Enhanced CD1d phosphatidylserine presentation using a single-domain antibody promotes immunomodulatory CD1d-TIM-3 interactions

Background: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. Methods: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. Results: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A′-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. Conclusion: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides

Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool

Calpain 3 Is a Rapid-Action, Unidirectional Proteolytic Switch Central to Muscle Remodeling

Calpain 3 (CAPN3) is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

DNA-protein alignment of V gene segments and anti-HIV VHHs obtained via the family approach (2C7/2C9 and 2B10)

Species of the Camelidae family possess a class of antibodies consisting only of heavy chains. The variable domains of these heavy chain antibodies (VHHs) can be used as process aids, as diagnostic tool or in a wide variety of therapeutic applications, such as prevention of HIV infection. The maturation of antibodies processes through a series of steps all aimed at optimizing the functionality. In this paper we describe new Lama glama V gene segments and discuss how the maturation of Camelid IgV and IgD VHHs differs from what is known for human and mouse VHs. Knowledge of this maturation pathway will eventually assist the selection and design of better VHHs against HIV

Found keyresidues in VHH structures

A high production yield and good stability are required for the large scale application of llama heavy chain antibody fragments (VHHs) against targets such as a microbicide against HIV. Many aspects of the production process are amenable to optimization by engineering. Codon usage, for example, can be improved, but it is also possible to remove proteolytic cleavage sites, chaperone binding sites, or to improve the packing of the protein by increasing the numbers of hydrophobic contacts or hydrogen bonds. However, not every amino acid can be mutated freely. Obviously, the cysteine bridge should stay intact at all cost. It also seems unwise to mutate residues involved in scaffolding the CDRs. We have made a large series of in silico studies, and tabulated and analyzed all known mutations in VHHs. No mutation information was available for eight residues that were predicted to be important for folding and production yield using the rules for in vitro folding of Vendruscolo. When we mutated these eight residues we observed thermostability differences ranging from -1 to -14 degrees in agreement with what one would expect from the selected modifications. The production yield of most of the mutants decreased as well. All known and predicted mutation effects were combined in a generic 'VHH-mutability' table. This table could be used to flawlessly predict the best producing variants when a series of HIV neutralizing VHH variants was obtained

Circular Dichroism plots

A high production yield and good stability are required for the large scale application of llama heavy chain antibody fragments (VHHs) against targets such as a microbicide against HIV. Many aspects of the production process are amenable to optimization by engineering. Codon usage, for example, can be improved, but it is also possible to remove proteolytic cleavage sites, chaperone binding sites, or to improve the packing of the protein by increasing the numbers of hydrophobic contacts or hydrogen bonds. However, not every amino acid can be mutated freely. Obviously, the cysteine bridge should stay intact at all cost. It also seems unwise to mutate residues involved in scaffolding the CDRs. We have made a large series of in silico studies, and tabulated and analyzed all known mutations in VHHs. No mutation information was available for eight residues that were predicted to be important for folding and production yield using the rules for in vitro folding of Vendruscolo. When we mutated these eight residues we observed thermostability differences ranging from -1 to -14 degrees in agreement with what one would expect from the selected modifications. The production yield of most of the mutants decreased as well. All known and predicted mutation effects were combined in a generic 'VHH-mutability' table. This table could be used to flawlessly predict the best producing variants when a series of HIV neutralizing VHH variants was obtained

DNA-protein alignment of V gene segments and anti-HIV VHHs

Species of the Camelidae family possess a class of antibodies consisting only of heavy chains. The variable domains of these heavy chain antibodies (VHHs) can be used as process aids, as diagnostic tool or in a wide variety of therapeutic applications, such as prevention of HIV infection. The maturation of antibodies processes through a series of steps all aimed at optimizing the functionality. In this paper we describe new Lama glama V gene segments and discuss how the maturation of Camelid IgV and IgD VHHs differs from what is known for human and mouse VHs. Knowledge of this maturation pathway will eventually assist the selection and design of better VHHs against HIV

Results of in silico analyses of 70 defined VHHs

Variable domains of camelid heavy chain antibodies (VHHs) are the smallest available antigen binding fragments. The GRAS organism Saccharomyces cerevisiae is often used for the production of VHHs. The production yield in this organism often is too low for commercially viable large scale application as, for example, a therapeutic agent. We compare two VHHs, one of which can be produced up to 150 mg/l while the other is produced in amounts that are hardly detectable, to shed light on molecular and biological origins of those VHH production yield differences. Sequence characteristics, structure properties, and growth conditions were tested. We found that codon optimization is much more important for the gene encoding the badly produced VHH then the good produced VHHs. Even more important is to avoid VHHs in with the FW4 is encoded by germline J segment 7 which sequence contains K and L on positions 120 and 123 respectively. Moreover we validated earlier findings that ethanol improves the yield of VHHs expressed in S. cerevisiae, even for VHHs that contain only one S-S bridge. Electron microscopy showed that bad yields of secreted VHHs coincide with increased intracellular levels of (most likely) aggregated VHHs; however the level of intracellular VHHs is heterogeneously distributed of the cells of the same population