Antigen-binding Pocket Research Articles

OP26 Disentangling the role of HLA proteins in shaping the T-cell repertoire in Inflammatory Bowel Disease using CDR3-QTL analysis

Abstract Background The HLA region leads in the number of the strongest genetic associations for chronic immune-mediated diseases, including Inflammatory Bowel Disease1. Despite decades of research, the role of HLA in modulating chronic T cell-mediated responses remains unclear. HLA proteins present antigens for recognition by T cell receptors (TCRs). TCRs bear six complementarity determining regions (CDR), with the most variable CDR3 of the beta chain (CDR3b) due to a V(D)J recombination. The importance of CDR3 is determined by its direct interaction and recognition of antigens. Recent work by Ishigaki et al.2 identified that HLA-DRB1 site 13 influences the amino acid composition of CDR3b. Building on this, we extended the mapping between HLA sites and the CDR3b region to detect HLA sites that significantly contribute to the amino acid composition of CDR3b in IBD TCR repertoires. Methods We performed a comprehensive CDR3-QTL association analysis based on multivariate multiple linear regressions on a large dataset of ~2,000 paired HLA and TCR repertoires of healthy donors (n = 767) and IBD patients (n = 1188: 786 CD and 402 UC) using the analytical framework of Ishigaki et al.2 Results We discovered multiple novel HLA-DRB1 and HLA-DQ sites with distinct influences on TCRs. HLA-DRB1 sites 11 and 57 and DQB1 sites 57 and 71, located in antigen-binding pockets, strongly influence CDR3 composition in the IBD cohort. Notably, these effects were more pronounced in Crohn’s disease than in ulcerative colitis. HLA-DRB1 sites 67 and 73 and site 55 in DQB1 significantly influenced CDR3 composition only in healthy donors. HLA-DRB1 sites 13 and 71 and DQB1 site 87 were observed in both datasets, suggesting shared mechanisms irrespective of disease.The structural analysis revealed that healthy-specific sites directly interact with TCR, while common and IBD-specific sites predominantly occupy antigen-binding pockets. Conclusion Our study extensively identifies novel HLA sites that influence the most variable region in TCR - CDR3b in IBD patients. Our findings propose two distinct mechanisms by which HLA influences the CDR3: (1) direct TCR binding in TCRs (2) antigen-driven modulation in IBD-specific and common sites. The majority of identified HLA sites align with the loci previously associated with autoimmune diseases, and have been previously linked to common infections, particularly Epstein-Barr virus infection, suggesting that HLA influences CDR3 composition through both antigen-driven and direct TCR interactions.

Crystal Structures of Antigen-Binding Fragment of Anti-Osteocalcin Antibody KTM219.

Osteocalcin is a useful biomarker for bone formation and bone-related diseases. KTM219 is an anti-osteocalcin C-terminal peptide antibody. The single-chain variable region (scFv) and antigen-binding fragment (Fab) of KTM219 are applicable to the Quenchbody (Q-body) immunoassay. Q-body is a new type of fluorescent immunosensor, which is scFv or Fab labeled with a fluorescent dye. When Q-body binds to its antigen, the fluorescence intensity increases. The highly sensitive detection of antigens by changes in fluorescence intensity is performed in a single step by mixing the sample and reagent. In this study, to reveal the recognition mechanism of the KTM219 antibody and to discuss the structural basis for Q-body, we solved the crystal structures of Fab of the anti-osteocalcin antibody KTM219 and its complex with the antigen osteocalcin C-terminal peptide (BGP-C7). Also, we solved the structure of a KTM219 Fab crystal grown in the presence of a fluorescent dye, carboxytetramethylrhodamine (TAMRA); however, tightly bound TAMRA was not found in the electron density map. We predicted the binding sites of TAMRA in the antigen-binding pocket by docking simulations. These results support the proposed Q-body mechanism. The crystal structures of KTM219 Fab would be useful for further development and improvement of Q-body fluorescent immunosensors.

Direct Blockade of the Norovirus Histo-Blood Group Antigen Binding Pocket by Nanobodies.

