Human Antibody Fab Research Articles

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

Case Studies Discussing the Pathology, Immunogenicity, and Proposed Mechanism of Toxicity of an Inhaled Anti-TGFβ Humanized Fab Antibody in Non-Human Primates and Mice.

Treatment of nonhuman primates and mice with a humanized antigen-binding fragment (Fab) antibody (UCBFab) inhibiting transforming growth factor β via daily inhalation for up to 13 weeks resulted in low systemic exposure but high local exposure in the lung. Target engagement was demonstrated by reduced levels of signal transducers, phosphoSMAD and plasminogen activator inhibitor-1 in the bronchoalveolar lavage fluid (BALF). Treatment was associated with a high frequency and titer of antidrug antibodies, indicating high local immunogenicity, and local pathology within the lung and draining lymph nodes. Microscopic changes were characterized by perivascular (PV) and peribronchiolar (PB) mononuclear inflammatory cell (MIC) infiltrates that were principally lymphocytic in nature and mixed inflammatory cell infiltrates and/or inflammation within the alveoli. Immunohistochemical investigation revealed a predominantly CD68-positive macrophage and CD3- and CD8>CD4-positive T-cell response in the alveoli, whereas within the airways, there was a variable mixture of CD3-positive T cells, CD20-positive B cells, and CD68-positive macrophages. Increased cellularity of the draining lymph nodes was also noted, indicating the presence of an immune response to the inhaled test article. Morphologic changes did not progress over time, and all changes partially recovered. Increased leukocytes (principally macrophages) in BALF cytology correlated with the changes seen by histopathology.

Structural basis of serum albumin recognition by SL335, an antibody Fab extending the serum half-life of protein therapeutics

Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.

Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library.

The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met. A phage library, with a functional size of 5.5 × 1010 individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques. Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (Kaff: 0.3 × 109M-1), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%). Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells.

A novel peptide-based sensor platform for detection of anti-Toxoplasma gondii immunoglobulins

Toxoplasma gondii is an intracellular protozoan parasite responsible for toxoplasmosis, which affects humans and animals. Serologic detection of anti-T. gondii immunoglobulins plays a crucial role in the clinical diagnosis of toxoplasmosis. In this work, a novel electrochemical immunosensor for detecting anti-T. gondii immunoglobulins is reported, based on immobilization of an in silico predicted peptide (PepB3), obtained from membrane protein of T. gondii, on the graphite electrode modified with poly(3-hydroxybenzoic acid). Indirect ELISA confirmed infection and binding specificity of peptide PepB3. Molecular modelling and simulations show this peptide binds to the T. gondii human Fab antibody in the surface antigen 1 (SAG1) binding site, remaining a stable complex during the molecular dynamic simulations, especially by hydrogen bonds and hydrophobic interactions. This electrochemical immunosensor was able to discriminate different periods of infection, using infected mouse serum samples, showing selectivity and discriminating infected and uninfected mouse serum.

Assessment of patients post reversal with idarucizumab.

Idarucizumab, a fully humanized Fab antibody fragment, is indicated for reversal of dabigatran's anticoagulant activity. Idarucizumab neutralizes the anticoagulant effects of dabigatran by binding to dabigatran and its metabolite. In the full analysis of 503 patients, idarucizumab fully reversed the anticoagulant effect of dabigatran in more than 98% of patients. Real-world clinical experience with idarucizumab for dabigatran reversal remains limited. We report 11 real-world clinical cases in which idarucizumab was administered for dabigatran reversal in the setting of bleed (bleeding cohort n = 5) or emergent procedure (emergent procedure cohort, n = 6). Coagulation tests and clinical outcomes were assessed before and after idarucizumab administration. Clinical outcomes included thromboembolic events and hemostasis. The median (IQR) aPTT (seconds) before versus after idarucizumab was 40.4 (36.1) versus 27.3 (6.2) (bleeding cohort) and 50.1 (13.4) versus 26.5 (8.1) (emergent procedure cohort). The median (IQR) INR before and after idarucizumab was 2.0 (1.1) versus 1.2 (0.1) (bleeding cohort) and 1.1 (0.5) versus 1.1 (0.3) (emergent procedure cohort). Hemostasis was achieved in 4/5 patients in the bleeding cohort and 5/6 patients in the emergent procedure cohort. Thrombotic events occurred in four patients with a median time (IQR) from idarucizumab administration of 7.4 (4.3-14.7) days. Idarucizumab achieved adequate dabigatran reversal as evident by normalization of aPTT, INR, and achieving hemostasis. However, our data demonstrates a high thrombotic risk associated with dabigatran reversal with idarucizumab than previously reported.

Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp

Data obtained recently in the United Kingdom following a nationwide infant immunization program against serogroup B Neisseria meningitidis (MenB) reported >80% 4CMenB vaccine-mediated protection. Factor H-binding protein (fHbp) is a meningococcal virulence factor and a component of two new MenB vaccines. Here, we investigated the structural bases underlying the fHbp-dependent protective antibody response in humans, which might inform future antigen design efforts. We present the co-crystal structure of a human antibody Fab targeting fHbp. The vaccine-elicited Fab 1A12 is cross-reactive and targets an epitope highly conserved across the repertoire of three naturally occurring fHbp variants. The free Fab structure highlights conformational rearrangements occurring upon antigen binding. Importantly, 1A12 is bactericidal against MenB strains expressing fHbp from all three variants. Our results reveal important immunological features potentially contributing to the broad protection conferred by fHbp vaccination. Our studies fuel the rationale of presenting conserved protein epitopes when developing broadly protective vaccines.

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 109 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes.

A human Fab exclusively binding to the extracellular domain of LMP2A

In the areas of North Africa, Southeast Asia as well as South China, Nasopharyngeal carcinoma (NPC) is among the most widespread cancers. Plenty of research findings confirmed that Epstein-Barr virus (EBV) played a crucial role in NPC. EBV-encoded Latent membrane protein 2A (LMP2A) which continuously expressed in cell membrane protein induced an epithelial-mesenchymal transition and increased the number of side population stem-like cancer cells in NPC. This reveals that LMP2A could contribute to the development and recurrence in NPC. Above evidences suggest that LMP2A could be the potential target molecule in the treatment of NPC. In the current study, a novel human antibody Fab (Fab29) against the extracellular domain of LMP2A was produced with success. Through immunofluorescence experiment it was proved that human antibody Fab29 exclusively combined the surface of SUNE cells (LMP2A-positive). Then flow cytometry result exhibited that the fluorescent intensities of SUNE cells and CNE cells were distinct (96.89% and 0.02% respectively). After that, it was shown by affinity test that the Fab29 fragment had high affinity (KD (M) 1.79E-09) with LMP2A. It was also revealed by immunohistochemical analysis that the Fab29 fragment could combine with LMP2A-positive human NPC tissues in comparison with the control group. Finally, the MTT result indicated that the Fab29 fragment could inhibit the proliferation of LMP2A-positive NPC cells. The inhibiting rate to SUNE cell proliferation reached a peak by Fab29 (19.67%) compared with unrelated Fab and CNE with Fab29 at a concentration of 500 μg/L in first 24 h and in the next 24 h the inhibition rate grew to 22.54%. In brief, it was shown that Fab29, a characteristic human antibody, could recognize LMP2A protein and inhibit the proliferation of LMP2A-expressing NPC cells in vitro.

Selective function-blocking monoclonal human antibody highlights the important role of membrane type-1 matrix metalloproteinase (MT1-MMP) in metastasis.

