Romain Rouet

General strategy for the generation of human antibody variable domains with increased aggregation resistance

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (VH and VL) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30–33, 35 in VH and 24, 49–53, 56 in VL). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (VH3 and Vκ1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant VH and VL domains revealed a surprising degree of structural conservation, indicating compatibility with VH/VL pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.

Aggregation, stability, and formulation of human antibody therapeutics

Many human monoclonal antibodies display poor biophysical properties, such as low stability and a propensity to aggregate. These unfavorable tendencies can be even more pronounced for human antibody fragments, which often require a considerable degree of optimization. In this review, we describe methods for analyzing aggregation and stability of human antibodies and antibody fragments. We also provide an overview of recent approaches to improve these properties through engineering and formulation.

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity.

Significance Antibodies are selected to bind microbial but not self-antigens, because binding to self would compete with binding microbes, shorten antibody half-life, and cause autoimmunity. Self-tolerance is actively acquired in part by discarding self-binding antibodies before the body is exposed to a microbe or vaccine. The experiments here provide evidence of an opposite mechanism, allowing antibodies that initially bind both foreign and self-antigens to acquire self/non-self discrimination during the course of an immune response through somatic hypermutation away from self-reactivity. In addition to selection for lower-affinity binding to self, antibody variants were selected with fewer binding sites available to bind self-antigen because most were occupied by N-linked carbohydrate, possibly explaining the frequent occurrence of N-linked glycosylation of antibody variable domains. The best-understood mechanisms for achieving antibody self/non-self discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgMlow IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgMlow IgD+ B cells form twice as many GC progeny as naïve IgMhi IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.

Stability engineering of the human antibody repertoire

Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This behavior hinders their production, and places limitations on the development of novel formulation conditions and therapeutic applications. Antibodies are highly diverse molecules, with much of the sequence variation observed within variable domain families and, in particular, their complementarity determining regions. This has complicated the development of comprehensive strategies for the stability engineering of the human antibody repertoire. Here we provide an overview of the field, and discuss recent advances in the development of robust and aggregation resistant antibody therapeutics.

Expression of high-affinity human antibody fragments in bacteria

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.Cpf1 is a promising addition to the CRISPR toolkit but displays wide variability of activity in different eurkaryotes. Here the authors identify temperature as a modulator of activity and use this to efficiently edit ectothermic vertebrate species.

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

Background: Camelid antibody domains are naturally stable and capable of cleft binding. Results: Protein engineering can endow human antibody domains with such properties. Conclusion: Our strategy does not require undesirable antibody framework changes. Significance: Robust building blocks for human therapeutic applications. Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.

I-motif DNA structures are formed in the nuclei of human cells

Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.I-motif DNA structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Now an engineered antibody that is selective for i-motif structures has been developed and used to detect i-motifs in the nuclei of human cells.

Structural basis for epitope masking and strain specificity of a conserved epitope in an intrinsically disordered malaria vaccine candidate

Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.

Bispecific antibodies with native chain structure

Two new methods overcome a major hurdle that blocked easy and efficient design and expression of human bispecific antibodies.