ALBERT

Description: Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen–deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

Description: Acquired thrombotic thrombocytopenic purpura (aTTP), a potentially fatal syndrome, is primarily caused by autoantibodies against the metalloprotease ADAMTS13. Most patients with aTTP harbor an immunoglobulin (Ig) G isotype in blood that targets the spacer domain of ADAMTS13. The precise epitopes of the anti-ADAMTS13 IgGs and the mechanism underlying their inhibition activity are not fully understood. We hypothesized that inhibitory IgG autoantibodies from aTTP patients achieve their inhibitory function by binding to a discontinuous epitope in the spacer domain of ADAMTS13. To test this hypothesis, we determined the binding epitope of one out of 〉100 unique human monoclonal antibody (mAb) fragments (single-chain Fv, scFv) isolated by phage display from aTTP patients. We developed a novel hydrogen-deuterium exchange-mass spectrometry technology (HX-MS) to identify the antibody binding sites at single amino acid residue resolution. Human ADAMTS13 inhibitory scFv 4-20 was expressed in E. coli Top10 cells and purified to homogeneity by Ni-chelating affinity chromatography. In the HX-MS experiment, the mAb was coupled to affi-gel 10 resin and used to bind recombinant ADAMTS13-MDTCS fragment expressed in a stably transfected Drosophila schneider 2 (S2) cell line. After exchange with deuterium (D2O) oxide for various periods of time, the reaction was stopped, the protein was eluted, and digested to peptide fragments with pepsin, and the peptides with or without deuterium bound were resolved and identified by fast HPLC and mass spectrometry. We find that mAb scFv4-20 binds to amino acid residues Arg636, Leu637, Arg639, and Leu640 spanning from Leu632 to Leu640 (in exosite 4) in the spacer domain of ADAMTS13. This sequence is highly conserved in the ADAMTS13 spacer domains from zebrafish to mammals. In addition, mAb scFv4-20 binds Arg660, Tyr661, and Tyr665 in exosite 3, previously shown to play an important role in substrate recognition and anti-ADAMTS13 autoantibody-mediated inhibition, as well as Lys608, upstream exosites 3 and 4. Apparently, mAb scFv4-20 inhibits plasma ADAMTS13 activity (IC50 ∼0.40 nM) by binding these non-linear surface residues in the spacer domain (Fig. 1A). In agreement, site-directed mutagenesis shows that complete deletion (Δ632LTEDRLPR639) or partial deletion (Δ632LTED635 or Δ636RLPR639), or replacement of these residues with alanines (632LTED635/4A or 636RLPR639/4A) abolished or dramatically reduced mAb scFv4-20 binding. A deletion or alanine substitution of the surface residues on exosite 4 also abolished or reduced ADAMTS13 proteolytic activity toward a fluorescein-labeled VWF73 peptide and multimeric VWF (Fig. 1B), indicating that the ADAMTS13 epitope for mAb scFv4-20 is also part of ADAMTS13’s substrate recognition site. We conclude that anti-ADAMTS13 autoantibodies work by physically blocking the well-conserved VWF binding site on ADAMTS13. These results demonstrate the powerful use of HX-MS technology to determine both linear and non-linear antibody binding epitopes. The results provide valuable information concerning the mechanism of autoantibody-mediated aTTP that may be exploited to develop targeted therapy by reengineering ADAMTS13 to avoid autoantibody inhibition. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

Description: Understanding anti-Rh(D) antibodies on a molecular level would facilitate the genetic analysis of the human immune response to Rh(D), lead to the design of therapeutically useful reagents that modulate antibody binding, and provide relevant information regarding the structural organization of Rh(D) epitopes. Previously, we described a Fab/phage display-based method for producing a large array of anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single alloimmunized donor. In the current study, we present a detailed analysis of 83 randomly selected clones. Sequence analysis showed the presence of 28 unique γ1 heavy chain and 41 unique light chain gene segments. These paired to produce 53 unique Fabs that had specificity for at least half of the major Rh(D) epitopes. Surprisingly, despite this diversity, only 4 closely related heavy chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21). Similarly, nearly all Vκ light chains (15/18) were derived from one germline gene (DPK9). λ light chains showed a more diverse VL gene usage, but all (23/23) used the identical Jλ2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In particular, 2 such clones differed by only 3 residues, which resulted in a change from epD2 to epD3 specificity. These results suggest a model in which footprints of anti-Rh(D) antibodies are essentially identical to one another, and Rh(D) epitopes, as classically defined by panels of Rh(D) variant cells, are not discrete entities. Furthermore, these data imply that the epitope specificity of an anti-Rh(D) antibody can change during the course of somatic mutation. From a clinical perspective, this process, which we term epitope migration, has significance for the design of agents that modulate antibody production and for the creation of mimetics that block antibody binding in the settings of transfusion reactions and hemolytic disease of the newborn.