The invasion-promoting MT1-MMP is a cell surface-associated collagenase with a plethora of critical cellular functions. There is a consensus that MT1-MMP is a key protease in aberrant pericellular proteolysis in migrating cancer cells and, accordingly, a promising drug target. Because of high homology in the MMP family and a limited success in the design of selective small-molecule inhibitors, it became evident that the inhibitor specificity is required for selective and successful MT1-MMP therapies. Using the human Fab antibody library (over 1.25×109 individual variants) that exhibited the extended, 23-27 residue long, VH CDR-H3 segments, we isolated a panel of the inhibitory antibody fragments, from which the 3A2 Fab outperformed others as a specific and potent, low nanomolar range, inhibitor of MT1-MMP. Here, we report the in-depth characterization of the 3A2 antibody. Our multiple in vitro and cell-based tests and assays, and extensive structural modeling of the antibody/protease interactions suggest that the antibody epitope involves the residues proximal to the protease catalytic site and that, in contrast with tissue inhibitor-2 of MMPs (TIMP-2), the 3A2 Fab inactivates the protease functionality by binding to the catalytic domain outside the active site cavity. In agreement with the studies in metastasis by others, our animal studies in acute pulmonary melanoma metastasis support a key role of MT1-MMP in metastatic process. Conversely, the selective anti-MT1-MMP monotherapy significantly alleviated melanoma metastatic burden. It is likely that further affinity maturation of the 3A2 Fab will result in the lead inhibitor and a proof-of-concept for MT1-MMP targeting in metastatic cancers.

Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma.

We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodis-tribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.

Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life

The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0→∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

Studies of Nontarget-Mediated Distribution of Human Full-Length IgG1 Antibody and Its FAb Fragment in Cardiovascular and Metabolic-Related Tissues

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097.

The secreted cystine-knot protein sclerostin was first identified from genetic screening of patients suffering from the rare bone-overgrowth diseases sclerosteosis and van Buchem disease. Sclerostin acts a negative regulator of bone growth through inhibiting the canonical Wnt signalling cascade by binding to and blocking the Wnt co-receptor LRP5/6. Its function in blocking osteoblastogenesis makes it an important target for osteoanabolic therapy approaches to treat osteoporosis, which is characterized by a progressive decrease in bone mass and density. In this work, the production, crystallization and preliminary X-ray diffraction data analysis of a sclerostin-neutralizing human Fab antibody fragment, AbD09097, obtained from a naive antibody library are reported. Crystals of the Fab AbD09097 belonged to space group P21, with unit-cell parameters a = 45.19, b = 78.49, c = 59.20 Å, β = 95.71° and diffracted X-rays to a resolution of 1.8 Å.

Preventing Primate GvHD Using a Novel Antagonistic Anti-CD28 Antibody Plus Rapamycin: Downregulation of CD8 Activation and Preservation of the Naïve Cell Phenotype Predicts GvHD-Free Survival

