Immunology

Monoclonal Antibodies – Definition, Types, Production, Side Effect, Applications

Antibodies produced by a single clone of cells (e.g., myeloma) are homogenous and referred to as monoclonal antibodies. In multiple myeloma, for...

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This article writter by MN Editors on October 31, 2022

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monoclonal antibody production
monoclonal antibody production
  • Antibodies produced by a single clone of cells (e.g., myeloma) are homogenous and referred to as monoclonal antibodies. In multiple myeloma, for instance, antibodies are produced by a single clone of plasma cells against a single antigenic determinant; these antibodies are hence monoclonal.
  • The monoclonal antibodies differ from polyclonal antibodies, which are heterologous and generated in response to antigen by many clones of plasma cells.
  • Kohler and Milstein (1975) were the first to disclose a method for the synthesis of monoclonal antibodies against a desired antigen; they were awarded the Nobel Prize in 1984 for their achievement.

Monoclonal Antibodies

  • Monoclonal Antibodies are cells generated from a single ancestral cell during cell division.
  • Monoclonals are a type of antibodies that are the identical offspring of a hybridoma, are highly specific to a particular body site, are created from a single clone, and may be grown forever.
  • Monoclonal antibodies identify and bind to antigens to distinguish between distinct epitopes, hence providing protection against disease-causing pathogens.
  • Monoclonal antibodies target receptors and other proteins located on the surface of normal and malignant cells that impact cell activity.
  • The specificity of monoclonal antibodies enables their binding to diseased cells by coupling a cytotoxic agent, such as a powerful radioactive substance, which then seeks to destroy cancer cells without hurting healthy cells.
  • When tumour cells that can reproduce indefinitely are merged with mammalian cells that manufacture a specific antibody, a fusion called hybridoma is formed that produces antibodies continually.
  • These antibodies are referred to be monoclonal because they originate from a single type of cell, the hybridoma cell.
  • Antibodies manufactured using traditional techniques and derived from preparations including several cell types are known as polyclonal Antibodies.
  • Antibodies against a certain antigen are synthesised artificially in order to bind to their target antigens.
  • Laboratory manufacture of monoclonal antibodies is derived from clones of a single cell, hence each antibody produced by the cell is identical.
  • Fusion of myeloma cells in cell culture with antibodies from mammalian spleen cells yields hybrid cells/hybridomas that produce huge quantities of monoclonal antibody.
  • The cell fusion produced two distinct cell types, one with the capacity for continuous growth and the other with the capacity to manufacture large quantities of purified antibody.
  • Hybrid cells create a single antibody that is purer than polyclonal antibodies produced by standard methods.
  • Monoclonal Antibodies are significantly more effective than conventional drugs due to the fact that conventional drugs target both the foreign substance and the body’s own cells, causing severe side effects, whereas monoclonal antibodies target only the foreign antigen/target molecule, with no or minimal side effects.
  • A high concentration of a specific monoclonal antibody in the blood indicates the presence of an aberrant protein.
  • Typically, this protein can be found via a screening blood test called “protein electrophoresis” and can be recognised during a physical examination.
  • A small population of plasma cells in the bone marrow is the source of aberrant monoclonal antibody production.

Types of monoclonal antibodies

Types of monoclonal antibodies based on composition

Monoclonal antibodies are synthetic proteins that function similarly to human antibodies within the immune system. There are four various techniques to make them, and their names reflect the materials used.

Types of monoclonal antibodies
Types of monoclonal antibodies

1. Murine monoclonal antibodies

  • These are manufactured from mouse proteins, and their names end in -omab.
  • Murine monoclonal antibodies derive their name from their origin in rodent hosts, typically mice and rats from the family Muridae.
  • In the past, murine antibodies of the immunoglobulin G (IgG) class have played a significant role in the development of contemporary antibody production procedures and have helped us understand the therapeutic and analytical potential of these immunoglobulins.
  • Frequently, murine antibodies are modified to minimise their immunogenicity towards human patients.
  • The advent of molecular biology tools that permit the sequencing and cloning of antibody sequences and the subsequent transfection of various mammalian expression systems spurred the initial efforts.
  • This adaptability provided researchers with more control over antibody sequences and, consequently, the immunogenicity burden imposed by native mouse monoclonal antibodies.
  • Initially, genetic modification of antibodies produced chimeric antibody structures with mouse Fab and human Fc domains. This combination of areas of various origin increased the compatibility of these antibodies produced by mice with human immune cells.
  • Nonetheless, more significant results have been achieved with humanization procedures, which have permitted the targeted modification of the mouse Fab region to increase its homology with native human antibodies.
  • Recent developments have also enabled the manufacture of antibody fragments using less complex and less expensive expression methods, such as bacteria (such as Escherichia coli) and yeast (e.g. Saccharomyces cerevisiae).
  • These antibody fragments can be combined in many different ways to form bispecific antibodies (BiTE, diabody, DART, among others) and, because of the lack of the Fc region, they pose a reduced risk of challenging the patient’s immune system and eliciting an immune response.

