Monoclonal antibody

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A general representation of the method used to produce monoclonal antibodies[1][2]

A monoclonal antibody (mAb, more rarely called moAb) 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.

Bispecific monoclonal antibodies
can also be engineered, by increasing the therapeutic targets of one monoclonal antibody to two epitopes.

It is possible to produce monoclonal antibodies that specifically bind to almost any suitable substance; they can then serve to detect or purify it. This capability has become an investigative tool in biochemistry, molecular biology, and medicine. Monoclonal antibodies are used in the diagnosis of illnesses such as cancer and infections[3] and are used therapeutically in the treatment of e.g. cancer and inflammatory diseases.

History

In the early 1900s,

Nobel Prize for Physiology or Medicine
for providing the theoretical basis for immunology.

By the 1970s,

hybridomas.[6] In 1975, Georges Köhler and César Milstein succeeded in making fusions of myeloma cell lines with B cells to create hybridomas that could produce antibodies, specific to known antigens and that were immortalized.[7] They and Niels Kaj Jerne shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery.[7]

In 1988, Gregory Winter and his team pioneered the techniques to humanize monoclonal antibodies,[8] eliminating the reactions that many monoclonal antibodies caused in some patients. By the 1990s research was making progress in using monoclonal antibodies therapeutically, and in 2018, James P. Allison and Tasuku Honjo received the Nobel Prize in Physiology or Medicine for their discovery of cancer therapy by inhibition of negative immune regulation, using monoclonal antibodies that prevent inhibitory linkages.[9]

The translational work needed to implement these ideas is credited to Lee Nadler. As explained in an NIH article, "He was the first to discover monoclonal antibodies directed against human B-cell–specific antigens and, in fact, all the known human B-cell–specific antigens were discovered in his laboratory. He is a true translational investigator, since he used these monoclonal antibodies to classify human B-cell leukemia and lymphomas as well as to create therapeutic agents for patients. . . More importantly, he was the first in the world to administer a monoclonal antibody to a human (a patient with B-cell lymphoma)."[10]

Production

Looking at slides of cultures of cells that make monoclonal antibodies
Monoclonal antibodies can be grown in unlimited quantities in flasks.
hybridoma
).
Bathing prepared slides in a solution

Hybridoma development

Much of the work behind production of monoclonal antibodies is rooted in the production of hybridomas, which involves identifying antigen-specific plasma/plasmablast cells that produce antibodies specific to an antigen of interest and

nucleic acids
, thus requiring supplementation to survive.

The selective culture medium is called

hybridoma
) cells. Unfused myeloma cells cannot grow because they lack HGPRT and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells referred to as hybridomas, are able to grow indefinitely in the medium because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or antigen microarray assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid.

The medium must be enriched during in vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-media conditioned by macrophages can be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, ascites is considered unethical.[14]

Novel mAb development technology

Several monoclonal antibody technologies have been developed recently,[15] such as phage display,[16] single B cell culture,[17] single cell amplification from various B cell populations[18][19][20][21][22] and single plasma cell interrogation technologies. Different from traditional hybridoma technology, the newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by PCR and produce in either bacterial or mammalian systems with recombinant technology. One of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals in the laboratory.

Purification

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. Cell culture sample contaminants consist primarily of media components such as growth factors, hormones and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture and thus the contaminants, one or the other method (in vivo or in vitro) may be preferable.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by

dialysis
.

Most of the charged impurities are usually

anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated along with the anions based on their isoelectric point (pI). In proteins, the isoelectric point (pI) is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge, and when the pH < pI, a protein has a net positive charge. For example, albumin
has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. Thus, at a pH between 4.8 and 6.1, the average charge of albumin molecules is likely to be more negative, while mAbs molecules are positively charged and hence it is possible to separate them. Transferrin, on the other hand, has a pI of 5.9, so it cannot be easily separated by this method. A difference in pI of at least 1 is necessary for a good separation.

Transferrin can instead be removed by

size exclusion chromatography. This method is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution
times.