Noroviruses are the leading cause of outbreaks of acute gastroenteritis. These viruses usually interact with histo-blood group antigens (HBGAs), which are considered essential cofactors for norovirus infection. This study structurally characterizes nanobodies developed against the clinically important GII.4 and GII.17 noroviruses with a focus on the identification of novel nanobodies that efficiently block the HBGA binding site. Using X-ray crystallography, we have characterized nine different nanobodies that bound to the top, side, or bottom of the P domain. The eight nanobodies that bound to the top or side of the P domain were mainly genotype specific, while one nanobody that bound to the bottom cross-reacted against several genotypes and showed HBGA blocking potential. The four nanobodies that bound to the top of the P domain also inhibited HBGA binding, and structural analysis revealed that these nanobodies interacted with several GII.4 and GII.17 P domain residues that commonly engaged HBGAs. Moreover, these nanobody complementarity-determining regions (CDRs) extended completely into the cofactor pockets and would likely impede HBGA engagement. The atomic level information for these nanobodies and their corresponding binding sites provide a valuable template for the discovery of additional "designer" nanobodies. These next-generation nanobodies would be designed to target other important genotypes and variants, while maintaining cofactor interference. Finally, our results clearly demonstrate for the first time that nanobodies directly targeting the HBGA binding site can function as potent norovirus inhibitors. IMPORTANCE Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics. We successfully developed and characterized four norovirus nanobodies that bound at the HBGA pockets. Compared with previously developed norovirus nanobodies that inhibited HBGA through disrupted particle stability, these four novel nanobodies directly inhibited HBGA engagement and interacted with HBGA binding residues. Importantly, these new nanobodies specifically target two genotypes that have caused the majority of outbreaks worldwide and consequently would have an enormous benefit if they could be further developed as norovirus therapeutics. To date, we have structurally characterized 16 different GII nanobody complexes, a number of which block HBGA binding. These structural data could be used to design multivalent nanobody constructs with improved inhibition properties.

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRβ1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.

Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

Structure of the Anti-C60 Fullerene Antibody Fab Fragment: Structural Determinants of Fullerene Binding

The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by - stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.

Application of the NZ‐1 Fab as a crystallization chaperone for PA tag‐inserted target proteins

An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy.

Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight β-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbβ3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.

Abstract 4079: Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers

Abstract Selective High Affinity Ligands (SHALs) are small molecules that can be designed to bind selectively and with high affinity to any protein target by linking together several ligands that recognize and bind to different sites on the protein’s surface. SH7139, our first SHAL therapeutic for treating advanced non-Hodgkin’s lymphoma, has shown remarkable efficacy in B-cell lymphoma xenograft models by providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.4 μg/kg. Functioning similar to an antibody-drug conjugate and a pro-drug, SH7139 targets and binds to a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope are expressed in approximately 85% of B-cell lymphomas. Upon binding to its HLA-DR target, SH7139 is carried into the cytoplasm where it is concentrated and subsequently metabolized, releasing toxic metabolites (derived from each of the linked ligands) that inhibit a series of cellular activities required for tumor cell function. In preparation for an IND application submission and advancing SH7139 into clinical trials, acute dose range finding (i.v. bolus) and multiple dose (i.v. bolus on days 1, 8, and 15) toxicology and safety studies have been conducted with SH7139 in rats and beagle dogs. In the rat studies, doses up to 30 mg/kg (NOAEL = 30 mg/kg, 11,854 fold higher than the efficacy dose), were well tolerated with no findings associated with the drug. Beagle dogs were found to be the most sensitive species (NOAEL = 0.3mg/kg, 395 fold higher than the efficacy dose), with doses at 1 and 10mg/kg producing a reversible allergic-type reaction. A maximum tolerated dose (MTD) was reached in rats (100 mg/kg). No attempt was made to identify the MDT in dogs. In vitro assays conducted to evaluate potential cardiac safety showed SH7139 had no effect on potassium (hERG) and calcium (hCaV1.2) ion channel function up to the highest concentration tested (25 μM). A detectable effect above background was only observed with the sodium channel hNaV1.5 at 25 μM, a plasma concentration that would only be achieved in patients administered a dose ~2,000 fold higher than the efficacy dose. SH7139 was found to be negative in inducing gene mutation in five tester strains of Salmonella and E. coli (AMES assay). In a CHO-K1 micronucleus assay, no genotoxicity was observed in the presence of S9 over the entire concentration range tested. In the absence of S9, a positive response (3.3 fold) just above the triggering threshold (3 fold) of the assay was observed at the highest SH7139 concentration (500 μM). Based on these results and others derived from mouse xenograft efficacy studies, a maximum recommended Phase I trial starting dose has been tentatively set at 0.5 μg/kg with a safety factor of 324. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Rod Balhorn, Monique Cosman Balhorn. Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2017-4079

