Antibody fragments

Trending: Antibody fragments

There is high interest in antibody fragments since binding works similarly compared to full-length immunoglobulins. Only the fragments are smaller and therefore offer deep tissue penetration and fast clearance. Fragments are used in many constructs; currently over a hundred have been developed.

Antibody fragments market

The antibody fragments market is rapidly developing. The market share of antibody fragments as therapeutics is expected to grow by around 5% each year. A few examples of antibody fragment types already on the market are:

• Ranibizumab (Fab fragments derived from bevacizumab)
• Brolucizumab (single chain variable fragment (scFv))
• Certolizumab (Fab’ fragment (apostrophe indicates that a part of the hinge is present))
• Caplacizumab (nanobody® (Nb))
• Blinatumomab (bispecific T-cell engager (BiTE))

The antibody fragments meaning for the terms and abbreviations above will be thoroughly explained in this article.

Introduction to antibody fragments

Every conventional antibody has the same characteristic Y-shape as shown in the figure below. The end of each arm enables binding to a specific antigen. This part is commonly used in antibody fragments. There are multiple antibody isotypes, subtypes, and allotypes but all of them consist in general of the same regions. These antibody regions (also referred to as portions, parts, or domains) are connected by hinges that can be split. After splitting, antibody fragments can be derived. These fragments can be used as is or can be used to engineer further constructs.

Hinge and linkers

The hinge region connects the heavy chains of the Fc region with the heavy chain of both arms. Between the heavy and light chains is an inter-chain. Both are disulfide, covalent bonds. These sulfate bonds, often written as -s-s- are cleavable by reducing agents such as the commonly used enzymes papain and pepsin. After cleaving these bonds the antibody is divided into fragments, and bonds can be indicated by -sh (sulfhydryl).

Antibody splitting, the production of antibody fragments

Did you know that there are multiple methods of splitting/cleaving antibodies? Methods can result in different fragments. See figure “Anatomy of an Antibody.” A few common examples of antibody cleaving are:

• Splitting antibodies with the Pepsin enzyme results in one F(ab’)2 fragment and a fragmented Fc domain for each antibody.
• Antibody fragments derived from Papain cleavage are two Fab fragments and an intact Fc domain. One F(ab’)2 fragment can be obtained from Papain which has been activated with cysteine.
• Ficin digestion will break down the Fc domain into numerous fragments, leaving two Fab regions or one F(ab’)2. Ficin is used for mouse monoclonal IgG1.

Important to know, is that not all methods can be used for all antibody isotypes and subclasses. Also, results per origin of antibody can differ.

Antibody fragment production methods

Another method besides enzymatic fragmentation to produce antibody fragments is recombinant DNA technology. Specific antibody fragments can be produced by expressing antibody genes in a suitable host, such as:

• Phage display
• Yeast display
• Bacterial display
• Mammalian cells

In addition to these methods, a few others are:

• Cell-free protein synthesis
• Chemical synthesis
• Protein subunit vaccines

The preferred method is depending on the antibody fragment type and application.

What are the different types of antibody fragments?

There are several types of antibody fragments.

1. Natural antibody fragments that are solely derived from cleaving.
2. Antibody fragment constructs from re-engineered fragments with linkers.
3. Antibody fragment constructs that use parts from multiple different antibodies, other compounds or molecules, or changed antibody structures.
4. Artificial antibody mimicking compounds.

Antibody fragments from enzyme cleaving

These antibody fragments can be made by only cleaving the hinge region at specific points.

• Fv (Fragment Variable)
• Fc (Fragment Crystallizable) (antibody tail region)
• Fab (Fragment antigen binding, consists of one constant and one variable domain of both light and heavy chain)
• Fab’ (Fab with part of hinge)
• (Fab’)2 (two Fab regions with hinge)
• rIgG / Half-IgG (only the left or right part of IgG, consisting of one heavy chain (HC) and one light chain (LC))

Other antibody fragments (in general inadvertently) created from cleavage:

• Fd’ (VH+CH1 including a piece of the hinge)
• Fc/2 (half Fc fragment)
• LC (only the light chain)
• pFc (fragmented Fc fragment)

Fab antibody

The Fab fragment interacts with the antigens. For most mammalian antibodies it is of a similar construct. A Fab fragment is made of a heavy and a light chain connected with a disulfide bond and noncovalent interactions such as salt linkages, hydrogen bonds & hydrophobic interactions. Good to know: the heavy chain only antibody lacks the light chain.

