Whitney Shatz

Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies

By enabling the simultaneous engagement of two distinct targets, bispecific antibodies broaden the potential utility of antibody-based therapies. However, bispecific-antibody design and production remain challenging, owing to the need to incorporate two distinct heavy and light chain pairs while maintaining natural nonimmunogenic antibody architecture. Here we present a bispecific-antibody production strategy that relies on co-culture of two bacterial strains, each expressing a half-antibody. Using this approach, we produce 28 unique bispecific antibodies. A bispecific antibody against the receptor tyrosine kinases MET and EGFR binds both targets monovalently, inhibits their signaling, and suppresses MET and EGFR-driven cell and tumor growth. Our strategy allows rapid generation of bispecific antibodies from any two existing antibodies and yields milligram to gram quantities of bispecific antibodies sufficient for a wide range of discovery and preclinical applications.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines

Background: Dual neutralization of IL-4 and IL-13 is a promising therapeutic approach for asthma and allergy. Results: Knobs-into-holes IgG1 and IgG4 bispecific antibodies targeting both cytokines were developed. Conclusion: Bispecific antibodies of both isotypes have comparable in vitro potencies, in vivo pharmacokinetics, and lung partitioning. Significance: Further extension of knobs-into-holes technology to human IgG4 isotype as reported here provides greater options for therapeutics. Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies.1,2 The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture

ABSTRACT Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho β that is being developed for type 2 diabetes and other obesity-linked disorders.

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

ABSTRACT To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.