Do you know your diabodies from your zybodies?

Antibodies are a highly important class of therapeutics used to treat a range of diseases. Given their success as therapeutics, a wide variety of alternative antibody formats have been developed – these are driving the next generation of antibody therapeutics.

To note, this is not an exhaustive list but rather intended to demonstrate the range of existing antibody formats.

Inspired by this article in The Guardian: “Rachel Roddy’s A-Z of pasta“

A – Antibodies

Antibodies – a fitting place to start this post. Antibodies are proteins produced by our immune systems to detect and protect against foreign pathogens. The ability of antibodies to bind molecules strongly and specifically – properties essential to their role in our immune defence – also make them valuable candidates for therapeutics. Antibody therapies have been developed for the treatment of various diseases, including cancers and viruses, and form a market estimated at over $100 billion¹.

Antibodies are comprised of a heavy chain and a light chain, forming a Y-shaped protein. Target (antigen) binding is mediated by 3 complementarity-determining regions (CDRs) on the variable domains of each chain. Traditional antibodies are symmetric, binding the same epitope with each ‘arm’.

B – Bispecific antibodies

B is for bispecific antibodies (BsAbs), a large class of alternative antibody formats, which are receiving substantial attention and progressing in the clinical pipeline. BsAbs are able to bind to two different target sites (epitopes), either two antigens or two epitopes on one antigen. There are a large number (>60) of different formats of BsAbs², which will make up many of the entries in this post.

• Advantages compared to traditional antibodies: bispecificity confers capabilities e.g., the ability to bring two molecules or cells into close proximity
• Limitations: production can be challenging due to the heavy chain and light chain pairing problems (however, many strategies have since been developed to overcome this); depending on the format, chain pairing & orientation can be important factors impacting efficacy²

To note, bispecificity may be spatial (binding two targets at one time) or temporal (binding two targets sequentially).

C – Conjugates

A key property of antibodies – the ability to bind strongly and specifically to an antigen of interest – is exploited in antibody-drug conjugates (ADCs). ADCs are comprised of small molecule drugs chemically linked to antibodies (note the linked antibody can be of an alternative format). The antibody delivers the drug to the desired site in the body by binding its target antigen. The ADC is then taken up into the bound cell and the drug is subsequently released following the intracellular cleavage or degradation of the linker connecting the antibody and drug³.

• Advantages compared to traditional antibodies: precise targeting to reduce off-target effects³
• Limitations: linker design; the conjugated drug must have a functional group that allows it to be chemically linked to the antibody; site-specific vs. random conjugation (the latter results in heterogeneity with different drug-antibody ratios); poor penetration of solid tumors when a drug is conjugated to a traditional antibody (due to the size of the antibody; can be overcome by conjugation to e.g., nanobodies)³

As of 2019, 4 ADCs have been approved by the FDA and >60 are being tested in clinical trials³. ADCs demonstrate great promise as targeted chemotherapies, delivering cytotoxins to tumour cells via antibody targeting to tumour-surface antigens.

D – Diabodies

Diabodies are a member of the ‘mix-and-match’ trend of bispecific alternative antibody formats. They are formed by two fragments, each comprising of a heavy chain variable domain (VH) and light chain variable domain (VL) domain. The VH and VL domains are connected by a linker that is too short for domains on the same chain to be paired and, as such, the two different fragments associate with each other, creating two different antigen-binding sites⁴.

Other versions of diabodies have been engineered, for example DARTs (which have enhanced stability due to the inclusion of a disulphide bond) and TandAbs (formed from two chains, each containing two pairs of VH and VL domains)^2,5–7.

F – Fragments

A common theme across alternative antibody formats, as we will continue to see, is the use of portions, or fragments, of traditional antibodies. Fragments can be employed ‘as-is’, be strung together or be linked to additional molecules as required for a particular application. There are a range of different fragments that can be derived from an antibody. One that comes up often in this post is the single chain variable fragment (scFv), which is formed of a VH and VL domain connected by a linker. In contrast to the diabody, this linker is long enough to allow the VH and VL domains, on the same polypeptide chain, to pair⁸.