▪Introduction: We have previously shown that blocking CD28:CD80/86 costimulation with CTLA4-Ig can be a successful strategy to inhibit GVHD. However, since CTLA4-Ig targets CD80/86, there remain concerns of off-target, immune activating effects, given that it can inhibit both positive signaling through CD28 and negative signaling through CTLA4. In order to specifically target CD28, we have developed a humanized anti-CD28 monovalent Fab antibody (FR104) and have tested its ability to protect against GVHD.Methods: NHP underwent MHC-mismatched transplantation after myeloablative radiation-based pre-transplant conditioning. They were transplanted with GCSF-mobilized PBSCs (4.2x10^8 +/- 1.1x10^8 TNC/kg and 1.9 x 10^7 +/- 0.5 x 10^7 CD3+ T cells/kg. GVHD prophylaxis consisted of either monotherapy with FR104 (5mg/kg given weekly) or with combination FR104 and rapamycin (rapamycin trough 5-15 ng/ml). These were compared to three control groups: no GVHD prophylaxis, rapamycin monotherapy and autologous HSCT. Clinical and histologic GVHD was monitored using our NHP grading scale, and longitudinal immunologic analysis was performed. Treatment was continued as tolerated with planned terminal analysis at 35 days in all survivors.Results: Untreated controls (n = 5) developed rapid, severe multi-organ GVHD (MST=7 days). Rapamycin monotherapy (n=6) partially protected against GVHD (MST =14 days). Monotherapy with FR104 showed a significant prolongation in survival compared to untreated animals (MST = 21 days, p=0.02 n = 3), with breakthrough GVHD (liver, skin predominant) ultimately occurring. In contrast to all other groups, combination therapy with FR104 and rapamycin (n=4) resulted in striking protection from GVHD with all animals reaching planned terminal analysis with minimal clinical disease (p=0.01 vs. untreated controls, p=0.01 vs. rapamycin monotherapy, Fig. 1).We have documented a major role for both T cell proliferation (Ki-67) and Granzyme B (Gran-B) overexpression in GVHD, with untreated controls demonstrating rampant CD8+ proliferation (81% + 5.4% Ki-67+ at day 7 vs. 4% pre-transplant) and Gran-B overexpression (81% + 2.7% Gran-Bvery high at day 7 vs. 0.5% + 0.5% pre-transplant). Monotherapy with either rapamycin or FR104 partially controlled proliferation (53% + 23% and 30% + 21.7%, respectively) and Gran-B overexpression (21% + 8.7% and 11% + 5%, respectively) on day 7. Combination therapy with FR104 + rapamycin resulted in synergistic control of both proliferation and Gran-B overexpression (0.6% + 0.2% Ki67+ and 2.3% + 1.3% Gran-B overexpression on Day 7), reverting the CD8+ activation phenotype to that observed in auto-HSCT controls (5.6% + 2.1% Ki67+, 9% + 3.2% Gran-B overexpression, Fig. 2). This reversion to the auto-HSCT phenotype was maintained until the terminal analysis, and the control of Day 7 proliferation and Gran-B overexpression correlated closely with GVHD-free survival.The control of CD28-mediated T cell activation in FR104-treated recipients resulted in significant preservation of the naïve CD8+ T cell (CD8 Tn) phenotype in these animals. Thus, in untreated controls and rapamycin monotherapy groups, the CD8 Tn population virtually disappeared post-transplant (2.1% + 0.7% and 6.6% + 1.5% at day 7, respectively compared to 33.6%+ 4.1% pre-transplant), consistent with widespread allo-stimulation and costimulation. In contrast, FR104 monotherapy resulted in partial preservation of CD8 Tn (24.3% + 2.8% at Day 7) and FR104 + rapamycin combination therapy promoted nearly complete preservation of CD8 Tn (48.3% + 0.9% at day +7), with maintenance of this naïve population for the duration of therapy (Fig. 3).Discussion: These results show, for the first time, that specific blockade of CD28 can inhibit NHP GVHD, and when coupled with rapamycin it can effectively control both the clinical and immunologic sequelae of this disease. CD28 blockade with FR104 + rapamycin significantly inhibited both CD8+ T cell proliferation and Granzyme B overexpression, and lead to robust preservation of the naïve T cell phenotype, consistent with efficient inhibition of allo-activation. Our results suggest that targeting the CD28:CD80/86 costimulation axis by selective CD28 blockade may be effective in inhibiting the T cell activation associated with GVHD, and is deserving of clinical evaluation for safety and efficacy in patients undergoing HCT. [Display omitted] [Display omitted] [Display omitted] DisclosuresVanhove:Effimune: Research Funding.

Bovine antibodies with ultra long H3 Complementarity Determining Regions

About 10% of the bovine antibody repertoire exhibit extremely long H3 complementarity determining regions (CDRs). These H3 CDRs are usually described as `loops' in the more familiar mouse and human antibody Fab structures, but the ultra long bovine H3 CDRs are actually small, cysteine-rich protein domains that vary in size from 44 to 64 amino acids. We have recently determined the structures for two bovine antibody Fab fragments, and will describe these, as well as compare them with two other previously determined bovine Fab structures (Wang et al., Cell, 2013). One new Fab has a relatively short H3 CDR region of 44 residues, with just one disulfide bond, while the other boasts one of the longest H3 CDRs, with 63 residues and four disulfide bonds. These H3 CDRs fold to form apparently rigid `stem' regions, that present the disulfide bonded `knob' domain far above the five other Fab CDR loops. Despite extreme diversity in sequence, length and disulfide bonding patterns, the CDRs share structural homology, both in their long stems and in the more variable knob regions.

Protein crystallization with microseed matrix screening: application to human germline antibody Fabs.

The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors

A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74–92% and 90–96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3–IGKV1, IGHV3–IGVL1, and IGHV3–IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.