Description: Introduction: We previously demonstrated prolonged circulation and improved efficacy of erythrocyte (red blood cell, RBC) coupled thrombomodulin (TM) fusion proteins as thromboprophylactic and anti-inflammatory agents. To further this therapeutic platform, we produced humanized analogues and investigated their efficacy in models where RBC-coupling may provide not only a pharmacokinetic advantage, but also a pharmacodynamic advantage by local delivery to RBC membranes. Previous fusions were constructed with mouse TM fused to single-chain variable fragments (scFv) of a rat-derived anti-glycophorin A antibody. Therefore, to produce clinically translatable therapeutic fusion proteins, modification of both cargo and targeting moiety are necessary for use in humans to render them non-immunogenic and capable of binding to human RBCs. We aimed to use human-like antibodies against high-prevalence antigens, fuse these antibodies to the extracellular domain of TM, confirm their binding and enzymatic activity, and demonstrate their efficacy in whole-blood models of human vascular pathology. Methods: An IgG Fab phage display library was prepared from a Rhesus macaque immunized with human RBCs. By panning on intact RBCs, Fab/phage specific for human RBCs were identified. We selected two clones from this library, one against a high-prevalence Band 3 antigen (Wright B (Wrb), Diego blood group, 〉106 copies/RBC) and one against a high-prevalence RhCE antigen (Rh17, Rhesus blood group, ~105 copies/RBC). The variable chain sequences of these antibodies were cloned into a scFv construct and fused to the extracellular domain of human TM. Fusion proteins were produced in S2 cells using a metallothionein promoter expression system. The soluble extracellular domain of human TM was similarly cloned and produced as a control. Binding of the fusions to RBCs was measured by indirect agglutination and by ELISA with immobilize erythrocyte ghosts. Activity of the TM fusions was measured by colorimetric cleavage of an APC substrate. Adverse effects of fusions on RBCs were investigated in a model of osmotic stress in hypotonic saline as well as mechanical integrity against agitation with glass beads. Assays of endothelial protection were performed on human umbilical vein endothelial cells (HUVEC) activated lipopolysaccharide (LPS), TNF-α, heme, and HMGB1. Culture supernatants were analyzed by ELISA for IL-6, IL-8, and vWF. An endothelialized, whole-blood microfluidic platform was developed using the Bio-Flux (Fluxion) microfluidic system. Models of vascular pathology included TNF-α activation of the endothelialized channels as well as localized, light-induced injury with hematoporphyrin. Results: We successfully produced fusion proteins of human TM and human-like scFv antibody derivatives capable of specifically binding to human RBCs (Figure 1, a). The fusions demonstrated affinities suitable for translation, and enabled comparison of different levels of surface loading. The scFv/TM fusions did not induce direct RBC agglutination, and did not have adverse effects on RBC integrity under osmotic and mechanical stress. The fusions maintained similar enzymatic activity to their soluble TM counterparts (Figure 1, b) and remained active when bound to RBCs (Figure 1, c). The TM fusions also demonstrated efficacy in protection of HUVECs against activation by inflammatory mediators such as LPS and thrombin (Figure 1, d-f), both as soluble proteins and when bound to RBCs. In a whole-blood endothelialized microfluidic system, the fusions reduced fibrin deposition and channel occlusion in activated and injured endothelium. Figure 1 - (a) hTM-Wrb binds specifically to human RBC with detectable binding at