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.
Clonal anergy is an enigmatic self-tolerance mechanism because no apparent purpose is served by retaining functionally silenced B cells bearing autoantibodies. Human autoantibodies with IGHV4-34*01 ...heavy chains bind to poly-N-acetyllactosamine carbohydrates (I/i antigen) on erythrocytes and B lymphocytes, cause cold agglutinin disease, and are carried by 5% of naive B cells that are anergic. We analyzed the specificity of three IGHV4-34*01 IgG antibodies isolated from healthy donors immunized against foreign rhesus D alloantigen or vaccinia virus. Each IgG was expressed and analyzed either in a hypermutated immune state or after reverting each antibody to its unmutated preimmune ancestor. In each case, the preimmune ancestor IgG bound intensely to normal human B cells bearing I/i antigen. Self-reactivity was removed by a single somatic mutation that paradoxically decreased binding to the foreign immunogen, whereas other mutations conferred increased foreign binding. These data demonstrate the existence of a mechanism for mutation away from self-reactivity in humans. Because 2.5% of switched memory B cells use IGHV4-34*01 and >43% of these have mutations that remove I/i binding, clonal redemption of anergic cells appears efficient during physiological human antibody responses.
We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 ...protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.
Spleen-resident dendritic cell (DC) populations occupy sentinel positions for the capture and presentation of blood-borne antigens. Here we found a difference in expression of the chemotactic ...receptor EBI2 (GPR183) on splenic DC subsets and that EBI2 regulated the positioning and homeostasis of DCs in the spleen. EBI2 and its main ligand, 7α,25-OHC, were required for the generation of the splenic CD4(+) DC subset and the localization of DCs in bridging channels. Absence of EBI2 from DCs resulted in defects in both the activation of CD4(+) T cells and the induction of antibody responses. Regulated expression of EBI2 on DC populations is therefore critical for the generation and correct positioning of splenic DCs and the initiation of immune responses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The adaptive immune system is tasked with producing antibodies that recognize a wide scope of potential pathogens, including those never before encountered, and concurrently avoiding formation of ...antibodies binding host tissues. The diverse repertoire of antibodies produced by V(D)J recombination inevitably includes autoantibodies that bind to self‐antigens, estimated to be as much as 70% of nascent antibodies on immature B cells. Early theoretical models of tolerance hypothesized that such self‐reactive clones could not possibly be allowed to survive and mature. However from the first direct view of the fate of nascent B cells carrying a self‐binding antibody it was clear that many “forbidden clones” circulate to secondary lymphoid tissues, where they adopt an IgMlow IgD+ cell surface phenotype and are prevented from secreting autoantibodies by a series of tolerance checkpoints referred to as “clonal anergy.” Since anergic B cells can be reactivated to secrete pathogenic autoantibodies in certain settings, the advantage of controlling self‐reactive antibodies by clonal anergy has until recently remained enigmatic. Here we review this topic and recent advances showing that anergic B cells are recruited into the germinal center to mutate away from self‐reactivity, undergoing “clonal redemption” into cells making antibodies with exquisite specificity for foreign immunogens.
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 ...(V H and V L) 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 V H and 24, 49–53, 56 in V L). 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 (V H3 and V κ1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V H and V L domains revealed a surprising degree of structural conservation, indicating compatibility with V H/V L 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.
Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying ...an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.
Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared ...mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.
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•Single-cell omics reveal rare cells making a common pathogenic human autoantibody•Antibody mutations cause pathogenicity by phase transition to insoluble aggregates•Lymphoma driver mutations are present in cells making pathogenic autoantibodies•Driver mutations dysregulate NF-κB signaling, cell cycle, and antibody mutation
Integrated proteomics and single-cell analysis reveal rogue cell clones with lymphoma driver mutations producing damaging antibodies with the propensity for phase transition into insoluble antibody-autoantigen complexes.
Here we report a method to reorganize the core structure of aliphatic unsaturated nitrogen‐containing substrates exploiting polyprotonation in superacid solutions. The superelectrophilic activation ...of N‐isopropyl systems allows for the selective formal Csp3−H activation/cyclization or homologation / functionalization of nitrogen‐containing substrates. This study also reveals that this skeletal reorganization can be controlled through protonation interplay. The mechanism of this process involves an original sequence of C−N bond cleavage, isopropyl cation generation and subsequent C−N bond and C−C bond formation. This was demonstrated through in situ NMR analysis and labelling experiments, also confirmed by DFT calculations.
The rearrangement of isopropyl‐containing nitrogen‐containing compounds allows the skeletal reorganization to cyclic and elongated functionalized systems. An unprecedented tandem C−N bond cleavage / C−C and C−N bond formation through the generation of a dication is shown.
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 IgM ˡᵒʷ 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 IgM ˡᵒʷ IgD+ B cells form twice as many GC progeny as naïve IgM ʰⁱ 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.
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