to develop large number of peptides, proteins and antibodies with the basis of physical linkage of phage phenotype (phage coat fusion protein expression) and genotype as single-strained DNA (ssDNA) of the related virion. This technique allows the development of phage display libraries containing up to 1010 phage variants, affinity assay of protein-ligand interactions, characterization, epitope recognition, enzyme substrate selection, antibody repertoire screening and favors the emergence of identical phage particles from Escherichia coli clone [32-36].

Figure 2: A schematic illustration of hybridoma technology.

Single B cell amplification

Currently, for the isolation of high affinity mAbs by somatic hypermutation and affinity maturation technique, the direct molecular cloning of identical pairs of antibody light chain lambda variable (VLλ), heavy chain (IgH) variable (VH) and light chain kappa variable (VLκ) genes from single antigen-specific plasma/plasmablast cells (ASPCs) with the help of polymerase chain reaction (PCR) is an alternative technique being used for the development of mAb from immunized animals [37-42]. Additionally, there are some drawbacks such as expensive equipment and technical skills which are required to isolate ASPCs. The VH and VL genes from the cells are manually amplified and construction of immunoglobin heavy and light (IgH and IgL) chains is also limiting steps. For the development of rapid and scalable generation of mAbs from single cell, a high-throughput single-cell-based immunoglobulin gene cloning technique was used by developing a non-contact magnetic power transmission system (MAGrahd) for automated single-cell-based cDNA synthesis, 3' end homopolymer tailing and a target-selective joint PCR (TS-jPCR) for IgH and IgL gene expression [43-53].

Advances in cancer therapy by mAbs

The advent of hybridoma technology revolutionized the areas of cell biology, immunology, biotechnology and medical research. Before the introduction of hybridoma technology, antibodies were produced by recurrent animal immunization with antigen of interest and the sera were used for the therapy. There were allergic responses and no clinical benefits to the patients of the administration of the crude sera. Therefore, by using the hybridoma technology, a large number of mAbs were produced against the target antigens with the help of mice splenocytes and immortal myeloma cells [1,54-56].

Until now, the mAbs have extensively been used for diagnostics and cancer therapy. However, the administrations of mouse mAbs produces HAMA in cancer patients, cause rapid clearance and are highly immunogenic. To overcome these limitations, the recent biomedical and technological advances in genetic engineering helped reduction of immunogenicity, enhanced production of antibody-drug conjugates, smaller antibody fragments in cancer imaging [57,58].

The recent advances such as use of

  1. Transgenic mice (first generation, 1970s),
  2. Chimeric antibody (second generation, 1980s),
  3. Humanized, fully humanized and
  4. Bispecific antibody (third generation, 1990s and 2000s) aided in the increase of mAbs half-life and therapeutic potential.
    1. The transgenic mouse antibody contains two identical heavy and light chains (CH, CL), and variable domains (VH, VL) further containing antigen binding site (CDRs), immunogenicity is generated by the constant (Fc) region of an antibody which is linked by disulphide (-S-S-) bonds.
    2. Chimeric mAbs are constructed by substituting constant region of mouse antibody with constant region of human IgG antibody.
    3. Humanized antibody contains >90% of human sequences and produced by the fusion of DNA for three CDRs of mouse variable domain into human IgG structure. Fully humanized antibodies are constructed with the use of transgenic mice having human Ig or phage display technique. It consisted of 100% human and therefore less immunogenic than its mouse counterparts, chimeric and humanized.
    4. Bispecific antibodies are comprised of two distinct antigen-binding regions and binds to two distinct antigens consecutively. Shorter antibody fragments have been developed such as monovalent scFv (30 kDa) and divalent (scFv) (60 kDa) with antigen binding specificity for the respective antibody [59-64].

A large number of mAbs have been patented for therapeutic use by the US Food and Drug Administration (FDA) and/or the European Union Health Authorities. These antibodies are administered against various fatal diseases such as autoimmunity, inflammatory responses, hematological malignancies, organ transplant rejection and human cancers respectively. Additionally, many other therapeutic mAbs have been in various phases of clinical trials against deadly infections such as HIV/AIDS, Ebola and Zika viral disease [64-69].

Conclusion

Since last 60 years, there has been a great deal of progress and advancements in mAb development. Various powerful techniques such as hybridoma technology, phage display and B-cell amplification with the help of PCR have been developed for the construction of numerous types of antibodies and antibody fragments with widespread structural modifications. The mAbs are really significant as they are highly specific and play a key role in specific immunity against target antigens. They have various applications in areas of cell biology, biochemistry, biotechnology, immunology and medicines. This study will help in understanding the current advancements of mAbs development and will benefit in further progress in future.

