Humanized antibody

Last updated

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. [1] [2] The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice). The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients (see also Human anti-mouse antibody). The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab (see Nomenclature of monoclonal antibodies).

Contents

Humanized antibodies are distinct from chimeric antibodies. The latter also have their protein sequences made more similar to human antibodies, but carry a larger stretch of non-human protein.

There are other ways to develop monoclonal antibodies. This list covers many of the monoclonals developed for use in humans.

Use of recombinant DNA in humanization process

The humanization process takes advantage of the fact that production of monoclonal antibodies can be accomplished using recombinant DNA to create constructs [3] capable of expression in mammalian cell culture. That is, gene segments capable of producing antibodies are isolated and cloned into cells that can be grown in a bioreactor such that antibody proteins produced from the DNA of the cloned genes can be harvested en masse. The step involving recombinant DNA provides an intervention point that can be readily exploited to alter the protein sequence of the expressed antibody. The alterations to antibody structure that are achieved in the humanization process are therefore all effectuated through techniques at the DNA level. Not all methods for deriving antibodies intended for human therapy require a humanization step (e.g. phage display) but essentially all are dependent on techniques that similarly allow the "insertion" or "swapping-out" of portions of the antibody molecule.

Distinction from "chimeric antibody"

Sketches of chimeric (top right), humanized (bottom left) and chimeric/humanized (bottom middle) monoclonal antibodies. Human parts are shown in brown, non-human parts in blue. Chimeric and humanized antibodies.svg
Sketches of chimeric (top right), humanized (bottom left) and chimeric/humanized (bottom middle) monoclonal antibodies. Human parts are shown in brown, non-human parts in blue.

Humanization is usually seen as distinct from the creation of a mouse-human antibody chimera. So, although the creation of an antibody chimera is normally undertaken to achieve a more human-like antibody (by replacing constant region of the mouse antibody with that from human) simple chimeras of this type are not usually referred to as humanized. Rather, the protein sequence of a humanized antibody is essentially identical to that of a human variant, despite the non-human origin of some of its complementarity-determining region (CDR) segments responsible for the ability of the antibody to bind to its target antigen.

Chimeric antibody names contain a -xi- stem. Examples of chimeric antibodies approved for human therapy include abciximab (ReoPro), basiliximab (Simulect), cetuximab (Erbitux), infliximab (Remicade) and rituximab (MabThera). There are also several examples of chimerics currently in clinical trials (e.g. bavituximab, see sortable list for additional examples).

Humanizing via a chimeric intermediate

The humanization process may also include the creation of a mouse-human chimera as an initial step. In this case, a mouse variable region is spliced to a human constant region. The chimera can then be further humanized by selectively altering the sequence of amino acids in the variable region of the molecule.

The alteration process must be "selective" to retain the specificity for which the antibody was originally developed. That is, since the CDR portions of the variable region are essential to the ability of the antibody to bind to its intended target, the amino acids in these portions cannot be altered without the risk of undermining the purpose of the development. Aside from the CDR segments, the portions of the variable regions that differ from those in humans can be corrected by exchanging the appropriate individual amino acids. This is accomplished at the DNA level through mutagenesis.

Naming of humanized chimeras includes the stem for both designations (-xi- + -zu-). Otelixizumab is an example of a humanized chimera currently in clinical trials for treatment of rheumatoid arthritis and diabetes mellitus. [4]

Humanization by insertion of relevant CDRs into human antibody "scaffold"

It is possible to produce a humanized antibody without creating a chimeric intermediate. "Direct" creation of a humanized antibody can be accomplished by inserting the appropriate CDR coding segments (so-called 'donor', responsible for the desired binding properties) into a human antibody "scaffold" (so-called 'acceptor'). As discussed above, this is achieved through recombinant DNA methods using an appropriate vector [3] and expression in mammalian cells. That is, after an antibody is developed to have the desired properties in a mouse (or other non-human), the DNA coding for that antibody can be isolated, cloned into a vector and sequenced (or the DNA can be sequenced directly using single-cell methods). The DNA sequence corresponding to the antibody CDRs can then be determined. Once the precise sequence of the desired CDRs are known, a strategy can be devised for inserting these sequences appropriately into a construct containing the DNA for a human antibody variant. [5] [6] [7] [8] [9] The strategy may also employ synthesis of linear DNA fragments based on the reading of CDR sequences. The process requires computer-modelling software to determine which of the antibody's amino acids can be changed from murine-sequence to human-sequence without the changes compromising the conformation of the binding site. In the United States, this software was developed, patented, and demonstrated, by Protein Design Labs, Inc. in Mountain View, California, in the 1980s and 1990s. [10]

Alemtuzumab is an early example of an antibody whose humanization did not include a chimeric intermediate. In this case, a monoclonal dubbed "Campath-1" was developed to bind CD52 using a mouse system. The hypervariable loops of Campath-1 (that contain its CDRs and thereby impart its ability to bind CD52) were then extracted and inserted into a human antibody framework. [1] Alemtuzumab is approved for treatment of B-cell chronic lymphocytic leukemia [11] and is currently in clinical trials for a variety of other conditions including multiple sclerosis. [12]

Derivation from sources other than mice

There are technologies that completely avoid the use of mice or other non-human mammals in the process of discovering antibodies for human therapy. Examples of such systems include various "display" methods (primarily phage display) as well as methods that exploit the elevated B-cell levels that occur during a human immune response.

