Vaughn V. Smider

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2+ cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants.

X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.

Protein evolution with an expanded genetic code

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

Synthesis of bispecific antibodies using genetically encoded unnatural amino acids.

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2+ cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2+ cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.

Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen–antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.

Selective Formation of Covalent Protein Heterodimers with an Unnatural Amino Acid

We report a strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here, we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical, and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.

Bispecific small molecule–antibody conjugate targeting prostate cancer

Significance We have developed a semisynthetic method for the production of bispecific antibody-like therapeutics consisting of a small molecule targeting moiety conjugated to an antibody. A highly selective prostate-specific membrane antigen-binding ligand was site specifically conjugated to a mutant α cluster of differentiation 3 (αCD3) Fab containing an unnatural amino acid with orthogonal chemical reactivity. The optimized conjugate showed potent in vitro activity, good serum half-life, and potent in vivo activity in prostate cancer xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors. Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ∼100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

Immunochemical termination of self-tolerance

The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr86) of murine tumor necrosis factor-α (mTNF-α) to p-nitrophenylalanine (pNO2Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr86 → Phe mutant. The antibodies generated against the pNO2Phe are highly cross-reactive with native mTNF-α and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.