Amonoclonal antibody (mAb, more rarely calledmoAb) is anantibody produced from acell lineage made bycloning a uniquewhite blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies are identical and can thus havemonovalent affinity, binding only to a particularepitope (the part of anantigen that is recognized by the antibody).[3] In contrast,polyclonal antibodies are mixtures of antibodies derived from multipleplasma cell lineages which each bind to their particular target epitope. Artificial antibodies known asbispecific monoclonal antibodies can also be engineered which include two different antigen binding sites (FABs) on the same antibody.
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 inbiochemistry,molecular biology, andmedicine. Monoclonal antibodies are used in the diagnosis of illnesses such ascancer and infections[4] and areused therapeutically in the treatment of e.g. cancer andinflammatory diseases.
In the early 1900s,immunologistPaul Ehrlich proposed the idea of aZauberkugel – "magic bullet", conceived of as a compound which selectively targeted a disease-causing organism, and could deliver a toxin for that organism. This underpinned the concept of monoclonal antibodies and monoclonal drug conjugates. Ehrlich andÉlie Metchnikoff received the 1908Nobel Prize for Physiology or Medicine for providing the theoretical basis for immunology.[citation needed]
By the 1970s,lymphocytes producing a single antibody were known, in the form ofmultiple myeloma – a cancer affectingB-cells. These abnormal antibodies orparaproteins were used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a givenantigen.[5]: 324 In 1973,Jerrold Schwaber described the production of monoclonal antibodies using human–mouse hybrid cells.[6] This work remains widely cited among those using human-derivedhybridomas.[7] In 1975,Georges Köhler andCésar 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.[8] They andNiels Kaj Jerne shared theNobel Prize in Physiology or Medicine in 1984 for the discovery.[8]
In 1988,Gregory Winter and his team pioneered the techniques tohumanize monoclonal antibodies,[9] 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 andTasuku 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.[10]
The translational work needed to implement these ideas is credited toLee 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)."[11]
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 andfusing these cells withmyeloma cells.[8] Rabbit B-cells can be used to form arabbit hybridoma.[12][13]Polyethylene glycol is used to fuse adjacent plasma membranes,[14] but the success rate is low, so a selective medium in which only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesizehypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for thesalvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless thede novo purine synthesis pathway is also disrupted. Exposing cells toaminopterin (afolic acid analogue which inhibitsdihydrofolate reductase) makes them unable to use the de novo pathway and become fullyauxotrophic fornucleic acids, thus requiring supplementation to survive.[citation needed]
The selective culture medium is calledHAT medium because it containshypoxanthine, aminopterin andthymidine. This medium is selective for fused (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).[citation needed]
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 asELISA or antigen microarray assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.[citation needed]
The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in theperitoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid calledascites fluid.[citation needed]
The medium must be enriched duringin 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 consideredunethical.[15]
Several monoclonal antibody technologies have been developed recently,[16] such asphage display,[17] single B cell culture,[18] single cell amplification from various B cell populations[19][20][21][22][23] 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 withrecombinant 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.[citation needed]
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 andtransferrins. In contrast, thein vivo sample is likely to have host antibodies,proteases,nucleases, nucleic acids andviruses. In both cases, other secretions by the hybridomas such ascytokines 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 orin vitro) may be preferable.[citation needed]
The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed byfiltration with a 0.45 μm filter. These large particles can cause a phenomenon calledmembrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is produced by a low-secreting cell line. The sample is therefore concentrated byultrafiltration ordialysis.[citation needed]
Most of the charged impurities are usuallyanions such as nucleic acids and endotoxins. These can be separated byion exchange chromatography.[24] Eithercation exchangechromatography is used at a low enoughpH that the desired antibody binds to the column while anions flow through, oranion 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 theirisoelectric 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.[citation needed]
Transferrin can instead be removed bysize 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 lowelution times.[citation needed]
A much quicker, single-step method of separation isprotein A/Gaffinity chromatography. The antibody selectively binds to protein A/G, so a high level of purity (generally >80%) is obtained. The generally harsh conditions of this method may damage easily damaged antibodies. 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 anagarose support. If the antigen is apeptide, it is commonly synthesized with a terminalcysteine, which allows selective attachment to a carrier protein, such asKLH 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.[citation needed]
Product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing.[25][26][27]
These variants are typically aggregates,deamidation products,glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions.[28] These seemingly minute structural changes can affect preclinical stability and process optimization as well as therapeutic product potency,bioavailability andimmunogenicity. The generally accepted purification method of process streams for monoclonal antibodies includes capture of the product target withprotein A, elution, acidification to inactivate potential mammalian viruses, followed byion chromatography, first withanion beads and then with cation beads.[citation needed]
Displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animalpharmacokinetic studies.[29][30] Knowledge gained during the preclinical development phase is critical for enhanced product quality understanding and provides a basis for risk management and increased regulatory flexibility. The recent Food and Drug Administration'sQuality 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.[31]
The production ofrecombinant monoclonal antibodies involves repertoirecloning,CRISPR/Cas9, orphage display/yeast display technologies.[32] Recombinant antibody engineering involves antibody production by the use ofviruses oryeast, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly differentamino acid sequences from which antibodies with desired specificities can be selected.[33] The phage antibody libraries are a variant of phage antigen libraries.[34] 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.[35] Fermentation chambers have been used for large scale antibody production.[citation needed]
While mouse and human antibodies are structurally similar, the differences between them were sufficient to invoke an immune response whenmurine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production ofhuman anti-mouse antibodies (HAMA).