Because the Fab region of antibody has high specificity, for drug development the combination of Fab and antigen is essential. When the combination Fab-antigen is found, the Fab fragment can be used for further development. This can be done in full-length immunoglobulin size, Fab2 fragment (both Fab domains), single Fab domain, or any of the other cleavable regions.

FC region of antibody

After cleaving, drug developers get the Fab and Fc region of antibody. The Fc portion of an antibody has a different function than the Fab region. What is the function of Fc region of antibody? The Fc region of antibody binds to cell surface receptors and certain proteins and can trigger the immune response.

Nanobody® from Heavy chain only antibody

A conventional antibody consists of two heavy chains and two light chains. For antigen binding, Fab fragment needs both the fragment binding region from the heavy and light chain. One single chain FV fragment does not offer the same binding. However, one antibody, the Heavy chain only antibody (HcAb) is significantly different. This antibody lacks light chains and can bind to an antigen as a single domain antibody (sdAb).

Antibody fragments with linkers

Not all antibody fragments can be used directly. The derived antibody fragments can be re-engineered with linkers to create many different designs. The most common ones are listed below. When the Fab and Fc region of an antibody are used, it can trigger effector functions. When only parts from the Fab region are used, it can still result in blocking or neutralizing pathogens, antigens, and receptors. It can inhibit pathways without causing an immune response. Full length immunoglobulins can be modified to lack certain effector functions, they are named Fc silenced antibodies.

• scFV (single-chain Fragment Variable) (VL and VH fragment)
• di-scFv (sometimes referred to as dscFv or diabody) (divalent scFV, two scFv fragments linked)
• tri-scFv (triabody) (three scFv fragments linked)
• scFab (Single chain Fab) (Fab element, connected by a linker instead of hinge)
• Minibody antibody(two scFv fragments linked to two CH3 regions)
• scFv-Fc (two scFv fragments linked to Fc region, but around 50 kDa lighter than a full-size IgG)

Different antibody fragment categories

Antibody fragments can be subdivided by design and by function. We list the common categories:

• Full-length antibodies symmetrical
• Full-length antibodies asymmetrical
• Antibody fragments with Fc domain
• Antibody fragments without Fc domain / Variable domain only Ab (Antibody)
• Monospecific antibodies
• Bispecific antibodies
• Monovalent antibodies
• Bivalent antibodies
• Multispecific antibodies (trispecific, tetraspecific (four-in-one) etc.)
• Multivalent antibodies (trivalent, tetravalent, pentavalent, hexavalent)
• CH1/CL fusion proteins
• Constant domain-extended bispecific fragments (antigen-binding fragments recombined with (parts of) fc region)
• Appended IgGs (IgG with appended antigen binding fragment(s))

Advantages of antibody fragments over full-size antibodies

As you can see, by linking antibody fragments, new constructs can be created. Antibody fragments can solve problems from full-size antibodies regarding function, biodistribution, and production. Advantages can be regarding a smaller molecular weight, short half-life, increased affinity, valency, and avidity. However, the above list of antibody fragments consists of monovalent, monospecific antibodies. Further engineering allows for multipecific and multivalent antibodies and antibody structures. In addition, new issues with antibody fragments such as a too short half-life, can be solved by fragment design.

Engineered antibody fragments and platforms

Many of these antibody fragments are developed as a platform. Drug developers can use this platform for their antigen binding part. Mostly this is done through a process of in-licensing or out-licensing. In general, this means that the licensor (the company owning the platform) grants the licensee the right to use their platform or technology in their pharmaceutical development and/or product.

List of engineered antibody fragments

Above are just a few schematic examples of monoclonal antibody fragments. There are many engineered antibody fragments. Although the antibody fragment list on this page is extensive, specific technologies or platforms might be missing. This is inevitable since new platforms and technologies are still being developed. In case you miss a specific platform, please let us know.

In general, this list comprehends all major fragment types and many of the platforms and technologies one must know. If an abbreviation has a clear meaning and if we know from a fragment that it is a platform or technology by a company, it’s written behind it.

Download antibody fragment list

Below is a list of common antibody fragments. You can download our Antibody Fragment List with 100+ fragments with images, more information, and relevant links for further research.

Or view first the condensed list without images, short description and (study) links.

Learn the language

When you understand the language, you can often understand what an antibody fragment looks like. Below is a short list of words and abbreviations and the corresponding meaning.

Also note that technologies can be adapted into different fragments, meaning formats don’t always look similar while sharing partially the same name. Examples are BEAT, KiH, and CrossMab.

Words and abbreviations definition list

“Charged” indicates that a part of the antibody is charged for the correct pairing of chains. This indicates that the construct is bispecific.