Display methods

These employ the selective principles of specific antibody production but exploit micro-organisms (as in phage display) or even cell free extracts (as in ribosome display). These systems rely on the creation of antibody gene "libraries" which can be wholly derived from human RNA isolated from peripheral blood. The immediate products of these systems are antibody fragments, normally Fab or scFv.

This means that, although antibody fragments created using display methods are of fully human sequence, they are not full antibodies. Therefore, processes in essence identical to humanization are used to incorporate and express the derived affinities within a full antibody.

Adalimumab (Humira) is an example of an antibody approved for human therapy that was created through phage display. [13] [14]

Antibodies from human patients or vaccine recipients

It is possible to exploit human immune reaction in the discovery of monoclonal antibodies. Simply put, human immune response works in the same way as that in a mouse or other non-human mammal. Therefore, persons experiencing a challenge to their immune system, such as an infectious disease, cancer or a vaccination are a potential source of monoclonal antibodies directed at that challenge. This approach seems especially apt for the development of anti-viral therapies that exploit the principles of passive immunity. Variants of this approach have been demonstrated in principle [15] and some are finding their way into commercial development. [16]

See also

Related Research Articles

<span class="mw-page-title-main">Antibody</span> Protein(s) forming a major part of an organisms immune system

An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

<span class="mw-page-title-main">Immunosuppressive drug</span> Drug that inhibits activity of immune system

Immunosuppressive drugs, also known as immunosuppressive agents, immunosuppressants and antirejection medications, are drugs that inhibit or prevent the activity of the immune system.

<span class="mw-page-title-main">Monoclonal antibody</span> Antibodies from clones of the same blood cell

A monoclonal antibody 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.

In immunology, antiserum is a blood serum containing antibodies that is used to spread passive immunity to many diseases via blood donation (plasmapheresis). For example, convalescent serum, passive antibody transfusion from a previous human survivor, used to be the only known effective treatment for ebola infection with a high success rate of 7 out of 8 patients surviving.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

<span class="mw-page-title-main">Phage display</span> Biological technique to evolve proteins using bacteriophages

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. The proteins that the phages are displaying can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those of other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

<span class="mw-page-title-main">Cancer immunotherapy</span> Artificial stimulation of the immune system to treat cancer

Cancer immunotherapy (immuno-oncotherapy) is the stimulation of the immune system to treat cancer, improving on the immune system's natural ability to fight the disease. It is an application of the fundamental research of cancer immunology and a growing subspecialty of oncology.

<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted:

<span class="mw-page-title-main">Protein A</span>

Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family. Through these interactions in serum, where IgG molecules are bound in the wrong orientation, the bacteria disrupts opsonization and phagocytosis.

The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary, names to monoclonal antibodies. An antibody is a protein that is produced in B cells and used by the immune system of humans and other vertebrate animals to identify a specific foreign object like a bacterium or a virus. Monoclonal antibodies are those that were produced in identical cells, often artificially, and so share the same target object. They have a wide range of applications including medical uses.

<span class="mw-page-title-main">Monoclonal antibody therapy</span> Form of immunotherapy

Monoclonal antibodies (mAbs) have varied therapeutic uses. It is possible to create a mAb that binds specifically to almost any extracellular target, such as cell surface proteins and cytokines. They can be used to render their target ineffective, to induce a specific cell signal, to cause the immune system to attack specific cells, or to bring a drug to a specific cell type.

<span class="mw-page-title-main">Fusion protein</span> Protein created by joining other proteins into a single polypeptide

Fusion proteins or chimeric (kī-ˈmir-ik) proteins are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Chimeric or chimera usually designate hybrid proteins made of polypeptides having different functions or physico-chemical patterns. Chimeric mutant proteins occur naturally when a complex mutation, such as a chromosomal translocation, tandem duplication, or retrotransposition creates a novel coding sequence containing parts of the coding sequences from two different genes. Naturally occurring fusion proteins are commonly found in cancer cells, where they may function as oncoproteins. The bcr-abl fusion protein is a well-known example of an oncogenic fusion protein, and is considered to be the primary oncogenic driver of chronic myelogenous leukemia.

A bispecific monoclonal antibody is an artificial protein that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen. Naturally occurring antibodies typically only target one antigen. BsAbs can be manufactured in several structural formats. BsAbs can be designed to recruit and activate immune cells, to interfere with receptor signaling and inactivate signaling ligands, and to force association of protein complexes. BsAbs have been explored for cancer immunotherapy, drug delivery, and Alzeimer's disease.

