Antibody fragments comprise a portion of a full-lenght antibody, as for example, the antigen binding or variable region of a full length antibody.  Antibody fragments that contain the idiotype (antigen-binding fragment)of the antibody molecule include fragments that include the Fab region, F(ab’)2 fragment and Fab’ fragments.

DAb (Domain Antibody): is either the variable domain of an antibody heavy chain (VH domain) or the variable domain of an antibody light chain (VL domain). Each DAb thus contains 3 or 6 naturally occurring complementarity determining regions (CDRs) from an antibody (Holt, “Domain antibodies: proteins for therapy” TRENDS Biotech. 21(11), 2003). It is thus 4 times smaller than a Fab and half the size of an scFV.

It has been known for years that VH and VL domains can exist in vivo as part of isolated antibody chains in some human diseases, such as heavy chain disease and as Bence-Jones proteins in multiple myeloma.

Advantages of Antibody Fragments: Antibody fragments have both pros and cons as therapeutics compared with full size mAb therapeutics. one advantage is that they are smaller and penetrate tissues and tumors more rapdily than mAbs. In addition, the small size has been suggested to permit binding to cryptic epitopes not accessible to full sized mAbs.

Use of antibody fragments in place of whole antibody is preferred due to favorable pharmacokinetic profile, i.e., their wide volume disrtribution and ability to reach tissue compartment at a faster rate when compared to IgG due to low moleular weight and less immunogenicity. Fernandes et al. (J. Chromatography B, 876 (2008) pp. 109-115). 

Disadvantages of Antibody Fragments:The cons are short ciruclating half-lives in humans, likely due to kideny clearance, ncessitating the need for derivitization with polyethylene glycol or other modifications.

Specific Types of Antibody Fragments

Fab fragment (VL-CL-CH1-VH): The “Fab region” and “fragment, antigen binding region,” interchangeably refer to portion of the antibody arms of the immunoglobulin “Y” that function in binding antigen. The Fab region is composed of one constant and one varible domain from each heavy and light chain of the antibody. Thus Fab molecules comprise the entire Ig light chain (both the variable and constant region of the light chain), but include only the variable region and first constant region domain (CH1) of the heavy chain. Fab fragments result from papain digestion that produces two identical antigen-binding fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Fab fragments behave as whole antibodies in terms of antigen recognition.

Fab fragments (fragment antigen bidning) are the antigen-binding domains of an antibody molecule, containing VH+CH1 and CL+VL. Between CL and CH1 and interchain disulfide bond is present. The molecule weight of the heterodimer is usually around 50 kDa. Fab fragments can be repared by papain digestions of whole antibodies. (Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15). 

When papaiin is used for antibody digestionk 2 Fab (50 kDa each) and 1 Fc (50 kDa) are obtained from one antibody. (Chakrabarti, “separation of monoclonal antibodies by analytical size exclusion chromatography” Antibody engineeering, Cahpt 7, 2018). 

The VH and CH1 domains form heterodimers with the light chain VL and CL domains to creat teh Fab (Fragment, antigen binding) domain. (Haixia Ru “PH and temperature-dependent mechaisms of non-native aggregation of anti-CD40 IGG1” Dissertion, Fall 2015. 

Fab’ (a Fab with the heavy chain hinge region): can be obtained by treating whole antibody with pepsin followed by reduction to yeild an intact light chain and a portion of the heavy chain. Two Fab’ fragments are obtained per antibody molecule.

F(ab’)2 (a dimer of Fab’ fragments jointed by the heavy chain hinge region) fragment has two antigen combining sites that results from pepsin treatment. It is a dimer of two Fab’ fragments held together by two disulfide bonds.

When pepsin is used for antibody digestion, a F(ab’)2 is obtained. Chakrabarti, “separation of monoclonal antibodies by analytical size exclusion chromatography” Antibody engineeering, Cahpt 7, 2018).

Fc or “fragment, crystallizable” region refers to a portion of the base of the immunoglobulin “Y” that functions in modulating immune cell activity. The Fc region is compoased of two heavy chains that contributes 2 or 3 constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils. In an experimental setting, Fc and Fab fragments can be generated by cleaving an immunoglobulin monomer with the enzyme papain into 2 Fab fragments and an Fc fragment.

The CH2 and CH3 domains can be isolated togatehr as the Fc (Fragment, crystallizable) fragment, with the FAB domains being removed via proteolysis. 

The Fc domain of IgG salvages the protein from endosomal degradation by binding to the neonatal Fc receptor (FcR) (FcRN), facilitating recycling. Proteins or peptides lacking a functional Fc may fail as drug products because they ahve a very short serum half-life due to fast renal clearance. Thus, so-called Fc-fusiohn technologies, in which the Ig Fc is fused genetically to a protein of interest, have emerged to confer antibody-like properties on proteins and petpides of therapeutic interest. (Levin, “Fc fusion as a platoform technology: potential for modulating immunogenicity” Trends in Biotechnology, 33(1), 2015, pp. 27-34). 

Fv is the minimum antibody fragment which contains a complete antigen-recognition and binding site. The region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent assocciation. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.

The minimal fragment (about 30kDa) that still contains the whole anitgen-binding site of a whole IgG antibody is composed of both the variable heavy chain (VH) and variable light chain (VL) domains. This heterodimer, called Fv fragment (for fragment variable) is still capable of binding the antigen. Normally, native Fv fragments are unstable since the non-covalently associated VL and VH domains tend to dissociate from one another at low protein concentrations. (Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15). Several methods aiming to stabilize Fv fragments have been developed, most commonly through the covalent attachment of a VH domain to a VL domain using a flexible peptide linker, resulting in a single chain molecule (scFV). (Nymalm, J Structural Biology 138 (2002) 171-186). 