2. Chimeric monoclonal antibodies

  • These proteins are a mixture of mouse and human DNA, and the therapy names end with -ximab.
  • Chimeric antibodies are structural chimaeras created by fusing the variable portions of one species, such as a mouse, with the constant regions of another, such as a human.
  • Chimerization of antibodies is required to minimise immunogenicity and increase serum half-life while manufacturing therapeutic monoclonal antibodies (mAbs).
  • Chimeric antibodies can be produced through very simple genetic engineering by fusing the immunoglobulin (Ig) variable portions of a selected mouse hybridoma with human Ig constant regions. They can be used as is or as a starting point for further humanization.
  • First, the spleen containing B cells is extracted from an immunised mouse. To create hybridoma and isolate a clone producing antigen-specific IgG, B cells are united with myeloma cells.
  • The DNA sequences encoding mouse VH and VL are extracted from the clone, along with the DNA sequences encoding human immunoglobulin constant sections from human cells.
  • Using genetic engineering, chimeric mouse/human genes are generated and transfected into mammalian cells.
  • Finally, the clone with the highest degree of chimeric IgG expression is chosen, and the IgGs are separated from the culture supernatant.
  • The use of chimeric monoclonal antibodies in medicines and immunoassays is crucial and potent. Switching the antibody constant regions to match the species of the host or secondary antibody could dramatically reduce background staining in in vitro applications such as immunohistochemistry investigations and ELISA assay development.
Chimeric monoclonal antibodies
Chimeric monoclonal antibodies

3. Humanized monoclonal antibodies

  • These are composed of small portions of mouse proteins fused to human proteins, and their names end in -zumab.
  • Humanized antibodies are non-human antibodies whose protein sequences have been changed to increase their closeness to antibody types naturally produced by people.
  • The term “humanization” is typically given to monoclonal antibodies intended for human administration (for example, antibodies developed as anti-cancer drugs).
  • Humanization may be required when the process of producing a specific antibody involves non-human immune system generation (such as that in mice).
  • The protein sequences of these antibodies are somewhat dissimilar from those of naturally occurring homologous antibodies in humans, making them potentially immunogenic when delivered to human patients (see also Human anti-mouse antibody).
  • Humanized antibodies’ International Nonproprietary Names finish in -zumab, as in omalizumab (see Nomenclature of monoclonal antibodies).
  • Humanized antibodies differ from hybridised antibodies. The protein sequences of the latter are likewise modified to be more comparable to those of human antibodies, but they contain a longer length of non-human protein.
  • The humanization method takes advantage of the fact that monoclonal antibody manufacturing may be carried out using recombinant DNA to generate constructs that are capable of expression in mammalian cell culture.
  • Thus, gene segments capable of making antibodies are identified and cloned into cells that can be cultured in a bioreactor, allowing for the mass harvesting of antibody proteins made from the DNA of the cloned genes.
  • The process involving recombinant DNA provides an intervention point that can be conveniently used to modify the antibody’s protein sequence.
  • Therefore, any modifications to antibody structure that occur during the humanization process are carried out using DNA-level approaches.
  • Not all methods for producing antibodies designed for human therapy require a humanization stage (e.g., phage display), but virtually all rely on processes that similarly permit the “insertion” or “swapping-out” of antibody molecule segments.

4. Human monoclonal antibodies

  • These are entirely human proteins whose amino acid sequences have been altered using molecular biology techniques.
  • This modifies the specificity, affinity, or biological roles, resulting in the acquisition of sequences not found in the human repertoire.
  • The therapeutic names for human mAbs end in -umab.