• Advantages compared to traditional antibodies: reduced size therefore better tissue/tumour penetration; cheaper to manufacture; lacking a fragment crystallisable (Fc) region and therefore do not activate the host immune system (can be beneficial or a downside, depending on the application); can be fused to other antibody formats to provide additional specificity⁸
• Limitations: reduced half-life; increased propensity for aggregation⁸

To note, there are further antibody fragments – e.g., Fab and F(ab)2 fragments, as well as Fc fragments – which have various therapeutic applications as well.

H – HSAbodies

H is for human serum albumin (HSA)-bodies. As their name would suggest, HSAbodies contain HSA, which is fused to two scFvs⁹. The HSA fusion confers improved pharmacokinetic properties (e.g., related to half-life and clearance) onto the scFvs.

I – ImmTACs

ImmTACs are employed to direct T cells to target cells, based on peptides presented by MHC complexes at the cell surface. They are formed from a scFv (that binds CD3, a T-cell co-receptor) fused to an affinity matured T-cell receptor^2,10. The latter retains the ability to bind MHC-presented peptides.

K – Knobs-into-holes

Knobs-into-holes represent another example of BsAbs: their name refers to mutations engineered to ensure correct heavy chain heterodimerisation. A “knob” mutation (inserting a large amino acid, Trp) is introduced into one heavy chain while a complementary “hole” mutation (to a small amino acid, e.g., Ser, Ala, or Val) is introduced into the other heavy chain¹¹. The “knob” makes homodimerization unfavorable, and the “knob-in-hole” combination promotes the desired heterodimerization.

L – LUZ-Y

Yet another BsAb format! LUZ-Y proteins are the result of another method for generating BsAbs, in which leucine zippers are used to promote heavy chain heterodimerisation (then subsequently removed by proteolysis)^2,12.

M – Minibodies

Minibodies incorporate the scFv fragment discussed at letter F: they are a dimer of a scFv-CH3 fusion construct¹³.

N – Nanobodies

Nanobodies are perhaps one of the better known entries in this post. They are a monomeric binder, with the same general structural architecture as human VH domains¹⁴. Despite their much smaller size and lack of light chain, they are able to bind target antigens with as high affinity as has been observed for traditional antibodies.

• Advantages compared to traditional antibodies: improved tissue/tumour penetration due to smaller size; greater water-solubility; cheaper to manufacture (can be expressed in bacterial systems, as nanobodies are monomeric and lack post-translational modifications)¹⁴
• Limitations: rapid clearance due to small size; higher risk of kidney toxicity; cannot activate host immune responses which are dependent on the presence of an Fc region¹⁴

As of 2020, 1 nanobody therapeutic has been approved by the FDA (for the treatment of thrombotic thrombocytopenic purpura) and multiple are being tested in clinical trials for the treatment of a range of diseases including cancers, rheumatoid arthritis and RSV infection¹⁴. In addition to their promise as therapeutics, nanobodies have multiple further applications, including in life sciences research (e.g., to stabilize proteins for solving structures by X-ray crystallography) and as diagnostics (e.g., medical imaging for cancer diagnosis).

To note, nanobodies can also be employed in alternative formats (e.g., bi- or tri-specific constructs comprised of different nanobodies, drug-conjugated).

O – Orthogonal Fabs

Orthogonal Fab‘s arose in response to the heavy/light chain pairing problems of designing BsAbs. Using a computational and rational design strategy, mutations were engineered into the VH/VL and CH1/CL interfaces to disfavour mispairing¹⁵.

Q – Quads

Another avenue for antibody design is to strengthen antibody-antigen interactions through increasing avidity. Avidity can be increased by increasing the number of binding sites, or the valency. This approach was taken in the design of Quads, in which antibody formats are assembled into tetramers, conferring tetra- or octa-valency¹⁶. Quad assembly is facilitated by fusing antibody formats to the self-assembling p53 tetramerization domain¹⁶.

S – SEEDbodies

SEEDbodies are asymmetric BsAbs, assembled around strand-exchange engineered domain (SEED) CH3 heterodimers, which are formed from alternating human IgG and IgA CH3 segments¹⁷.