Recombinant DNA has been explored since the late 1980s to increase residence times. In one approach called "CDR grafting",[36] 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 throughcell culture yielded part-mouse, part-human antibodies.[37][38]
Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation offully human products to reduce the side effects of humanised or chimeric antibodies. Several successful approaches have been proposed:transgenic mice,[39]phage display[17] and single B cell cloning.[16]
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. SevenUniversity 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.[40]
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 theWestern blot and immunodot blot tests. Inimmunohistochemistry, 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.[citation needed]
Antibodies can also be used to purify their target compounds from mixtures, using the method ofimmunoprecipitation.[citation needed]
Therapeutic monoclonal antibodies act through multiple mechanisms, such as blocking of targeted molecule functions, inducingapoptosis in cells which express the target, or by modulating signalling pathways.[41][42][43]
One possible treatment forcancer involves monoclonal antibodies that bind only to cancer-cell-specificantigens and induce animmune response against the target cancer cell. Such mAbs can be modified for delivery of atoxin,radioisotope,cytokine or other active conjugate or to designbispecific antibodies that can bind with theirFab regions both to target antigen and to a conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with itsFc region.
MAbs approved by the FDA for cancer include:[45]
Monoclonal antibodies used forautoimmune diseases includeinfliximab andadalimumab, which are effective inrheumatoid arthritis,Crohn's disease,ulcerative colitis andankylosing spondylitis by their ability to bind to and inhibitTNF-α.[46]Basiliximab anddaclizumab inhibitIL-2 on activatedT cells and thereby help prevent acuterejection of kidney transplants.[46]Omalizumab inhibits humanimmunoglobulin E (IgE) and is useful in treating moderate-to-severe allergicasthma.[citation needed]
Monoclonal antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine likeCiteAb. Below are examples of clinically important monoclonal antibodies.
| Main category | Type | Application | Mechanism/Target | Mode |
|---|---|---|---|---|
| Anti- inflammatory | infliximab[46] | inhibitsTNF-α | chimeric | |
| adalimumab | inhibitsTNF-α | human | ||
| ustekinumab | blocksinterleukinIL-12 andIL-23 | human | ||
| basiliximab[46] |
| inhibitsIL-2 on activatedT cells | chimeric | |
| daclizumab[46] |
| inhibitsIL-2 on activatedT cells | humanized | |
| omalizumab | inhibits humanimmunoglobulin E (IgE) | humanized | ||
| Anti-cancer | gemtuzumab[46] |
| targets myeloid cell surface antigenCD33 onleukemia cells | humanized |
| alemtuzumab[46] | targets an antigenCD52 onT- andB-lymphocytes | humanized | ||
| rituximab[46] |
| targets phosphoproteinCD20 onB lymphocytes | chimeric | |
| trastuzumab |
| targets theHER2/neu (erbB2) receptor | humanized | |
| nimotuzumab |
| EGFR inhibitor | humanized | |
| cetuximab |
| EGFR inhibitor | chimeric | |
| sasanlimab | Targets theprogrammed cell death protein 1 (PD-1) receptor | humanized | ||
| panitumumab |
| EGFR inhibitor | human | |
| bevacizumab &ranibizumab |
| inhibitsVEGF | humanized | |
| Anti-cancer and anti-viral | bavituximab[47] |
| immunotherapy, targetsphosphatidylserine[47] | chimeric |
| Anti-viral |
| immunotherapy, targets spike protein ofSARS-CoV-2 | human | |
| bamlanivimab/etesevimab[49] |
| immunotherapy, targets spike protein ofSARS-CoV-2 | human | |
| Sotrovimab[50] |
| immunotherapy, targets spike protein ofSARS-CoV-2 | human | |
| Other | palivizumab[46] |
| inhibits an RSV fusion (F) protein | humanized |
| abciximab[46] |
| inhibits the receptorGpIIb/IIIa onplatelets | chimeric |
In 2020, the monoclonal antibody therapiesbamlanivimab/etesevimab andcasirivimab/imdevimab were givenemergency use authorizations by the USFood and Drug Administration to reduce the number of hospitalizations, emergency room visits, and deaths because ofCOVID-19.[48][49] In September 2021, the Biden administration purchasedUS$2.9 billion worth of Regeneron monoclonal antibodies at $2,100 per dose to curb the shortage.[51]
As of December 2021,in vitro neutralization tests indicate monoclonal antibody therapies (with the exception ofsotrovimab andtixagevimab/cilgavimab) were not likely to be active against the Omicron variant.[52]
Over 2021–22, twoCochrane reviews found insufficient evidence for using neutralizing monoclonal antibodies to treat COVID-19 infections.[53][54] 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.[54]
In March 2024,pemivibart, a monoclonal antibody drug, received an emergency use authorization from the US FDA for use aspre-exposure prophylaxis to protect certain moderately to severely immunocompromised individuals against COVID-19.[55][56]
Several monoclonal antibodies, such asbevacizumab andcetuximab, can cause different kinds of side effects.[57] These side effects can be categorized into common and serious side effects.[58]
Some common side effects include:
Among the possible serious side effects are:[59]
This article incorporates text from this source, which is in thepublic domain. This article incorporates text from this source, which is in thepublic domain. This article incorporates text from this source, which is in thepublic domain.Engineeredmonoclonal antibodies andantibody mimetics | ||
|---|---|---|
| Whole antibody |  | |
| Fab fragment | ||
| Variable fragment | ||
| Smaller units | ||
| Intracellular | ||
| Antibody mimetics | ||
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