A much quicker, single-step method of separation is protein A/G affinity chromatography. The antibody selectively binds to protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to support purification. The antibody-containing medium is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

Antibody heterogeneity

Product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing.[citation needed]

These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions.[24] These seemingly minute structural changes can affect preclinical stability and process optimization as well as therapeutic product potency, bioavailability and immunogenicity. The generally accepted purification method of process streams for monoclonal antibodies includes capture of the product target with protein A, elution, acidification to inactivate potential mammalian viruses, followed by ion chromatography, first with anion beads and then with cation beads.[citation needed]

Quality by Design initiative attempts to provide guidance on development and to facilitate design of products and processes that maximizes efficacy and safety profile while enhancing product manufacturability.[27]

Recombinant

The production of

CRISPR/Cas9, or phage display/yeast display technologies.[28] Recombinant antibody engineering involves antibody production by the use of viruses or yeast, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.[29] The phage antibody libraries are a variant of phage antigen libraries.[30] These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy and their detectability in diagnostic applications.[31]
Fermentation chambers have been used for large scale antibody production.

Chimeric antibodies

While mouse and human antibodies are structurally similar, the differences between them were sufficient to invoke an immune response when

murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production of human anti-mouse antibodies
(HAMA).

Recombinant DNA has been explored since the late 1980s to increase residence times. In one approach called "CDR grafting",[32] mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this "chimeric" or "humanised" DNA through cell culture yielded part-mouse, part-human antibodies.[33][34]

Human antibodies

Approaches have been developed to isolate human monoclonal antibodies.[15]

Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of fully human products to reduce the side effects of humanised or chimeric antibodies. Several successful approaches have been proposed: transgenic mice,[35] phage display[16] and single B cell cloning.[15]

Cost

Monoclonal antibodies are more expensive to manufacture than small molecules due to the complex processes involved and the general size of the molecules, all in addition to the enormous research and development costs involved in bringing a new chemical entity to patients. They are priced to enable manufacturers to recoup the typically large investment costs, and where there are no price controls, such as the United States, prices can be higher if they provide great value. Seven University of Pittsburgh researchers concluded, "The annual price of mAb therapies is about $100,000 higher in oncology and hematology than in other disease states", comparing them on a per patient basis, to those for cardiovascular or metabolic disorders, immunology, infectious diseases, allergy, and ophthalmology.[36]

Applications

Diagnostic tests

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. Proteins can be detected using the Western blot and immuno dot blot tests. In immunohistochemistry, monoclonal antibodies can be used to detect antigens in fixed tissue sections, and similarly, immunofluorescence can be used to detect a substance in either frozen tissue section or live cells.

Analytic and chemical uses

Antibodies can also be used to purify their target compounds from mixtures, using the method of immunoprecipitation.

Therapeutic uses

Therapeutic monoclonal antibodies act through multiple mechanisms, such as blocking of targeted molecule functions, inducing apoptosis in cells which express the target, or by modulating signalling pathways.[37][38][39]

Cancer treatment

One possible treatment for

Fab regions both to target antigen and to a conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with its Fc region
.

ADCC: antibody dependent cell-mediated cytotoxicity; CDC: complement-dependent cytotoxicity; MAb: monoclonal antibody; scFv, single-chain Fv fragment.[40]

MAbs approved by the FDA for cancer include:[41]

Autoimmune diseases

Monoclonal antibodies used for

rejection of kidney transplants.[42] Omalizumab inhibits human immunoglobulin E (IgE) and is useful in treating moderate-to-severe allergic asthma
.

Examples of therapeutic monoclonal antibodies

Monoclonal antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine like CiteAb. Below are examples of clinically important monoclonal antibodies.

Main category Type Application Mechanism/Target Mode
Anti-
inflammatory
infliximab[42] inhibits
TNF-α
chimeric
adalimumab inhibits
TNF-α
human
ustekinumab blocks interleukin IL-12 and IL-23 human
basiliximab[42]
  • acute
    rejection
    of kidney transplants
inhibits
T cells
chimeric
daclizumab[42]
  • acute
    rejection
    of kidney transplants
inhibits
T cells
humanized
omalizumab
  • moderate-to-severe allergic asthma
  • chronic ideopathic
    urticaria
inhibits human immunoglobulin E (IgE) humanized
Anti-cancer
gemtuzumab[42]
targets myeloid cell surface antigen CD33 on leukemia cells humanized
alemtuzumab[42]
  • B cell leukemia
targets an antigen
B-lymphocytes
humanized
rituximab[42]
  • non-Hodgkin's lymphoma
  • rheumatoid arthritis
targets phosphoprotein
B lymphocytes
chimeric
trastuzumab
  • breast cancer with
    HER2/neu
    overexpression
targets the
HER2/neu
(erbB2) receptor
humanized
nimotuzumab
  • approved in
    squamous cell carcinomas
    , Glioma
  • clinical trials for other indications underway
EGFR inhibitor humanized
cetuximab
  • approved in
    colorectal carcinoma
EGFR inhibitor chimeric
panitumumab
  • approved in
    colorectal carcinoma
EGFR inhibitor human
bevacizumab & ranibizumab
  • Anti-angiogenic
    cancer therapy
inhibits VEGF humanized
Anti-cancer and anti-viral bavituximab[43] immunotherapy, targets phosphatidylserine[43] chimeric
Anti-viral

