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Definitions:

VHH refers to an antibody single variable domain (VH or VL) polypeptide that specifically binds antigen. While the antigen binding unit of a naturally occuring antibody in humans and most other mammals is generally known to be comprised of a pair of V regions (VL/VH), camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain sequences. (see Hamers-Casterman, Nature, 363, 1993). 

VHHs were discovered in 1993 at the University of Brussels. Only a fraction of the immune repertoire of the animals of the camelid family comprise the heavy chain antibodies. The other fraction are normal classical antibodies. The heavy chains of these so called VHH bind their antigen by one single domains, the variable domain of the heavy immunoglobuilin chain, referred to as VHH. With a MW of about 12 kDa, the VHH domain is the smallest known intact binding fragment dervied from a funcitonal immunoglobuilin.

antibody single domain or single domain antibody refers to antibody fragments that include either a VH or VL domain of antibody, that is, a single monomeric variable antibody domain. Unlike whole antibodies, single domains do not exhibit complement system triggered cytotoxicity, as they lack an Fc region. Antibody single domains often have peptide chains of about 110 amino acids and MW in the range of 12-15 kDa. They are much smaller than intact antibodies which generally are about 150-160 kDa, being composed of two heavy chains and two light chains. Antibody single domains are also smaller than Fab fragments (about 50 kDa; one light chain and half a heavy chain) and single-chain variable fragments (about 25 kDa; a light chain variable domain and a heavy chain variable domain). The small size of antibody single domains allows better solubility and better permeability in tissues, as well as greater heat-resistance and stability towards detergents and urea. Small size often leaves also to a short serum half-life, as smaller molecuels are eliminated renally more readily. Nonetheless, small size facilitates binding to hidden epitopes that may not be accessible to intact antibodies or larger antibody fragments. (Castanheira, WO 2013/043070). 

A dAb is either the variable domain of an antibody heavy cahin (VH domain) or teh variable domain of an antibody light chain (VL domain) each dAb therefore contains three of the six natturally occurring complementary determining regions (CDRs) from an antibody. Although it might seem surprising that three CDR regions are sufficient to confer antigen-binding specificity and high affinity, Darwinian evolution has itself arrived at that very solution in camelids, which product antibodies including only a heavy chain. The antigen ginding site of these antibodies consists of a single unpaired varable domain (referred to as VHH). Holt, “Domain antibodies: proteins for therapy” TRENDS Biotech. 21(11), 2003).

Types of Domain Antibodies: VHH/Domain antibodies (dAb) (also known as “domain antibody” or “single domain antiboy” or “single variable domain” or “immunogloublin single variable domain or dAb or nanobody®)

Single domain (SdAb): One class of antibodies are single domain antibodies (or “nanobodies”). Unlike standard human antibodies which are exclusively expressed as paired species containing both heavy and light chains, single domain antibodies contain either only the H chain variable region or only the L chain variable region, while preserving the antigne-binding properties of conventional antibodies. They are able to demonstrate high specificity and affinity similar to standard IgG antibodies but are much smaller in size. which is useful in a number of appliations such as biosensor, diagnostic and therapeutic applications. Although single domain antibodies exist as monomers, they can dimerize to form dimers. For example, light chain dimers known as Bence-Jones proteins have previously been described. (Christei (US 16/497,788, published as US 20200033363). See also particular types of antibodies purified in outline 

The single antigen domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies which need to interact with another variable domain (.e.g. through a VH/VL interaction to form a functional antigen binding domain. Domain antibodies will often comprise at least one intradomain disulfide bridge between cysteine 22 and cysteine 92. Sometimes, nanobodies comprises a disulfide bond between CDR3 and Cys45 in the framework region FR2. There may also be a disulfide bond between CDR2 and CDR3 for example between cystein 45 and CDR3 or between cysteine 50 and CDR3. (Schotte US13/266503 and US2012/0157664)

Camedlid VHH antibodies:

In 1993 Hamers-Casterman discovered a novel class of IgG antibodies in Camelidae (camels, dromedaries and LLamas). These antibodies are devoid of light chains and thus called “heavy-chain” IgGs or HCab. They have a MW of about 95 kDa instead of the about 160kDa for conventional IgG antibodies. Their binding domains consist only of the heavy-hain variable domains. Since the first constant domain (H1) is absent (spliced out during mRNA processing due to loss of a splice consensus signal, the variable domain (VHH) is immediately followed by the hinge region, the CH2 and CH3 domains. Although HCAbs are devoid of light chains, they have an authentic antigen binding repertoire.(Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15).

