Humanized antibody
Encyclopedia
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. 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
). Not all monoclonal antibodies designed for human administration need be humanized since many therapies are short-term interventions (see List of monoclonal antibodies to get an overview of the various types of antibodies developed for therapeutic administration in humans). The International Nonproprietary Name
s of humanized antibodies end in -zumab, as in omalizumab
(see Nomenclature of monoclonal antibodies
).
Humanized antibodies are distinct from chimeric antibodies. The latter also have their protein sequences made more similar to human antibodies, but the fraction of foreign protein is larger than in humanized ones.
to create constructs
capable of expression
in mammal
ian cell culture
. That is, gene segments capable of producing antibodies are isolated and cloned
into cells that can be grown in a tank
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.
. So, although the creation of an antibody chimera is normally undertaken to achieve a more human-like antibody (by substituting the mouse Fc
region of the 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).
.
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
.
. 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. The strategy may also employ synthesis
of linear DNA fragments based on the reading of CDR sequences.
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. Alemtuzumab is approved for treatment of B-cell chronic lymphocytic leukemia and is currently in clinical trials for a variety of other conditions including multiple sclerosis
.
) as well as methods that exploit the elevated B-cell levels that occur during a human immune response.
) 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.
. Variants of this approach have been demonstrated in principle and some are finding their way into commercial development.
Peptide sequence
Peptide sequence or amino acid sequence is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group...
have been modified to increase their similarity to antibody variants produced naturally in humans. 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
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients (see also Human anti-mouse antibody
Human anti-mouse antibody
Human anti-mouse antibody is an antibody found in humans which reacts in immunoglobins found in mice.-The HAMA response:Antibody treatment is a type of therapy that is used to treat certain types of cancer and immune disorders. Antibodies are proteins which are naturally formed by the body in...
). Not all monoclonal antibodies designed for human administration need be humanized since many therapies are short-term interventions (see List of monoclonal antibodies to get an overview of the various types of antibodies developed for therapeutic administration in humans). The International Nonproprietary Name
International Nonproprietary Name
An International Nonproprietary Name is the official nonproprietary or generic name given to a pharmaceutical substance, as designated by the World Health Organization...
s of humanized antibodies end in -zumab, as in omalizumab
Omalizumab
Omalizumab is a humanized antibody drug approved for patients with moderate-to-severe or severe allergic asthma, which is caused by hypersensitivity reactions to certain harmless environmental substances...
(see Nomenclature of monoclonal antibodies
Nomenclature of monoclonal antibodies
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...
).
Humanized antibodies are distinct from chimeric antibodies. The latter also have their protein sequences made more similar to human antibodies, but the fraction of foreign protein is larger than in humanized ones.
Use of recombinant DNA in humanization process
The humanization processes takes advantage of the fact that production of monoclonal antibodies can be accomplished using recombinant DNARecombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
to create constructs
Expression vector
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
capable of expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
in mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
. That is, gene segments capable of producing antibodies are isolated and cloned
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
into cells that can be grown in a tank
Bioreactor
A bioreactor may refer to any manufactured or engineered device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This...
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
Phage display
Phage display is a method 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. Phage Display was originally invented by George P...
) 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"
Humanization is usually seen as distinct from the creation of a mouse-human antibody chimeraFusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
. So, although the creation of an antibody chimera is normally undertaken to achieve a more human-like antibody (by substituting the mouse Fc
Fragment crystallizable region
The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system...
region of the 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
Complementarity determining region
Complementarity determining regions are regions within antibodies or T cell receptors where these proteins complement an antigen's shape. Thus, CDRs determine the protein's affinity and specificity for specific antigens...
(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
Basiliximab
Basiliximab is a chimeric mouse-human monoclonal antibody to the α chain of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants...
(Simulect), cetuximab
Cetuximab
Cetuximab is a chimeric monoclonal antibody, an epidermal growth factor receptor inhibitor, given by intravenous infusion for treatment of metastatic colorectal cancer and head and neck cancer.- Distribution :Cetuximab is manufactured and distributed in North America by ImClone and Bristol-Myers...