casirivimab/imdevimab[44]

immunotherapy, targets spike protein of SARS-CoV-2 human
bamlanivimab/etesevimab[45] immunotherapy, targets spike protein of SARS-CoV-2 human
Sotrovimab[46] immunotherapy, targets spike protein of SARS-CoV-2 human
Other palivizumab[42]
  • RSV infections in children
inhibits an RSV fusion (F) protein humanized
abciximab[42]
  • prevent
    coronary angioplasty
inhibits the receptor
platelets
chimeric

COVID-19

In 2020, the monoclonal antibody therapies

emergency use authorizations by the US Food and Drug Administration to reduce the number of hospitalizations, emergency room visits, and deaths.[44][45] In September 2021, the Biden administration purchased US$2.9 billion worth of Regeneron monoclonal antibodies at $2,100 per dose to curb the shortage.[47]

As of December 2021, in vitro neutralization tests indicate monoclonal antibody therapies (with the exception of sotrovimab and tixagevimab/cilgavimab) were not likely to be active against the Omicron variant.[48]

Over 2021–22, two Cochrane reviews found insufficient evidence for using neutralizing monoclonal antibodies to treat COVID-19 infections.[49][50] The reviews applied only to people who were unvaccinated against COVID‐19, and only to the COVID-19 variants existing during the studies, not to newer variants, such as Omicron.[50]

Side effects

Several monoclonal antibodies, such as bevacizumab and cetuximab, can cause different kinds of side effects.[51] These side effects can be categorized into common and serious side effects.[52]

Some common side effects include:

  • Dizziness
  • Headaches
  • Allergies
  • Diarrhea
  • Cough
  • Fever
  • Itching
  • Back pain
  • General weakness
  • Loss of appetite
  • Insomnia
  • Constipation[53]

Among the possible serious side effects are:[53]

See also

  • List of monoclonal antibodies

References

  1. ^ Gelboin HV. "Cytochrome P450 Mediated Drug and Carcinogen Metabolism using Monoclonal Antibodies". home.ccr.cancer.gov. Archived from the original on 15 October 2004. Retrieved 2 April 2018.
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  40. ^ Takimoto CH, Calvo E. (1 January 2005) "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management Archived 4 October 2013 at the Wayback Machine
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  42. ^ a b "Bavituximab - Avid Bioservices". AdisInsight. Springer Nature Switzerland AG.
  43. ^ a b "Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19". U.S. Food and Drug Administration (FDA) (Press release). 21 November 2020. Retrieved 21 November 2020. Public Domain This article incorporates text from this source, which is in the public domain.
  44. ^ a b "FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19". U.S. Food and Drug Administration (FDA) (Press release). 9 February 2021. Retrieved 10 February 2021. Public Domain This article incorporates text from this source, which is in the public domain.
  45. ^ "Emergency Use Authorization letter" (PDF). U.S. Food and Drug Administration (FDA). 16 December 2021. Retrieved 6 January 2022. Public Domain This article incorporates text from this source, which is in the public domain.
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  50. ^ "Monoclonal antibodies to treat cancer". American Cancer Society. Retrieved 19 April 2018.
  51. ^ "Monoclonal antibody drugs for cancer: How they work". Mayo Clinic. Retrieved 19 April 2018.
  52. ^ a b Ogbru O (12 October 2022). Davis CP (ed.). "Monoclonal Antibodies: List, Types, Side Effects & FDA Uses (Cancer)". MedicineNet. Retrieved 19 April 2018.

Further reading

External links