Abstract 1171: Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors

Abstract SH7139, the first of a new class of cancer therapeutics developed for treating non-Hodgkin’s lymphoma, is unusual in that both targeting and pro-drug functionalities have been incorporated into the same small molecule. Functioning similar to an antibody-drug conjugate, SH7139 targets a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope within the β-subunit are reported to be over-expressed in approximately 85% of B-cell lymphomas. Upon binding to HLA-DR molecules located on the tumor cell’s surface, SH7139 is transported into the cytoplasm where it is concentrated and subsequently metabolized. A series of metabolic products derived from the SHAL’s recognition elements (three small molecules that are linked together to create SH7139 and to provide targeting selectivity) are generated as the drug is broken down, each of which inhibits one or more activities required for tumor cell survival. Preclinical studies with SH739 have demonstrated remarkable efficacy in treating B-cell lymphoma xenografts in mice, providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.41 μg/kg. Biopsy tissue binding studies conducted with SH7129, a biotinylated form of SH7139, and streptavidin-horse radish peroxidase detection have shown the drug binds to a significant fraction of tumors obtained from patients diagnosed with multiple myeloma and each of the B-cell lymphoma subtypes tested to date (DLBC, Burkitt’s, Mantle Cell, Follicular, MALT, and CLL). SH7129 was also observed to bind to tumor biopsies obtained from of a number of patients diagnosed with peripheral T-cell and nodular sclerosis Hodgkin’s lymphomas, a result consistent with observations reported by others that HLA-DRs are expressed in a subset of these lymphomas. HLA-DR expression has also been reported to occur in or be linked to a number of other types of cancer, including melanoma, cervical, ovarian, pancreatic and lung cancers. SH7129 staining of tumor microarrays have shown biopsy cores from a subset of patients diagnosed with each of these cancers also bind SH7139. While in vivo efficacy has only been tested in Burkitt’s (Raji), Mantle cell (Granta-519), and T-cell (Jurkat, a cell line control lacking HLA-DR and showing no efficacy) lymphoma xenografts, these tissue binding results suggest that in addition to the majority of the NHL subtypes, nodular sclerosis Hodgkin’s lymphoma, multiple myelomas, as well as a subset of melanomas, ovarian, cervical, pancreatic, and lung cancers may also respond to SH7139 therapy. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Monique Cosman Balhorn, Rod Balhorn. Therapeutic applications of the selective high affinity ligand SH7139 may extend beyond NHL to many other types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2017-1171

Identification of amino acids in antigen-binding site of class II HLA proteins independently associated with hepatitis B vaccine response