Due to structural differences compared to VHs of normal heterotetrameric IgGs, VH domains of the heavy chain dimer IgGs are called variable domain of the heavy-chain of heavy chain antibody (VHH). (Honda, US 2005/0037421).

The crystal and solution structures of several dAbs have been solved and show that isolated human VH and camelid VHH dAbs adopt a beta-sheet structure similar to a VH or VL immunoglobuilin fold in a conventional antibody. By contrast, some of the CDR structures seen in camel VHH dAbs are different fom those in naturally paired VH domains. A comparison of the structures of camelid CDRH1 and CDRH2 with the set of loops found in human and mouse antibodies has revealed significant differences in their main chain conformations. In addition, the CDRH3 region of naturally occurring camelid VHH antibodies are, on average, longer than the CDRH3 region s of naturally paired VH domains. Camelid VHH sequences also contain amino acid substitutiosn in their framework regions taht are not observed in paired VH domains and which might have evolved to accommodate the absence of the VL and constant CH1 domains. Holt, “Domain antibodies: proteins for therapy” TRENDS Biotech. 21(11), 2003).

Diversity of Single Domain Antibodies

Single domain antibodies interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH-VL combinatorial diversity, these heavy chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions (Muyldermans “Single domain camel antibodies: current status” Reviews in Mol. Biotech., 74 (2001) 277-302).

Analysis of the differences in amino acid sequence between the VHs of these camel heavy chain only antibodies and the VH domains form conventional human antibodies has been used to design an altered human VH domain. This “camelized VH” is a small and efficient recognition unit (“Riechmann “Single domain antibodies: comparison of camel VH and camelised human VH domains” J. Immunol. Methods, 231 (1999), 25-38).

The VH germline segments are highly diveeast of the single domain was at least fivefold higher than the scFv, and the ability of the single domain intraody to inhibit huntington aggregation, which has been implicated in the pathogenesis of Huntington’s disease was confiremd in a cell free in vitro assay as well as in a mammalian cell culture model of HD. ytoplasmic expression levels in y of this variable L chain doamin and it was found to retain full binding activity to huntingtin. Crse, leading to a broad structural repertoire of the antigen-binding loops. Seven VHH subfamilies are known, of which five have been confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrated that the rearranged VHHs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and high incidence of nucleotide inertions or deletions. These diversificaiton processes are driven by hypermutation and recombination hotspots embedded in the VHH germline genes at the regions affecting the structure of the antigen-binding loops. (Nguyen, EMBO Journal, 19(5), 2000, pp. 921-930)

The diversity of antibody repertoire of camelids is determeined by the CDR1-3 in the VH or VHH regions. The CDR3 in the camel VHH region is characterized by its relatively long lenght aeveraging 16 amino acids. For example, compared to the CDR3 of mouse VH having an average of 9 amino acids, the CDR3 of camel IGG is very long. (Honda US 2005/0037421).

The simpler structure of camelid VHH does not decrease their affinity or specificty due to the presence of an extremely variable CDR (Victor de Lrenzo Prieto, US2008/0280346).

Advantages of Single Domain Antibodies:

Advantages of VL:

Colby (J. Mol. Biol. 2004, 342, 901-912) discloses using yeast surface display to affinity mature an scFv pool against huntington form a non-immune human antibody library. Colby then analyzed the location of the binding site of the mutant with the highest affinity and discovered that the paratope was mapped exclusively to the light chain domain of the scFv. A single domain antibody was then constructed consisting soley of this variable light chain domain and was found to retain full binding activity to huntingtin. A potential advantage of using single-domain intrabodies rather than scFv for treatment of HD via gene therapy arises form the redued mass of the smaller intrabody. A hallmark of HD is the formation of intranuclear inclusions, so the ability of an intrabody to diffuse into the nuecleus an d and prevetn aggregation may be critical. While scFvs, with a MM of 25-30 kDa are close to the MM cutt-off of nuclear pores, single domain intrabodies are only half that size and should more readily diffuse into the nuclear subcompartment. 