References

  1. Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517): 495-497.
  2. Abdullah MA, Rahmah A, Sinskey AJ, Rha CK (2008) Cell Engineering and Molecular Pharming for Biopharmaceuticals. Open Med Chem J 2: 49-61.
  3. Crommelin DJA, Sidelar RD (2002) Pharmaceutical Biotechnology: An Introduction for Pharmacist and Pharmaceutical Scientist. New York: John Wiley & Sons, USA.
  4. Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100(8): 3451-3461.
  5. De Castro LN, Von Zuben FJ (1999) Artificial immune systems: Part I-basic theory and applications. Universidade Estadual de Campinas, Dezembro de, Technical Report, 210.
  6. Rauta PR, Nayak B, Das S (2012) Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunology letters 148(1): 23-33.
  7. Ochsenbein AF, Zinkernagel RM (2000) Natural antibodies and complement link innate and acquired immunity. Immunology today 21(12): 624-630.
  8. Mak TW, Saunders ME (2005) The immune response: basic and clinical principles. Academic Press, USA.
  9. Roopenian DC, Akilesh S (2007) FcRn: the neonatal Fc receptor comes of age. Nature Reviews Immunology 7(9): 715-725.
  10. Burmeister WP, Huber AH, Bjorkman PJ (1994) Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372(6504): 379-383.
  11. Israel EJ, Taylor S, Wu Z, Mizoguchi E, Blumberg RS, et al. (1997) Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells. Immunology 92(1): 69-74.
  12. Chaudhury C, Mehnaz S, Robinson JM, Hayton WL, Pearl DK, et al. (2003) The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197(3): 315-322.
  13. Schlachetzki F, Zhu C, Pardridge WM (2002) Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier. Journal of neurochemistry 81(1): 203-206.
  14. Raghavan M, Bonagura VR, Morrison SL, Bjorkman PJ (1995) Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants. Biochemistry 34(45): 14649-14657.
  15. Wang J, Iyer S, Fielder PJ, Davis JD, Deng R (2015) Projecting human pharmacokinetics of monoclonal antibodies from nonclinical data: comparative evaluation of prediction approaches in early drug development. Biopharm Drug Dispos 37(2): 51-65.
  16. Loisel S, Ohresser M, Pallardy M, Daydé D, Berthou C, et al. (2007) Relevance, advantages and limitations of animal models used in the development of monoclonal antibodies for cancer treatment. Critical reviews in oncology/hematology 62(1): 34-42.
  17. Hérodin F, Thullier P, Garin D, Drouet M (2005) Nonhuman primates are relevant models for research in hematology, immunology and virology. Eur Cytokine Netw 16(2): 104-116.
  18. Martin PL, Weinbauer GF (2010) Developmental toxicity testing of biopharmaceuticals in nonhuman primates previous experience and future directions. Int J Toxicol 29(6): 552-568.
  19. Strohl WR, Strohl LM (2012) Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry. Woodland Publishing Ltd, Cambridge, UK.
  20. Reichert JM (2012) Marketed therapeutic antibodies compendium. MAbs 4(3): 413-415.
  21. Smith SL (1996) Ten years of Orthoclone OKT3 (muromonab-CD3): a review. J Transpl Coord 6(3): 109-119.
  22. Walsh G (1999) Biopharmaceuticals: Biochemistry and Biotechnology. New York: John Wiley & Sons.
  23. Pedersen JT, Henry AH, Searle SJ, Guild BC, Roguska M, et al. (1994) Comparison of surface accessible residues in human and murine immunoglobulin Fv domains. Implication for humanization of murine antibodies. J Mol Biol 235(3): 959-973.
  24. Roguska MA, Pedersen JT, Keddy CA, Henry AH, Searle SJ, et al. (1994) Humanization of murine monoclonal antibodies through variable domain resurfacing. Proc Natl Acad Sci USA 91(3): 969-973.
  25. Hamilton RG (2001) The Human IgG Subclasses. Calbiochem Corp.
  26. Rayner LE, Hui GK, Gor J, Heenan RK, Dalby PA, et al. (2015) The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands. J Biol Chem 290(13): 8420-8438.
  27. Dangl JL, Wensel TG, Morrison SL, Stryer L, Herzenberg LA, et al. (1988) Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies. EMBO J 7(7): 1989-1994.
  28. Roux KH, Strelets L, Michaelsen TE (1997) Flexibility of human IgG subclasses. J Immunol 159(7): 3372-3382.
  29. Steinitz M, Klein G, Koskimies S, Makel O (1977) EB virus-induced B lymphocyte cell lines producing specific antibody. Nature 269(5627): 420-422.
  30. Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, et al. (2004) An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus Nat Med 10(8): 871-875.
  31. Kwakkenbos MJ, Diehl SA, Yasuda E, Bakker AQ, van Geelen CM (2010) Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nat Med 16(1): 123-128.
  32. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705): 1315-1317.
  33. Kaplan G, Gershoni JM (2012) A general insert label for peptide display on chimeric filamentous bacteriophages. Anal Biochem 420(1): 68-72.
  34. Hoffmann S, Funke SA, Wiesehan K, Moedder S, Glück JM, et al. (2010) Competitively selected protein ligands pay their increase in specificity by a decrease in affinity. Mol Biosyst 6(1): 126-133.
