The therapeutic potential of monoclonal antibodies (mAbs) has revolutionized modern medicine, laying the foundation for molecularly-based therapeutics. The hybridoma technique had expedited the development of the monoclonal antibody, paving way for an important class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Licensed monoclonal antibodies available in the market today are typically of the immunoglobulin G (IgG) subclass. Chimeric, humanised and fully human versions of IgG monoclonal antibodies (mAbs) can now be obtained by recombinant engineering and has become the most rapidly growing class of biopharmaceuticals. Clearly, the emergence of therapeutic antibodies presents a number of advantages.

1. Most therapeutic antibodies are humanized or human IgGs and their derivatives, which are well tolerated by the host.
2. Therapeutic antibodies bind specifically to target epitopes and interact with effector arms of the immune system to deliver a precise therapeutic action with limited off-target toxicities.
3. Therapeutic antibodies have long serum half-lives with both intra- and extra-vascular biodistribution

The modular nature of antibodies combined with the advent of novel recombinant technologies enables remarkable diversity and flexibility in the production of mAbs suitable for therapeutics. It is now possible to construct mAb formats with predetermined properties on a large scale. The current spectrum of therapeutic mAb formats can be divided into naturally occurring antibody formats and antibody fragments.

Therapeutic antibodies with naturally occurring antibody isotypes

The naturally occurring human immunoglobulins (Ig) or antibody isotypes that are suitable for therapeutics include IgG (IgG1, IgG2, IgG3 and IgG4), IgM and IgA (with IgA1 and IgA2). Presently the entire class of clinical-grade therapeutic antibodies are engineered recombinant IgG monoclonal antibodies (mAbs) and possess the same basic structure. The isotypes, IgG1 and IgG3 elicit effector mechanisms such as antibody‐dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement‐dependent cytotoxicity (CDC), whereas IgG2 and IgG4 trigger a more subtle response, and only in certain cases. Currently, IgG1 predominantly make up the marketed monoclonal antibodies, followed by IgG2 and IgG4.

Factors such as effector functions and structural stability, along with prior experience and availability of a particular IgG subclass in a company’s development portfolio determine preference for one IgG class over the other.  Some examples for IgG1 FDA approved monoclonal therapeutic antibodies are Adalimumab, Bevacizumab and Daratumumab.  Apart from IgGs, the high avidity of IgM and its effective complement activation and agglutination make IgM a potential candidate for future immunotherapy. Human IgM, HA-1A (Centoxin) is in phase 3 clinical trials to treat sepsis. While other human IgMs such as Mab 16.88, PAT-SC1, L612, rHIgM22 are in phase I for the treatment of colorectal cancer, gastric cancer, melanoma and multiple sclerosis, respectively. Also, more evidence suggests the role IgM NAbs in treating neurological disorders as they recognize lipid components of neural membranes. Interestingly, human IgAs might be used as a therapeutic antibody and have demonstrated positive signs in therapy for infectious diseases, inflammatory diseases, anti-tumor and in replacement therapy. But, as of now, none have advanced to the clinical trials.

Surprisingly, monoclonal antibodies from sharks, camels, llamas have also shown potential diagnostic and therapeutic values. All these animals produce unusual, diminutive antibodies that have a single antigen-binding domain and are only about half the size of the conventional monoclonal antibodies (i.e., 12-15 kDa as compared to 150 kDa of IgGs). The small antibodies produced by sharks and the camel family differ from the conventional versions of monoclonal antibodies not only in size but also in their structure and binding ability (see Figure).  This discovery of antibody isotypes in other animals paved the way to a novel class of therapeutics called the nanobody (Nbs) to treat various indications. Caplacizumab, a nanobody was developed, inspired from the monoclonal antibodies present in Camelidae and has been approved for treating acquired thrombotic thrombocytopenic purpura (aTTP), in conjunction with plasma exchange and immunosuppression.

Engineered Therapeutic Antibody Fragments

A growing trend in the vast number of formats of antibody fragments indicates a huge market potential for Ab fragments in the field of mAb therapeutics in the upcoming years. IgG derivatives such as fragment antigen-binding (Fab), the single-chain fragment variable (scFv), single-domain antibodies, and the fragment crystallizable (Fc) domains are now being exploited for therapeutic applications (Figure). Antibody fragments are designed to retain the antigen-binding domains and have low molecular weights compared to whole IgG formats, thus offering a couple of advantages.

1. Different Ab fragments can be linked to generate molecules of varying specificities, valencies and functionalities to achieve a broad range of biological effects. For example, bispecific antibody formats such as tandem scFvs are generated by linking two scFv fragments with a helical linker to target two antigens simultaneously. Similarly, more compact formats with higher stabilities have been designed, such as Diabodies, DART®s and TandAbs, that have modifications in arrangement and linker chemistry. Several other multispecific and multivalent formats including Fv-Fc fusions and F(ab’)₂ are also under development for clinical therapy (Figure and Table). Out of these, immune cell engager bispecific antibodies, such as BiTEs (bispecific T-cell engager) and ICEs (innate cell engagers) are of special interest in cancer immunotherapy. Read more: Bispecific Antibodies: Coming-of-age in Antibody Therapeutics: Part 1 and Part 2
2. Lower immunogenicity and no risk of unwanted immune activation and antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).
3. Small size improves tissue penetration and allows access to challenging, cryptic epitopes
4.  Ease of manufacture as they can be generated using microbial expression systems, along with higher yields and lower and lower production costs

However, on the downside, antibody fragments have lower thermostability than their parent mAbs and can aggregate leading to a risk of immunogenicity. The newer generations of antibody fragments use improved linker technology to overcome these shortcomings. Also, mAb-fragment drugs will require a higher or a more frequent dosing regimen as their small size will lead to rapid renal clearance. This can be overcome by the addition of moieties such as polyethene glycol or albumin that can increase the serum half-life.

Conclusion

The diversity of antibody formats has definitely broadened the therapeutic index of therapeutic monoclonal antibodies and have also expanded the scope of biopharmaceutical applications. The development of bispecific antibodies and other multispecific/multivalent antibody formats can now be used to target inaccessible proteins that were earlier thought to be impossible. For example, multi-specific antibody fragments have been designed that can cross the blood-brain barrier (BBB). Similarly, a fusion of antibody fragments to cell-penetrating peptides (CPPs) has shown the ability to target intracellular proteins. This shows great promise not only for therapeutic applications in oncology and auto-immune diseases but also neurodegenerative diseases, infections and anti-venom treatments.

Evidentic provides therapeutic clinical-grade aliquots of EU-licenced commercial drugs as aliquots. These can be used to benchmark quality characterization of whole IgG, Fv, Fab and Fc domains for the development of new antibody formats.

Read more: Monoclonal Antibody Therapy

References:

• Bates A, Power CA. David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies (Basel). 2019;8(2):28. Published 2019 Apr 9. doi: 3390/antib8020028

• Bannas P, Hambach J, Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol. 2017;8:1603. Published 2017 Nov 22. doi: 10.3389/fimmu.2017.01603

• Xu X, Ng SM, Hassouna E, Warrington A, Oh SH, Rodriguez M. Human-derived natural antibodies: biomarkers and potential therapeutics. Future Neurol. 2015;10(1):25-39. doi 2217/fnl.14.62