ScFvs (single chain Fv fragments) have a VH domain fused to a VL domain via polypeptide linker. Single-chain antigen binding proteins are composed of an antibody VL tethered to a VH by a designed peptide that links the carboxy terminus of the VL to the VH. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequence were used to construct the recombinant genes that were expressed in E coli. (Bir, “Single-chain antigen binding proteins” 1988). Single chain Fv (scFv) fragments are comprised of the variable domains of the VH and VL chains of an antibody tethered together by a polypeptide linker. The linker is long enough to allow the VH and the VL domains to fold into a functional Fv doamin enabling the scFv to recognized and bind its target with the similar or increased affinity of the parent antibody. A commonly used linker comprises glycine and serine residues to provide flexibility and protease resistance. (Hagal, WO 02/059264)

Typically, scFv monomers are designed with the C terminal end of the VH domain tethered by a polypeptide linker to the N terminal residue of the VL. Optionally an inverse orientation is employed: The C terminal end of the VL domain is tethered to the N terminal residue of VH through a polypeptide linker. The polypeptide linker is typically about twelve amino acids in lenght so that the scFvs can not fold into a functional Fv domain and instead associate with a second scFv to form a diabody. Further reducing the lengh of the linkers to less than three amino acids forces the scFv association into trimers or tetramers, depending on the linker lenght, composition and Fv domain orientations. (Hagal, WO 02/059264)

–ScFvs-CH3 Domain:

ScFvs, comprised of the variable regions of the immunoglobulin H and L chain, covalently connected by a speptide linker are small prtoeins which generally retain specificity and affinity for antigen in a single polypetpide and can provdie a convenient building block for large, antigen-specific molecuels. However, due to their small size scFvs themselves have limited utility as tumor-targeting agents in vivo, generally demonstrating very rpaid clearance from the circultation in sanimal models. (Hu, Cancer Research 56, 305503061, 1996). 

Hu, Cancer Research 56, 305503061, 1996) dislose an engineered antibody fragmnet (VL-VH-CH3 or “minibody”) with bivalent binding to carcinembryonic antigen (CEA) produced by genetic fusion of an anti-CEA single chain antibody (scFv) to the human IgG1 CH3 domain. 

Diabodies (scFv dimers): Diabodies are scFv dimers in which each chain consists of a variable heavy (VH) domain connected to a variable light (VL) domain using a peptide linker that is too short to permit paring betwen domains on the same chain. Consequently, pairing occurs between complementary domains of two different chains, creating a stable non-covalently bound dimer with two binding sites. The contstruction of single-chain (sc) Fv fragments with short linkers (less than or equal to 10 residues) greatly restrict intra-chain pairing of variable domains. Interchain pairing of such scFv may occur to form a bivalent (scFv)2 fragmetn, the so called “diabody”. (Zu “high leavel secreution of a humanized bispecific diabody from Escherichia coli” Biotechnology, 1996, pp. 192-196, 14(2)). 

Hodgson (WO/2007/088371) discloses a synthetic bispecific single chain tetravalent diabodiy (bsctDB) in which the variable light and variable heavy regions from D1.3 (anti-lysozyme) and A5B7 (anti-CEA (carcinoembryonic antigen) were linked on a single polypeptide chain. This DNA sequence was then cloned into a plasmid which was trasnformed into E coli.

–Application in immuno-oncology:

Diabodies have been used in immuno-oncology. For example, Affimed develops TandAbs® which have the ability to bring NK and T cells into proximity and trigger a signal cascade that leads to the destruction of cancer cells. Due to their tetravalent architecture (which provides for four binding domains), the TandAbs bind to their targets with high affinity and have half lives that allow intravenous administration.  

Reusch (MAbs 2015; 7(3), 584-604) discloses a humanized tetravalent bispecific CD19/CD3 tandem diabody (TanAb) consisting solely of Fv domains. The tanab has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells.

VHH/Domain antibodies (dAb): See outline

Heavy chain antibodies (HCabs; VHHH; Nanobodies): are a new class of novel therapeutic proteins dervied from natrually occurring single chain antibodies. They are based on the observation that Camelidae (see above) are the only mammals which lack light chains but still have the full antigen binding capacity of conventional antibodies. In these heavy chain only antibodies, antigen binding occurs through a single variable domain (VHHH) which is the smallest functional antibody fragment of a naturally occurring heavy chain antibody. Nanobodies have a number of important competitive advantages which differentiate them from conventional monoclonal antibodies and small molecule drugs which include high affinity, small size, high stabiliy and braod applicability (See Ablynx Annual Report 2008)

The hetero-tetrameric H and L chain IgG antibody format formed the central dogma for antibody structure until 1993 when a novel antibody structure was discovered in the camelid species. Camelus dromedarius. These novel antibodies were shown to be heavy chain homodimers, which did not associate with a light chain, and lacked the CH1 domain. In camels, heavy chain antibodies constitute 75% of circulating antibodies. Camedlid species also display conventional hetero-tetrameric antibodies with identical paris of heavy and light chains. To distinguish them from conventional antibody heavy chain variable regions (VH), the variable domain of heavy chain antibodies has been denoted VHH. The unique structure and novel characteristics of this new class of antibodies are of commercial significant with VHH fragments forming a new class of therapeutics used to treat human diseases. The extended CDR-H3 region characteristic of VHH fragments, and their preference for binding protein clefts, makes them an interesting tool in the pharmaceutical industry. (Griffin, J. of Immunological Methods, 405 (2014) 35-46).