Expanding nucleic acid-encoded medicine

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The US Food and Drug Administration (FDA) has approved two mRNA vaccines from Moderna and Pfizer-BioNTech against SARS-CoV-2. The success of mRNA vaccines paves the way for a new class of biologics made from protein antigens encoded by nucleic acids. Direct intramuscular or intradermal introduction of a plasmid containing the protein-coding DNA sequence allows for the in situ production of target proteins. Thus, non-viral plasmid DNA and mRNA vectors turn the patient’s body into a biological factory, eliminating many steps of biologics production and providing a promising biologics delivery strategy. These biologics can be proteins, peptides, or antibodies. In this issue of Molecular Therapy, Helble et al. engineer SARS-CoV-2 monoclonal antibody (MAb) 2196 encoded by DNA (DMAb-2196) and delivered via electroporation.¹ By employing a chain-swap methodology, they aim to identify features that enhance expression through rational design and structural modeling. The development of an antibody frequency score (AFS) based on natural antibody sequence variations represents an innovative approach for achieving higher expression of antibody mutants. Their findings demonstrate that even a single amino acid change can significantly increase in vivo expression of protein payloads, paving the way for more effective DMAb therapies.

The development of MAbs has revolutionized modern medicine, with the global antibody therapeutics market valued at over $200 billion. Despite their therapeutic potential, the production, storage, and distribution of recombinant antibodies present significant challenges. Developing biosimilars to antibody drugs is also significantly more expensive than generics based on small molecules. The advent of DNA-encoded delivery platforms promises to overcome these hurdles by enabling in vivo expression of antibodies directly within the host. DMAb offers a promising alternative by bypassing the need for extensive antibody purification and quality control. DMAb not only simplifies the production process but also potentially ensures sustained antibody secretion for varying durations. A major limitation of in vivo nucleic acid-encoded antibody delivery is achieving sufficient circulating antibody levels to ensure therapeutic efficacy, as antibody bioavailability must be maintained above the required threshold to exert its therapeutic effect. Thus, improvements in expression levels are pivotal to the therapeutic efficacy. The study by Helble et al. addresses this challenge using protein engineering to enhance in vivo expression. The pipeline involves sequence optimization and a unique antibody chain-swapping method. By selectively mismatching antibody variable heavy (V_H) and variable light (V_L) chains, the authors hypothesize that alternative pairings might yield better expression profiles. They identify sequence features and motifs that modulate antibody expression, which are then sourced to develop the AFS. The AFS, derived from analyzing millions of V_H/V_L sequences, helps find mutations that can improve in vivo expression. The application of AFS to DMAb-2196 demonstrates its potential for significantly enhancing antibody expression. Single and combination mutations identified through AFS enhance in vivo expression up to 2-fold over the wild-type antibody, without affecting binding affinity. These findings highlight the importance of V_H/V_L chain swapping and AFS to advance the field of DMAb therapeutics.

The transfer of DNA-encoded protein cargoes has been attempted since the 1990s, but currently, no FDA-approved peptides, proteins, or antibodies are delivered by DNA plasmids for human use. Multiple classes of biologics can be delivered by DNA plasmids in vivo, with antibodies being the most challenging (Figure 1). Pemivibart, an MAb medication authorized for pre-exposure prophylaxis against SARS-CoV-2, is used intravenously with doses of 4,500 mg.² The recommended intramuscular dose for tixagevimab (the parental antibody to DMAb-2196) is 300 mg, along with cilgavimab (300 mg). To deliver DMAb-2196, 50 μg DNA per mouse was used. Immune checkpoint blockade (ICB) antibodies are used intravenously with doses of 200 mg (pembrolizumab) or 840 mg (atezolizumab). DNA plasmids encoding antibodies targeting p53 mutants R175H and E285K are delivered intramuscularly using electroporation at 200 μg³ or intratumorally using lipid nanoparticles (LNPs) at 40 μg in mouse xenograft tumor models.⁴ For neoantigen-based cancer vaccines, the dose for each synthetic long peptide (SLP) is 300 μg⁵; the mRNA dose is 25 μg (BNT122)⁶ or 1,000 μg (V940)⁷; the DNA dose is 1,000 μg (GNOS-PV02).⁸ For SARS-CoV-2 vaccines, the protein dose in Nuvaxovid is 5 μg; the mRNA dose is 30 μg (Comirnaty) or 100 μg (Spikevax). Adjuvants are needed for vaccines based on neoantigens and protein antigens, and ICB is needed for cancer therapies. Peptide hormones can be expressed using DNA plasmids, particularly for those with no atypical amino acid residues, and covalently linked to an Fc (fragment crystallizable) of an antibody. For instance, dulaglutide is a glucagon-like peptide-1 receptor agonist (GLP-1 agonist) consisting of GLP-1 (7-37) fused to the human immunoglobulin G4 Fc.⁹ The typical starting dosage of dulaglutide is 750 μg injected subcutaneously. The latter three classes of biologics are “low-hanging fruit” for DNA delivery, as the projected protein doses are less than 1,000 μg per delivery.

Figure 1.

Biologic classes can be encoded by DNA plasmids

These biologics include monoclonal antibodies, neoantigens, protein antigens, and peptide hormones. LNPs in mRNA vaccines against SARS-CoV-2 have immunostimulant activities; Nuvaxovid includes a saponin-based adjuvant named Matrix-M (purple). In addition to immunostimulants/adjuvants (LNPs for mRNA; interleukin-12 for DNA; polyinosinic-polycytidylic acid stabilized with lysine and carboxymethylcellulose for SLP), antibodies targeting either programmed cell death protein-1 or programmed death ligand-1 are adjunctive to cancer vaccines based on neoantigens (red). Created in BioRender.com.

The rapid development of anti-SARS-CoV-2 mRNA vaccines during the pandemic underscores the transformative impact of nucleic acid-encoded medicine. However, the development process with mRNA production and LNP formulation is fraught with complexities, including manufacturing, biochemical and biophysical analyses, and quality control assessments. The study by Helble et al. presents a promising DNA-based approach overcoming the limitations of in vivo mRNA-encoded antibody delivery and pushes the boundaries of DMAb and other DNA-encoded medicine. The continued exploration and optimization of protein designs, plasmid vectors, and delivery by electroporation or LNPs for the highest expression possible will be crucial for the widespread adoption and success of DNA-encoded biologics in clinical settings.

Declaration of interests

The authors declare no competing interests.