Background & aimsGenetic factors in class II human leukocyte antigen (HLA) have been reported to be associated with inter-individual variation in hepatitis B virus (HBV) vaccine response. However, the mechanism underlying the associations remains elusive. In particular, the broad linkage disequilibrium in HLA region complicates the localization of the independent effects of genetic variants. Thus, the present study aimed to identify the most probable causal variations in class II HLA loci involved in the immune response to HBV vaccine. MethodsWe performed a case-control study to assess whether HLA-DRB1, -DQB1, and -DPB1 4-digit alleles were associated with the response to primary HBV vaccination in 574 healthy Japanese students. To identify causative variants, we next assessed independently associated amino acid variants in these loci using conditional logistic regression analysis. Furthermore, to clarify the functional effects of these variants on HLA proteins, we performed computational structural studies. ResultsHLA-DRB1∗01:01, HLA-DRB1∗08:03, HLA-DQB1∗05:01, and HLA-DPB1∗04:02 were significantly associated with sufficient response, whereas HLA-DPB1∗05:01 was associated with poor response. We then identified amino acids independently associated with sufficient response, namely, leucine at position 26 of HLA-DRβ1 and glycine-glycine-proline-methionine at positions 84–87 of HLA-DPβ1. These amino acids were located in antigen-binding pocket 4 of HLA-DR and pocket 1 of HLA-DP, respectively, which are important structures for selective binding of antigenic peptides. In addition, the detected variations in HLA-DP protein were responsible for the differences in the electrostatic potentials of the pocket, which can explain in part the sufficient/poor vaccine responses. ConclusionHLA-DRβ1 position 26 and HLA-DPβ1 positions 84–87 are independently associated with anti-HBs production against HBV vaccine. Our results suggest that HBsAg presentation through these HLA pocket structures plays an important role in the inter-individual variability of HBV vaccination.

Selection of a potential diagnostic biomarker for HIV infection from a random library of non-biological synthetic peptoid oligomers

Non-biological synthetic oligomers can serve as ligands for antibodies. We hypothesized that a random combinatorial library of synthetic poly-N-substituted glycine oligomers, or peptoids, could represent a random “shape library” in antigen space, and that some of these peptoids would be recognized by the antigen-binding pocket of disease-specific antibodies. We synthesized and screened a one bead one compound combinatorial library of peptoids, in which each bead displayed an 8-mer peptoid with ten possible different amines at each position (108 theoretical variants). By screening one million peptoid/beads we found 112 (approximately 1 in 10,000) that preferentially bound immunoglobulins from human sera known to be positive for anti-HIV antibodies. Reactive peptoids were then re-synthesized and rigorously evaluated in plate-based ELISAs. Four peptoids showed very good, and one showed excellent, properties for establishing a sero-diagnosis of HIV. These results demonstrate the feasibility of constructing sero-diagnostic assays for infectious diseases from libraries of random molecular shapes. In this study we sought a proof-of-principle that we could identify a potential diagnostic antibody ligand biomarker for an infectious disease in a random combinatorial library of 100 million peptoids. We believe that this is the first evidence that it is possible to develop sero-diagnostic assays – for any infectious disease – based on screening random libraries of non-biological molecular shapes.

CD74/DQA1 dimers predispose to the development of arthritis in humanized mice.

Rheumatoid arthritis (RA) is associated with the presence of certain HLA class II genes. However, why some individuals carrying RA non-associated alleles develop arthritis is still unexplained. The trans-heterodimer between two RA non-associated HLA genes can render susceptibility to develop arthritis in humanized mice, DQA1*0103/DQB1*0604, suggesting a role for DQ α chains in pathogenesis. In this study we determined the role of DQA1 in arthritis by using mice expressing DQA1*0103 and lacking endogenous class II molecules. Proximity ligation assay showed that DQA1*0103 is expressed on the cell surface as a dimer with CD74. Upon immunization with type II collagen, DQA1*0103 mice generated an antigen-specific cellular and humoral response and developed severe arthritis. Structural modelling suggests that DQA1*0103/CD74 form a pocket with similarity to the antigen binding pocket. DQA1*0103 mice present type II collagen-derived peptides that are not presented by an arthritis-resistant DQA1*0103/DQB1*0601 allele, suggesting that the DQA1*0103/CD74 dimer may result in presentation of unique antigens and susceptibility to develop arthritis. The present data provide a possible explanation by which the DQA1 molecule contributes to susceptibility to develop arthritis.

