Fab-drug Conjugates Development

For antibody-conjugated drug delivery system, the smaller antigen-binding fragments Fab are preferred because of their relatively high penetration speed into solid-tumor tissue. Armed with decades of working experience in the generation of antibody-drug conjugate (ADC), Creative Biolabs provides customized Fab fragment-drug conjugates (Fab-based FDCs) development services for worldwide clients. Our approaches enable you to make informed decisions on all components of the FDC structure so that the optimal candidate is developed for your target of interest in terms of stability and efficacy. Once the optimized FDC has been generated, the chosen candidate can be seamlessly transferred into our manufacturing stream, allowing the accelerated production of small batches of non-GMP FDCs.

Fab Fragments

Fab fragments comprise the constant and variable domains of immunoglobulin (Ig), linked by a single disulfide bond present at the C-terminus. A popular route to Fab fragments is the proteolytic digestion of Ig. By carefully altering experimental parameters, it is possible to produce either Fab or F(ab)₂ fragments from a variety of IgG scaffolds. Popular enzyme choices include pepsin, papain, and ficin. However, this method only applies to the circumstance where yields are not a priority. For extra functionality and complexity, it is better to engineer and express Fab fragments by various expression systems. Mammalian cells are the preferred expression system for the protein of this size (∼50 kDa) and complexity. The choice of approach should be determined on a case-by-case basis.

Fab-based FDCs Examples

There have been many reports about successful Fab-based FDCs generation. For example, an anti-CD20 Fab with two residual hinge thiols was conjugated to produce a homogenous FDC with a drug to antibody ratio (DAR) of 2 using doxorubicin containing sizable (2 kDa) PEG chains. Using a current-generation payload, scientists reported an anti-HER2 trastuzumab Fab conjugated to monomethyl auristatin E (MMAE) via bis-sulfone disulfide bridging chemistry. Developing a protein-A based solid-phase conjugation method, scientists produced another trastuzumab Fab drug conjugate, dual labeled with a cleavable and non-cleavable auristatin-based payload (DAR 3) with an in vitro potency of 0.7-0.9 nM. The most potent Fab-based FDC to date, with an EC50 of ~100 pM acts as a bispecific antibody re-targeting cytotoxic T-cells (shown in Figure 1). The half-life of the FDC was extended to around 5 times the typical 1 h half-life of the unmodified Fab, resulting in a good pharmacokinetics (PK) profile.

Fig.1 Structure of p-acetylphenylalnine (pAcF) and oxime ligation of pAcF containing αCD3 Fab with P-linkers. (Kim, 2013)

Conjugation Technologies

There are a range of Fab drug conjugation technologies available with each having their own benefits, though no single option is optimal for every payload and antibody. Fab fragments present multiple native sites for payload conjugation. Recent techniques for modifying Fab fragments target the disulfide bond present at the C-terminus of the fragment. Besides, engineered approaches such as introduction of a cysteine residue at a specific location are also available. Creative Biolabs offers access to a variety of conjugation technologies for evaluation.

Cysteine Conjugation

Cysteine conjugation following the reduction of the single disulfide bond present at the C-terminus of Fab fragment is a widely used strategy for preparing Fab-based FDCs. Various rebridging methods have been developed for the generation of homogenous Fab-based FDC. Creative Biolabs has extensive experience in the use of maleimides and other commonly used conjugation technologies for conjugation to cysteines. For more information about rebridged Fab-based FDCs, please refer to Fab-drug conjugation by rebridging methods.

Lysine Conjugation

Lysine-based conjugation is one of the most widely used non-specific conjugation strategies. The side-chain amino groups of lysine residues are good nucleophiles and can be used as sites for drug conjugation. Creative Biolabs has extensive experience in producing Fab-based FDCs using the lysine conjugation approach.

Tyrosine Conjugation

Comparing to the above two conjugation approaches, surface accessible tyrosine residues are rare and their phenolic hydroxyl groups are chemically active to serve as conjugation sites. Creative Biolabs also offers a sophisticated novel strategy to conjugate toxins to Fab via the native amino acid tyrosine.

Other Approaches

Besides, Creative Biolabs also supplies a range of Fab modification methods for the development of Fab-based FDCs. These include standard and novel chemical modifications, the incorporation of unnatural amino acids, enzymatic approaches such as transglutaminase-based methods.

With expertise and dedication, advanced FDC design platforms and services from Creative Biolabs will be your best companion in creating customized FDCs. Please contact us for more information and a detailed quote.

Related Services

• ScFv-drug Conjugates Development
• SIP-drug Conjugates Development
• sdAb-drug Conjugates Development
• Scaffold-drug Conjugates Development

Reference

1. Kim, C. H.; et al. Bispecific small molecule-antibody conjugate targeting prostate cancer. Proceedings of the National Academy of Sciences. 2013, 110(44): 17796-17801.

For Research Use Only. NOT FOR CLINICAL USE.