“Knob-in-Hole”, abbreviated to KiH, is a technology for correct pairing, meaning the antibody is probably bispecific and made of two distinct half antibodies. KiH can be used in heavy chain pairing and heavy and light chain pairing.

“Di” indicates two fragments are used.

“Bi”, “Bis”, indicates bispecific antibody.

“Tri” often indicates three binding sites. The construct can be a full length Ig construct with an additional Fab or Fv fragment, or three fragments combined.

“Tand” stands often for “Tandem” meaning two or more fragments are linked next to each other.

“Fab2” or “Fv2” can indicate that there are two binding sites. This is used often when the conventional arms-shaped construct is used. The Fv or Fab fragments don’t necessarily have to be in conventional format, they can also be linked.

“Fv” indicates only the VH and VL fragments are used.

“Fab” indicates the VH+CH1 and VL+CL fragments are used. This can be with an original hinge or in single chain (cs) format.

“IgG” the well-known abbreviation for Immunoglobulin G and in antibody format it often indicates that a full-length IgG antibody is (partly) present.

“Nb” and “VHH” indicate that a nanobody® is present in the construct, where the latter is always of camelid type. The first however is often also of camelid origin, since the only other known nanobody® VNAR, derived from shark, is not yet commonly used. Nb, VHH, and VNAR are never of human origin.

“Sc” means Single chain, it suggests that a linker is used to combine fragments.

“Fc” means that the Fc region of the antibody is used.
“mFc” indicates that a monomeric Fc domain is used, meaning one CH2 and one CH3 domain.

“CH3” often indicates only the CH3 domain of the Fc region is used.

“(H), (L), (HC), (LC)” can be used to indicate where the additional fragment is connected. For example: scFv(LC)-IgG translates to a full-length IgG with added scFv fragments to the light chains.

“IgG2” can have several meanings. In general, it refers to the second antibody subclass of IgG. In antibody fragments, it can also mean two IgG antibodies connected to each other.

“IgG-IgG” can point out that two IgGs are linked together. From this you cannot say for sure if the construct is bispecific and how the antibodies are linked.

“IgG1/IgG2” can refer to a bispecific antibody with one half from IgG1 and the other half from IgG2.

“κ” specifically indicates that a kappa-chain is used, one of the two existing types of light chains in human immunoglobulins. The other type is lambda (λ). Light chains of sigma (σ) type exist in certain animals, but not in humans.

“λ” specifically indicates that a lambda light chain is present in the antibody format.

“dsFv” of “dsscFv” indicates that disulfide bonds are used in Fv or scFv fragments to improve stability.

“Cognate” indicates that the light chain originated from the same source as the heavy chain is used. You see this in bispecific antibodies.

“Common” indicates a bispecific antibody construct with two different heavy chains but a common light chain. Meaning one heavy chain lacks its cognate light chain.

“HCAb” indicates Heavy Chain only antibody. This is commonly not the same as a conventional antibody that lacks the light chain but is an immunoglobulin type on its own. From this type nanobodies® are derived, meaning the format probably contains Nb fragments. HCAb do not occur naturally in humans, only in camelids and sharks.

“T” The presence of a T in the name can indicate a relation to T-cells, for instance: 1 Fab-IgG TDB indicates a “T cell-Dependent Bispecific antibody” and “TCB” is short for “T-Cell Bispecific”, like in the antibody format 2:1 TCB.

Combinations with other proteins or other molecules can be indicated in the name. An example of this:
“scFv-HSA-scFv” is an antibody format made of two scFv fragments linked to one HSA (Human Serum Albumin) protein.

Frequently asked questions about antibody fragments

Antibody fragments are a relatively new topic in life science. As a Clinical Research Organization (CRO) for first in-human clinical trials we are pioneering with drug developers in this field. Therefore, we would like to answer some of the frequently asked questions. Is your question not listed or would you like more information? Don’t hesitate to contact us today.

Clinical trials for antibody fragments

After drug discovery, the phases of drug development begin. Choosing the fragment design influences Biodistribution (BD) and Pharmacokinetics (PK). It can be challenging to reproduce and establish results from the preclinical phase in clinical trials. At TRACER we specialize in first-in-human clinical trials. Often our Phase 0 imaging trials in-human can replace extensive animal research. This can decrease time and costs in drug development and provide drug developers with visual BD/PK data. Contact TRACER today to discuss the possibilities for your antibody (fragment) or compound.

Contact TRACER

Download Antibody Fragment List including images, short description and (study) links.

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