Types of monoclonal antibodies based on functions

1. Naked monoclonal antibodies

  • Naked mAbs are antibodies that have neither a medication nor a radioactive substance attached. They work independently.
  • This is the most prevalent class of mAbs used to treat cancer.
  • The majority of naked mAbs bind to antigens on cancer cells, but some also bind to antigens on non-cancerous cells or even free-floating proteins.
  • Unconjugated mAbs can function in various ways.
    • Some enhance an individual’s immune response to cancer cells by adhering to them and serving as a flag for the immune system to kill them. Alemtuzumab (Campath®), for instance, is used to treat certain patients with chronic lymphocytic leukaemia (CLL). Alemtuzumab binds to the CD52 antigen, which is present on lymphocyte cells (which include the leukaemia cells). Once bound, the antibody recruits immune cells for their destruction.
    • Some naked mAbs enhance the immune response by targeting checkpoints in the immune system. (See Immune Checkpoint Blockers and Their Adverse Effects.)
    • Other bare mAbs mostly function by binding to and inhibiting antigens on cancer cells (or other adjacent cells) that aid in the growth or spread of cancer cells. Trastuzumab (Herceptin) is an example of an antibody targeting the HER2 protein. Occasionally, this protein is abundant on the surface of breast and stomach cancer cells. When HER2 is active, it promotes the growth of these cells. Trastuzumab binds to these proteins and prevents their activation.

2. Conjugated monoclonal antibodies

  • Combining a chemotherapeutic medication or a radioactive particle with a conjugated mAb.
  • These mAbs serve as homing devices that transport one of these chemicals straight to cancer cells.
  • The mAb circulates throughout the entire body until it locates and binds to the target antigen. The material is subsequently transported to the location where it is required most.
  • This reduces the damage to healthy cells in other organs.
  • Occasionally, conjugated mAbs are referred to as tagged, labelled, or loaded antibodies.

a. Radiolabeled antibodies

  • Attached to radiolabeled antibodies are tiny radioactive particles. Ibritumomab tiuxetan (Zevalin) is a radiolabeled mAb example.
  • This is an antibody against the antigen CD20, which is present on B lymphocytes.
  • Radioactivity is delivered directly to cancer cells by the antibody. It is composed of a monoclonal antibody (rituximab) and a radioactive material (Yttrium-90).
  • This sort of antibody treatment is sometimes referred to as radioimmunotherapy (RIT).
  • Because the mAb searches for the target, the medication and radiation are delivered directly to the target cells, then the radiation impacts the target and adjacent cells to a certain amount.

b. Chemolabeled antibodies

  • Attached to these mAbs are potent chemotherapeutic (or other) medicines.
  • Examples include:
    • Attached to the chemotherapy medication MMAE is the antibody Brentuximab vedotin (Adcetris), which targets the CD30 antigen (present on cells).
    • Attached to the antibody Ado-trastuzumab emtansine (Kadcyla, commonly known as TDM-1) that targets the HER2 protein is the chemotherapy medication DM1.

3. Bispecific monoclonal antibodies

  • These medications are composed of portions of two distinct mAbs, thus they can simultaneously bind to two distinct proteins.
  • For instance, blinatumomab (Blincyto) is used to treat some forms of leukaemia.
  • A portion of blinatumomab binds to the CD19 protein present on some leukaemia and lymphoma cells.
  • Another component binds to CD3, a protein found on T cells, which are immunological cells.
  • By attaching to both of these proteins, this medication is believed to cause the immune system to attack cancer cells by bringing cancer cells and immune cells together.

Method of production of monoclonal antibodies

By fusing myeloma cells with antibody-producing cells, hybridomas are generated that manufacture monoclonal antibodies. These hybridomas generate nearly limitless numbers of useful antibodies for research and diagnostics. In this process, mouse splenic lymphocytes and mouse myeloma cells are united to create hybrid cells or hybridomas. Myeloma cells supply hybrid cells with immortality, while splenic plasma cells are responsible for antibody production. These hybridomas can manufacture monoclonal antibodies forever when maintained in culture. The hybridoma cells are created in the subsequent ways:

Method of production of monoclonal antibodies
Method of production of monoclonal antibodies
  1. First, an animal (such as a mouse) is inoculated with the target antigen.
  2. Lymphocytes from the spleen are then merged with mouse myeloma cells lacking the enzyme hypoxanthine phosphoribosyl transferase and cultured in vitro (HPRT).
  3. The inclusion of some compounds, such as polyethylene glycol, facilitates cell fusion. The fused cells are cultivated in a culture medium (HAT media) that supports the growth of the hybrid cells but not the proliferation of the parent cells.
  4. Finally, the resultant clones of cells are screened to determine whether or not they produce an antibody to the target antigen.
  5. These clones are then chosen for cultivation in perpetuity. The hybridomas may be maintained continuously and will manufacture monoclonal antibodies indefinitely.