Advantages of VHHs

Since the VH region of a heavy chain dimer IgG does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is mutated to hydrophilic amino acids residues. As a result, VHH have exellent solubility. Furthermore, VHHs derived form acmels and LLmas have very high thermostability compared to mouse heterotetrameric antibodies. The use of VHH derived form these species can provide, for example, molecles that maintain their antigen binding ability even at 90C. (Honda US 2005/0037421).

Single Domain Antibodies as Multimers (e.g., Dimers)

(Castanheira, WO 2013/043070) discloses more than one single dimain of an immunoglobulin as a dimer, trimer, tetramer, etc such as an anti-TNF alpha polypeptide that includes two antibody single domains in the form of a dimer. Dimer formats include, e.g., VH-VH dimers, VL-VL dimers, and VH-VL dimers. VL single dimains that showed particularly high affintiy bidning to human TNF alpha were sequenced. Two VL domains showing high affintiy binding were selected for combination as dimers. As a spacer between the two VL domains, either a 8 amino acid GlySer linker, 13 amino acid GlySer linker or 18 amino acid GlySer linker was used. Dimer combinations were cloned and expressed and purified. 

VL-VL Dimers:

It has been shwon that free immunoglobulin light chains assocaite as dimers, thus burying the hydrophobic surface that are normally involved in the interactions with the heavy chain. (Nymalm, J Structural Biology 138 (2002) 171-186). An unusual mode of domain-domain association is the VL-VL interaction, found in most cases of Bence-Jones disease and many myelomas lacking the H chain. This closely mimics the interace geometry of VL/VH, although its ontact area is 18% less. (Burrone, J. Mol. Biol. (2003), 333, 355-365). 

Christ (US 16/497,788, published as US 20200033363) discloses immunoglobulin homodimers that include identical light chain variable region (VL) monomers able to bind to an antigen. The single domain immunoglobulin homodimer is produce using V and J segments of the human antibody light chain family which are amplified by PCR and cloned into the phage display vector pHEN1. V segment diversity was introduced at CDR1 and CDR2 positions while J segment diversity was introduced by PCR using degenerate TRIM oligonucleotides. Combinatorial diversity was generated through recombination of the V and J segments using splice overlap extension PCR followed by cloning into display vector and transformation into E coli. This resulted in a library of phage cosntructs with each bacteriophage displaying multiple copies of the receptor on the tip of its filamentous surface. These multiple copies of each receptor were found to be able to dimerize, for example, upon binding an antigen. The phage library was for example panned against hen egg-white lysozyme (HEL) which had been biotinylated and the HEL antigen was captured using nuetravidin coated wells. For rounds 2 and 4 of the selection, amgnetic streptavidin beads were used as an alternative means of capture to prevent the selection of binder against the capture reagents. After four rounds of selection, binders were identified by soluble fragment ELISA. This selection step allowed the identification of antigen specific phage clones. Regions encoding single-domain immunoglobulin homodimer from selected clones were amplified by PCR and subsequently cloned into the periplasmic expression vector which were transformed into E. coli. The single domain homidmers were purified using Protein L affintiy resin. Next, a dimierization screening step using size exclusion chromatography coupled with multi-angle laser lgiht scattering (SECMALLS) was used to measure the MW of the immunoglboullin-antigen complex. By measuring the MW of the immunoglobulin-antigen complex, immunoglobulin alone and antigen alone, a stoichiometry of immunoglobulin-antigen complext was found to be 2:1. The measured MW were consistent with two Ig domains bound to a single HEL molecule in the immunoglobulin-antigen complex. 

VH-VH Dimers:

Functional VH dimers selected from phage display libraries can be secreted efficiently from both bacteria and mammalian cells in different formats. (Burrone, J. Mol. Biol. (2003), 333, 355-365).

The novel iimmunoglobulin isotype novel antigen recetpor (IgNAR) found in cartilagenous fish is composed of a heavy cahin homodimer that does not assocaite with light chains. The variable regions of igNAR function as independent domains similar to those found in the heavy chain immunoglobulins of Camelids. (Dooley, Molecular Immunogloby 40, 2003, 25-33). 

Isolation/Purification (See also purification of antibodies)

The variable fragments of heavy chains of immunoglobulins devoid of light chains can be prepared starting from immunoglobulins obtainable by purification from serum of camelids according to the process for the purification as described in WO94/04678.