<span class="mw-page-title-main">Glypican 3</span> Protein-coding gene in the species Homo sapiens

Glypican-3 is a protein that, in humans, is encoded by the GPC3 gene. The GPC3 gene is located on human X chromosome (Xq26) where the most common gene encodes a 70-kDa core protein with 580 amino acids. Three variants have been detected that encode alternatively spliced forms termed Isoforms 1 (NP_001158089), Isoform 3 (NP_001158090) and Isoform 4 (NP_001158091).

Zinc finger protein chimera are chimeric proteins composed of a DNA-binding zinc finger protein domain and another domain through which the protein exerts its effect. The effector domain may be a transcriptional activator (A) or repressor (R), a methylation domain (M) or a nuclease (N).

A rabbit hybridoma is a hybrid cell line formed by the fusion of an antibody producing rabbit B cell with a cancerous B-cell (myeloma).

Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of 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 in human medicine and research. In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity.

Passive antibody therapy, also called serum therapy, is a subtype of passive immunotherapy that administers antibodies to target and kill pathogens or cancer cells. It is designed to draw support from foreign antibodies that are donated from a person, extracted from animals, or made in the laboratory to elicit an immune response instead of relying on the innate immune system to fight disease. It has a long history from the 18th century for treating infectious diseases and is now a common cancer treatment. The mechanism of actions include: antagonistic and agonistic reaction, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC).

References

  1. 1 2 Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature. 332 (6162): 323–7. Bibcode:1988Natur.332..323R. doi: 10.1038/332323a0 . PMID   3127726. S2CID   4335569.
  2. ↑ Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA (Dec 1989). "A humanized antibody that binds to the interleukin 2 receptor". Proc Natl Acad Sci U S A. 86 (24): 10029–33. Bibcode:1989PNAS...8610029Q. doi: 10.1073/pnas.86.24.10029 . PMC   298637 . PMID   2513570. (This is an early example of the use of the term "humanized antibody".)
  3. 1 2 Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I (May 1997). "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells". J Immunol Methods. 204 (1): 77–87. doi:10.1016/S0022-1759(97)00034-3. PMID   9202712.
  4. ↑ Clinical Trials page list for otelixizumab
  5. ↑ Kashmiri SV, De Pascalis R, Gonzales NR, Schlom J (May 2005). "SDR grafting—a new approach to antibody humanization". Methods. 36 (1): 25–34. doi:10.1016/j.ymeth.2005.01.003. PMID   15848072.
  6. ↑ Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling". J Biochem. 144 (1): 115–20. doi:10.1093/jb/mvn052. PMID   18424812.
  7. ↑ Zhang, Yi-Fan; Ho, Mitchell (2016-09-26). "Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma". Scientific Reports. 6: 33878. doi:10.1038/srep33878. ISSN   2045-2322. PMC   5036187 . PMID   27667400.
  8. ↑ Zhang, Yi-Fan; Ho, Mitchell (April 2017). "Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples". mAbs. 9 (3): 419–429. doi:10.1080/19420862.2017.1289302. ISSN   1942-0870. PMC   5384799 . PMID   28165915.
  9. ↑ Zhang, Yi-Fan; Sun, Yaping; Hong, Jessica; Ho, Mitchell (January 2023). "Humanization of the Shark VNAR Single Domain Antibody Using CDR Grafting". Current Protocols. 3 (1): e630. doi:10.1002/cpz1.630. ISSN   2691-1299. PMC   9813873 . PMID   36594750.
  10. ↑ Fletcher, L. PDL's mAb technology finds right timing. Nat Biotechnol 19, 395–396 (2001). https://doi.org/10.1038/88010
  11. ↑ DrugBank entry for alemtuzumab
  12. ↑ Clinical Trials pages for alemtuzumab
  13. ↑ Kempeni J. (Nov 1999). "Preliminary results of early clinical trials with the fully human anti-TNFalpha monoclonal antibody D2E7". Ann Rheum Dis. 58 (Suppl 1): I70–2. doi:10.1136/ard.58.2008.i70. PMC   1766582 . PMID   10577977.
  14. ↑ Rau R (Nov 2002). "Adalimumab (a fully human anti-tumour necrosis factor alpha monoclonal antibody) in the treatment of active rheumatoid arthritis: the initial results of five trials". Ann Rheum Dis. 61 (Suppl 2): 70–3. doi:10.1136/ard.61.suppl_2.ii70. PMC   1766697 . PMID   12379628.
  15. ↑ Stacy JE, Kausmally L, Simonsen B, Nordgard SH, Alsøe L, Michaelsen TE, Brekke OH (Dec 2003). "Direct isolation of recombinant human antibodies against group B Neisseria meningitidis from scFv expression libraries". J Immunol Methods. 283 (1–2): 247–59. doi:10.1016/j.jim.2003.09.015. PMID   14659916.
  16. ↑ http://www.theraclone-sciences.com/pdf/Theraclone_ISTAR.pdf Archived 2016-03-03 at the Wayback Machine Example of method using human patients as source of monoclonal antibodies
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.