Human monoclonal antibodies, such as chimeric antibodies, have been generated with modification of the original process for therapeutic application, since mouse monoclonal antibodies are not acceptable. The chimeric antibodies consisting of human constant regions and mouse variable regions are being prepared for use in treatment of leukaemia. Chimeric antibodies are also utilised to destroy tumour cells either by delivering poisons, such as diphtheria to tumour cells, or by killing tumour cells through complement-mediated cytotoxicity.

A typical monoclonal antibody production Steps

  1. Immunization of mice and isolation of splenocytes – Mice are inoculated with an antigen, and their blood is afterwards tested for antibody production. The splenocytes that produce antibodies are then extracted for in vitro hybridoma formation.
  2. Preparation of myeloma cells – Myeloma cells are immortalised cells that, when joined with spleen cells, can result in a hybridoma with limitless growth. The cells of myeloma are prepared for fusion.
  3. Fusion – Myeloma cells and isolated splenocytes are fused together to create hybridomas in the presence of polyehtylene glycol (PEG), which triggers the fusion of cell membranes.
  4. Clone screening and picking – Antigen specificity and immunoglobulin class are considered during the screening and selection of clones.
  5. Functional characterization – Confirm, confirm, and describe (by ELISA, for instance) each potentially high-producing colony.
  6. Scale up and wean – Scale up the clones that produce the appropriate antibodies and wean them off the selection agent (s).
  7. Expansion – Increase the number of clones yielding the necessary antibodies (e.g. bioreactors or large flasks).
monoclonal antibody production
monoclonal antibody production

Recombinant Monoclonal Antibody Production

Recombinant Monoclonal Antibody Production
Recombinant Monoclonal Antibody Production

How do monoclonal antibody drugs work?

Different functions are designed into monoclonal antibodies. A medication may truly function through multiple mechanisms. Examples include:

  • Flagging cancer cells: Some immune system cells rely on antibodies to identify an attack’s target. Monoclonal antibody-coated cancer cells may be easier to identify and eliminate.
  • Triggering cell-membrane destruction: Some monoclonal antibodies can stimulate an immune system response that can damage a cancer cell’s outer membrane.
  • Blocking cell growth: Certain monoclonal antibodies obstruct the connection between a cancer cell and proteins that stimulate cell development – an essential action for the growth and survival of cancer.
  • Preventing blood vessel growth: A blood supply is required for a malignant tumour to grow and survive. Some monoclonal antibody medicines prevent the formation of new blood vessels by inhibiting protein-cell interactions.
  • Blocking immune system inhibitors: The production of proteins that regulate the activity of immune system cells prevents the immune system from becoming overactive. Monoclonal antibodies can inhibit this mechanism, allowing your immune system cells to attack cancer cells without interference.
  • Directly attacking cancer cells: Certain monoclonal antibodies may directly target the cell. When some of these antibodies bind to a cell, a chain of events within the cell may trigger its self-destruction.
  • Delivering radiation treatment: Due of its propensity to bind to cancer cells, a monoclonal antibody can be developed as a delivery vehicle for other therapies. When a monoclonal antibody is coupled with a small radioactive particle, it transports the radiation treatment directly to cancer cells and may reduce radiation’s side effects on healthy cells.
  • Delivering chemotherapy: In a similar fashion, some monoclonal antibodies are coupled with a chemotherapeutic medication to deliver the treatment directly to cancer cells while avoiding healthy cells.
  • Binding cancer and immune cells: Some medications combine two monoclonal antibodies, one of which adheres to a cancer cell and the other to a particular immune system cell. This link may stimulate the immune system to fight cancer cells.
How do monoclonal antibody drugs work?
How do monoclonal antibody drugs work?