  35. Zhang J, Li H, Wang X, Qi H, Miao X, et al. (2012) Phage-derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF. Biotechnol Prog 28(4): 981-989.
  36. Schüller C, Wiebe JC, Pegel A, Kramer K, Skerra A, et al. (2010) A system for repertoire cloning and phage display of murine and leporid antibody fragments. J AOAC Int 93(1): 66-79.
  37. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302(5909): 575-581.
  38. Babcook JS, Leslie KB, Olsen OA, Salmon RA, Schrader JW (1996) A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA 93(15): 7843-7848.
  39. Jin A, Ozawa T, Tajiri K, Obata T, Kondo S, et al. (2009) A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 15(9): 1088-1092.
  40. Love JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol 24(6): 703-707.
  41. Wrammert J, Smith K, Miller J, Langley WA, Kokko K (2008) Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453(7195): 667-671.
  42. Rawstron AC (2006) Immunophenotyping of plasma cells. Current Protocols in Cytometry 6-23.
  43. Kurosawa N, Yoshioka M, Isobe M (2011) Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells. BMC Biotechnol 11: 39-46.
  44. Coronella JA, Telleman P, Truong TD, Ylera F, Junghans RP (2000) Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells. Nucleic Acids Res 28(20): E85
  45. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, et al. (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329(1-2): 112-124.
  46. Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biol 8: 76.
  47. Smith K, Garman L, Wrammert J, Zheng NY, Capra JD, et al. (2009) Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4(3): 372-384.
  48. Yoshioka M, Kurosawa N, Isobe M (2011) Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells. BMC Biotechnol 11: 75.
  49. Ravetch JV, Siebenlist U, Korsmeyer S, Waldmann T, Leder P (1981) Structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell 27(3 Pt 2): 583-591.
  50. Hieter PA, Maizel JV, Leder P (1982) Evolution of human immunoglobulin kappa J region genes. J Biol Chem 257(3): 1516–1522.
  51. Schäble KF, Zachau HG (1993) The variable genes of the human immunoglobulin kappa locus. Biol Chem Hoppe Seyler 374(11): 1001-1022.
  52. Corbett SJ, Tomlinson IM, Sonnhammer EL, Buck D, Winter G (1994) Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, minor D segments or D-D recombination. J Mol Biol 270(4): 587-597.
  53. Kawasaki K, Minoshima S, Nakato E, Shibuya K, Shintani A, et al. (1997) One-megabase sequence analysis of the human immunoglobulin lambda gene locus. Genome Res 7(3): 250-261.
  54. Reichert JM, Valge-Archer VE (2007) Development trends for monoclonal antibody cancer therapeutics?Nat Rev Drug Discov 6(5): 349-356.
  55. Dimitrov DS, Marks JD (2009) Therapeutic antibodies: current state and future trends-is a paradigm change coming soon? Methods Mol Biol 525: 1-27.
  56. Reichert JM (2011) Antibody-based therapeutics to watch in 2011. MAbs 3(1): 76-99.
  57. Winter G, Milstein C (1991) Man-made antibodies. Nature 349(6307): 293-299.
  58. Lonbergm N (2008) Fully human antibodies from transgenic mouse and phage display platforms. Curr Opin Immunol 20(4): 450-459.
  59. Sharkey RM, Goldenberg DM (2006) Targeted therapy of cancer: new prospects for antibodies and immunoconjugate. CA. Cancer J Clin 56(4): 226-243.
  60. Hudson PJ, Souriau C (1993) Engineered antibodies. Nat Med 9(1): 129-134.
  61. Beck A, Haeuw JF, Wurch T, Goetsch L, Bailly C, et al. (2010) The next generation of antibody-drug conjugates comes of age. Discov Med 10(53): 329-339.
  62. Chames P, Baty D(2009) Bispecific antibodies for cancer therapy. Curr Opin Drug Discov Devel 12(2): 276-283.
  63. Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10(5): 317-327.
  64. Even-Desrumeaux K, Fourquet P, Secq V, Baty D, Chames P (2012) Single-domain antibodies: a versatile and rich source of binders for breast cancer diagnostic approaches. Mol Biosyst 8(9): 2385-2394.
  65. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5): 646-674.
  66. Pavlou AK, Belsey MJ (2005) BioPharma licensing and M&A trends. Nature Reviews Drug Discovery 4(4): 273-274.
  67. Adler MJ, Dimitrov DS (2012) Therapeutic antibodies against cancer. Hematol Oncol Clin North Am 26(3): 447-481.
  68. Chan AC, Carter PJ (2010) Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol 10(5): 301-316.
  69. Helmout M (2005) Monoclonal antibodies as therapeutic agents: advances and challenges. Iran J Immunol 2(1): 3-20.
© 2014-2016 MedCrave Group, All rights reserved. No part of this content may be reproduced or transmitted in any form or by any means as per the standard guidelines of fair use.
Creative Commons License Open Access by MedCrave Group is licensed under a Creative Commons Attribution 4.0 International License.
Based on a work at http://medcraveonline.com
Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version | Opera |Privacy Policy