T – Tri-specific antibodies

Expanding on BsAbs, there are also trispecific antibodies! Trispecific antibodies are able to bind to three different targets. A common application of multispecific antibodies is cancer immunotherapy, and trispecific antibodies are no exception. For example, one trispecific antibody has been developed to engage a cancer cell (via surface-expressed CD38) and activate a T cell response – the latter is achieved by the binding of two receptors on the T cell surface, CD3 (which activates the T cell) and CD28 (which induces signaling that blocks T cell death)^18,19.

V – V(H/L)-IgGs

Another ‘mix-and-match’ example, V(H)-IgG and V(L)-IgG are formed from variable fragment (Fv) domains linked to IgG molecules – at the N terminus of the IgG heavy chain and at the N terminus of the IgG light chain, respectively².

Z – Zybodies

And finally (after missing out a few letters), we have reached Z. Zybodies take the concept of multispecificity to another level. They are comprised of a traditional antibody fused to modular recognition domains (MRDs), which could be e.g., short peptides or specific domains²⁰. MRDs can be fused to the N or C terminus of the heavy or light chain, enabling up to penta-specificity – zybodies have been demonstrated to engage 5 targets at one time²⁰.

End. These examples serve to demonstrate the great diversity of antibody formats. I expect there will be entries for the missing letters very soon (if they do not exist already)!

References

1.        Grilo, A. L. & Mantalaris, A. The Increasingly Human and Profitable Monoclonal Antibody Market. Trends Biotechnol. 37, 9–16 (2019).

2.        Spiess, C., Zhai, Q. & Carter, P. J. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. 67, 95–106 (2015).

3.        Birrer, M. J., Moore, K. N., Betella, I. & Bates, R. C. Antibody-Drug Conjugate-Based Therapeutics: State of the Science. J. Natl. Cancer Inst. 111, 538–549 (2019).

4.        Holliger, P., Prospero, T. & Winter, G. ‘Diabodies’: Small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. U. S. A. 90, 6444–6448 (1993).

5.        Johnson, S. et al. Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion. J. Mol. Biol. 399, 436–449 (2010).

6.        Arndt, M. A. E., Krauss, J., Kipriyanov, S. M., Pfreundschuh, M. & Little, M. A Bispecific Diabody That Mediates Natural Killer Cell Cytotoxicity Against Xenotransplantated Human Hodgkin’s Tumors. 94, 2562–2568 (1999).

7.        Kipriyanov, S. M. et al. Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J. Mol. Biol. 293, 41–56 (1999).

8.        Bates, A. & Power, C. A. David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8, (2019).

9.        http://commercial.cancerresearchuk.org/sites/default/files/NT_CEA%20Antibodies_November%202014_FV.pdf

10.      Oates, J. & Jakobsen, B. K. ImmTACs: Novel bi-specific agents for targeted cancer therapy. Oncoimmunology 2, 20–23 (2013).

11.      Ridgway, J. B. B., Presta, L. G. & Carter, P. ‘Knobs-into-holes’ engineering of antibody C(H)3 domains for heavy chain heterodimerization. Protein Eng. 9, 617–621 (1996).

12.      Wranik, B. J. et al. LUZ-Y, a novel platform for the mammalian cell production of full-length IgG-bispecific antibodies. J. Biol. Chem. 287, 43331–43339 (2012).

13.      Hu, S. Z. et al. Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res. 56, 3055–3061 (1996).

14.      Jovčevska, I. & Muyldermans, S. The Therapeutic Potential of Nanobodies. BioDrugs 34, 11–26 (2020).

15.      Lewis, S. M. et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 32, 191–198 (2014).

16.      Miller, A., Carr, S., Rabbitts, T. & Ali, H. Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats. MAbs 12, 1–11 (2020).

17.      Davis, J. H. et al. SEEDbodies: Fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng. Des. Sel. 23, 195–202 (2010).

18.      Garfall, A. L. & June, C. H. Three is a charm for an antibody to fight cancer. Nature 575, 450–451 (2019).

19.      Wu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat. Cancer 1, 86–98 (2020).

20.      LaFleur, D. W. et al. Monoclonal antibody therapeutics with up to five specificities Functional enhancement through fusion of target-specific peptides. MAbs 5, 208–218 (2013).