Side Effects of monoclonal antibody drugs

Common side effects

  • In general, the most frequent adverse effects of monoclonal antibody medicines are allergic reactions, such as hives and itching.
  • Signs and symptoms of the flu, including chills, tiredness, fever, and muscular aches.
  • Nausea, vomiting.
  • Diarrhea.
  • Skin rashes.
  • Reduced blood pressure

Serious side effects

Serious yet uncommon adverse effects of monoclonal antibody treatment include:

  • Infusion reactions: Extreme allergic reactions are possible and only rarely result in death. Before beginning monoclonal antibody therapy, you may be given medication to prevent an allergic reaction. Infusion responses typically occur during or shortly after treatment administration, so your healthcare team will constantly monitor you. You may be required to spend a few hours in the treatment centre for monitoring.
  • Heart problems: Certain monoclonal antibodies augment the danger of hypertension, congestive heart failure, and heart attacks.
  • Lung problems: Some monoclonal antibodies are connected with an increased likelihood of inflammatory lung disease.
  • Skin problems: In some instances, skin sores and rashes can lead to dangerous infections. Additionally, serious sores can emerge on the tissue lining your cheeks and gums (mucosa).
  • Bleeding: Some monoclonal antibody drugs are associated with an increased risk of internal bleeding.

Applications of Monoclonal antibodies

  • Diagnostic tests: Once monoclonal antibodies for a particular substance have been produced, they can be used to detect its presence. The Western blot and immuno dot blot tests can be used to detect proteins. Monoclonal antibodies can be used in immunohistochemistry to detect antigens in fixed tissue sections, and immunofluorescence can detect a substance in either frozen tissue sections or living cells.
  • Analytic and chemical uses: Using immunoprecipitation, antibodies can also be used to purify their target compounds from mixtures.
  • Cancer treatment: Monoclonal antibodies that bind only to cancer-cell-specific antigens and induce an immune response against the target cancer cell are one potential treatment for cancer. These mAbs can be modified for the delivery of a toxin, radioisotope, cytokine, or other active conjugate, or for the design of bispecific antibodies that can bind with their Fab regions to both the target antigen and a conjugate or effector cell. The Fc region of every intact antibody can bind to cell receptors or other proteins. Example: Alemtuzumab, Bevacizumab, Cetuximab, Dostarlimab, etc.
  • Autoimmune diseases: Infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn’s disease, ulcerative colitis, and ankylosing spondylitis due to their ability to bind to and inhibit TNF-, are monoclonal antibodies used to treat autoimmune diseases. Basiliximab and daclizumab prevent acute rejection of kidney transplants by inhibiting IL-2 on activated T cells. Omalizumab inhibits human immunoglobulin E (IgE) and is effective in the treatment of moderate to severe allergic asthma. Example: infliximab, adalimumabm, ustekinumab, basiliximab, etc.
  • COVID-19: Monoclonal antibodies are also used for the treatment of covid-19.
Monoclonal Antibodies for Immunotherapy
Monoclonal Antibodies for Immunotherapy

References

  • Bayat AA, Yeganeh O, Ghods R, Zarnani AH, Ardekani RB, Mahmoudi AR, Mahmoudian J, Haghighat-Noutash F, Jeddi-Tehrani M. Production and characterization of a murine monoclonal antibody against human ferritin. Avicenna J Med Biotechnol. 2013 Oct;5(4):212-9. PMID: 24285995; PMCID: PMC3838765.
  • Casadevall A, Cleare W, Feldmesser M, Glatman-Freedman A, Goldman DL, Kozel TR, Lendvai N, Mukherjee J, Pirofski LA, Rivera J, Rosas AL, Scharff MD, Valadon P, Westin K, Zhong Z. Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies. Antimicrob Agents Chemother. 1998 Jun;42(6):1437-46. doi: 10.1128/AAC.42.6.1437. PMID: 9624491; PMCID: PMC105619.
  • Holzlöhner P, Hanack K. Generation of Murine Monoclonal Antibodies by Hybridoma Technology. J Vis Exp. 2017 Jan 2;(119):54832. doi: 10.3791/54832. PMID: 28117810; PMCID: PMC5407676.
  • Yokoyama, W. M., Christensen, M., Santos, G. D., Miller, D., Ho, J., Wu, T., … Neethling, F. A. (2013). Production of Monoclonal Antibodies. Current Protocols in Immunology, 102(1), 2.5.1–2.5.29. doi:10.1002/0471142735.im0205s102
  • Mallbris L, Davies J, Glasebrook A, Tang Y, Glaesner W, Nickoloff BJ. Molecular Insights into Fully Human and Humanized Monoclonal Antibodies: What are the Differences and Should Dermatologists Care? J Clin Aesthet Dermatol. 2016 Jul;9(7):13-5. Epub 2016 Jul 1. PMID: 27672407; PMCID: PMC5022998.
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