Treatment of norovirus particles with citrate

Human norovirus is a dominant cause of acute gastroenteritis around the world. Several norovirus disinfectants label citric acid as an active ingredient. In this study, we showed that norovirus virus-like particles (VLPs) treated with citrate buffer caused the particles to alter their morphology, including increased diameters associated with a new ring-like structure. We also found that epitopes on the protruding (P) domain on these particles were more readily accessible to antibodies after the citrate treatment. These results suggested that citrate had a direct effect on the norovirus particles. Using X-ray crystallography, we showed that the P domain bound citrate from lemon juice and a disinfectant containing citric acid. Importantly, citrate binds at the histo-blood group antigen binding pocket, which are attachment factors for norovirus infections. Taken together, these new findings suggested that it might be possible to treat/reduce norovirus infections with citrate, although further studies are needed.

Abstract 5498: Multiple mechanisms of action may contribute to the lymphoma cell-killing activity of SH7139

Abstract The tridentate selective high affinity ligand (SHAL) SH7139 is selectively cytotoxic at sub-nanomolar concentrations to lymphoma cells over-expressing HLA-DR10. Recent studies suggest SH7139 is unusual in that multiple mechanisms may contribute to its killing of targeted tumor cells. Electron microscopy data suggest one mechanism involves the triggering of a cell-signaling event that leads to the induction of apoptosis when SH7139 binds to a unique site on HLA-DR10 (the same region/epitope recognized by the Lym-1 antibody). A second mechanism appears to involve the internalization of SH7139 by tumor cells and the subsequent release of it's three individual recognition elements/ligands by metabolic cleavage of the amide bonds that link them to a scaffold (a unique feature of SHALs). Analogous to the process of toxin delivery by antibody-drug conjugates, once these ligands are released inside the cell, they can each inhibit a different key pathway required for tumor cell survival. Additional metabolic conversions of these ligands by the tumor cells to toxic metabolites may also contribute to cell killing through the inhibition of a number of other critical cellular functions. The third proposed mechanism is based on the fact that the target receptor HLA-DR10 participates in the activation of T-cells. The binding of SH7139 to the antigen binding pocket of HLA-DR10 may trigger an immune response that targets the cancer cells with bound drug. All three proposed mechanisms are currently under study. If multiple diverse mechanisms of action contribute to SH7139's cytotoxicity, it will be extremely difficult for lymphoma cells to develop a resistance to the drug. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843-02 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Monique Cosman Balhorn, Saphon Hok, Rod Balhorn. Multiple mechanisms of action may contribute to the lymphoma cell-killing activity of SH7139. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2015-5498

The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites.

New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a 'reagentless' fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity.

HLA-DRB1*07:01 Is Associated With Asparaginase Allergies In Children With Acute Lymphoblastic Leukemia

HLA-DRB1*07:01 Is Associated With Asparaginase Allergies In Children With Acute Lymphoblastic Leukemia

Improved Detection of Domoic Acid Using Covalently Immobilised Antibody Fragments

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.

Structure of a double-stranded DNA (6–4) photoproduct in complex with the 64M-5 antibody Fab

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation have been implicated in mutagenesis and cancer. The crystal structure of double-stranded DNA containing the (6-4) photoproduct in complex with the anti-(6-4)-photoproduct antibody 64M-5 Fab was determined at 2.5 Å resolution. The T(6-4)T segment and the 5'-side adjacent adenosine are flipped out of the duplex and are accommodated in the concave antigen-binding pocket composed of six complementarity-determining regions (CDRs). A loop comprised of CDR L1 residues is inserted between the flipped-out T(6-4)T segment and the complementary DNA. The separation of strands by the insertion of the loop facilitates extensive and specific recognition of the photoproduct. The DNA helices flanking the T(6-4)T segment are kinked by 87°. The 64M-5 Fab recognizes the T(6-4)T segment dissociated from the complementary strand, indicating that the (6-4) photoproduct can be detected in double-stranded DNA as well as in single-stranded DNA using the 64M-5 antibody. The structure and recognition mode of the 64M-5 antibody were compared with those of the DNA (6-4) photolyase and nucleotide-excision repair protein DDB1-DDB2. These proteins have distinctive binding-site structures that are appropriate for their functions, and the flipping out of the photolesion and the kinking of the DNA are common to mutagenic (6-4) photoproducts recognized by proteins.