(Erbitux), infliximab
Infliximab
Infliximab is a monoclonal antibody against tumour necrosis factor alpha . It is used to treat autoimmune diseases. Remicade is marketed by Janssen Biotech, Inc...
(Remicade) and rituximab
Rituximab
Rituximab, sold under the trade names Rituxan and MabThera, is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells...
(MabThera). There are also several examples of chimerics currently in clinical trials (e.g. bavituximab
Bavituximab
Bavituximab is a chimeric monoclonal antibody designed for the treatment of cancers and viral infections.-Mechanism of action:Bavituximab binds to phosphatidylserine which is exposed on the surface of certain atypical animal cells, including tumour cells and cells infected with any of six different...
, see sortable list for additional examples).
Humanizing via a chimeric intermediate
The humanization process may, however, include the creation of a mouse-human chimera in an initial step (mouse Fab spliced to human Fc). Thereafter the chimera might be further humanized by the selective alteration of the sequence of amino acids in the Fab portion of the molecule. The process must be "selective" to retain the specificity for which the antibody was originally developed. That is, since the CDR portions of the Fab 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 Fab sequence that differ from those in humans can be corrected by exchanging the appropriate individual amino acids. This is accomplished at the DNA level using mutagenesisMutagenesis
Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...
.
Naming of humanized chimeras includes the stem for both designations (-xi- + -zu-). Otelixizumab
Otelixizumab
Otelixizumab, also known as TRX4, is a monoclonal antibody, which is being developed for the treatment of type 1 diabetes and other autoimmune diseases. The antibody is being developed by Tolerx, Inc...
is an example of a humanized chimera currently in clinical trials for treatment of rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...
and diabetes mellitus
Diabetes mellitus
Diabetes mellitus, often simply referred to as diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced...
.
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 (responsible for the desired binding properties) into a human antibody "scaffold". As discussed above, this is achieved through recombinant DNA methods using an appropriate vector 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 sequencedDNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
. 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. The strategy may also employ synthesis
Gene synthesis
Artificial gene synthesis is the process of synthesizing a gene in vitro without the need for initial template DNA samples. The main method is currently by oligonucleotide synthesis from digital genetic sequences and subsequent annealing of the resultant fragments...
of linear DNA fragments based on the reading of CDR sequences.
Alemtuzumab
Alemtuzumab
Alemtuzumab is a monoclonal antibody used in the treatment of chronic lymphocytic leukemia , cutaneous T-cell lymphoma and T-cell lymphoma...
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
CD52
CD52 is a protein present on the surface of mature lymphocytes, but not on the stem cells from which these lymphocytes were derived.It also is found in monocytes and dendritic cells....
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. Alemtuzumab is approved for treatment of B-cell chronic lymphocytic leukemia and is currently in clinical trials for a variety of other conditions including multiple sclerosis
Multiple sclerosis
Multiple sclerosis is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms...
.
Antibodies for human therapy derived without using 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 displayPhage display
Phage display is a method 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. Phage Display was originally invented by George P...
) 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 displayPhage display
Phage display is a method 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. Phage Display was originally invented by George P...
) or even cell free extracts (as in ribosome display
Ribosome display
Ribosome display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step...
). 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
Adalimumab
Adalimumab is the third TNF inhibitor, after infliximab and etanercept, to be approved in the United States. Like infliximab and etanercept, adalimumab binds to TNFα, preventing it from activating TNF receptors; adalimumab was constructed from a fully human monoclonal antibody, while infliximab...
(Humira) is an example of an antibody approved for human therapy that was created through phage display.
Antibodies from human patients or vaccinees
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 immunityPassive immunity
Passive immunity is the transfer of active humoral immunity in the form of readymade antibodies, from one individual to another. Passive immunity can occur naturally, when maternal antibodies are transferred to the fetus through the placenta, and can also be induced artificially, when high levels...
. Variants of this approach have been demonstrated in principle and some are finding their way into commercial development.