Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp-lpr/lpr (MRL/lpr) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo.

Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells. MGD006 is designed to be rapidly cleared, and therefore requires continuous delivery. In a mouse model of continuous administration, MGD006 eliminated engrafted KG-1a cells (an AML-M0 line) in human PBMC (peripheral blood mononuclear cell)-reconstituted NSG/β2m(-/-) mice at doses as low as 0.5 μg/kg per day for ~7 days. MGD006 binds to human and cynomolgus monkey antigens with similar affinities and redirects T cells from either species to kill CD123-expressing target cells. MGD006 was well tolerated in monkeys continuously infused with 0.1 μg/kg per day escalated weekly to up to 1 μg/kg per day during a 4-week period. Depletion of circulating CD123-positive cells was observed as early as 72 hours after treatment initiation and persisted throughout the infusion period. Cytokine release, observed after the first infusion, was reduced after subsequent administrations, even when the dose was escalated. T cells from animals with prolonged in vivo exposure exhibited unperturbed target cell lysis ex vivo, indicating no exhaustion. A transient decrease in red cell mass was observed, with no neutropenia or thrombocytopenia. These studies support clinical testing of MGD006 in hematological malignancies, including AML.

Background: Acute myeloid leukemia (AML) blast and leukemic stem cells highly express CD123, which is associated with high-risk disease and disease progression. CD123 expression on normal hematopoietic stem cells is minimal, enabling a strategy of preferential ablation of AML with a CD123-targeted approach. Flotetuzumab (MGD006/S80880) is a novel T-cell redirecting (CD123 x CD3) bispecific DART® protein being tested in a phase 1 study in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: This phase 1 dose-escalation study is designed to define the safety profile, maximum tolerated dose and schedule (MTDS), and preliminary anti-leukemic activity of flotetuzumab. Relapsed/refractory (R/R) AML or intermediate-2/high-risk MDS patients (pts) are treated on 28-day cycles at doses from 3-1000ng/kg/day on one of 2 dosing schedules (4-day on/3-day off or a continuous 7-day on schedule). To mitigate cytokine-release syndrome (CRS), a one-step lead-in dose (LID) (100ng/kg/day for 4 days) or two-step LID (30 ng/kg/day for 3 days followed by 100ng/kg/day for 4 days) was instituted during Cycle 1/Week 1 (C1W1), followed by the cohort target dose (300-1000ng/kg/day) on either of the dosing schedules on W2-4. Cycle 2 and beyond, all pts were treated on a 4-day on/3-day off schedule at the cohort target dose for a maximum of 12 cycles, with 2 cycles after a complete response or complete remission with incomplete blood count recovery. Steroid-sparing, anti-cytokine therapy was used, if clinically indicated, to manage CRS symptoms. Disease status was assessed by International Working Group (IWG) criteria. Samples were collected for pharmacokinetics, anti-drug antibodies (ADA) and cytokine analysis, including IL-2, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and GM-CSF. Results: 45 pts with R/R AML/MDS (89% AML and 11% MDS) have been treated with flotetuzumab, median age of 64 (29-84), and 44% female. The MTDS has been reached at 500ng/kg/day. Overall flotetuzumab has demonstrated manageable toxicity; drug-related adverse events ≥G3 were observed in 20/45 (44%) pts; infusion-related reaction/CRS (IRR/CRS), the most common toxicity, was observed in 34/45 (76%) pts (G3 in 6/45, 13%). The most frequent CRS symptoms were pyrexia (15), chills (10), tachycardia (10), and hypotension (4). Cytokine levels were higher in pts with CRS than in pts without (median IL-6, 116.2 vs. 67.9 pg/mL; IL-8, 191.1 vs. 144.6 pg/mL; IL-10, 867.6 vs. 348.7 pg/mL) and were generally higher with increasing CRS grade. A two-step LID during week 1 appeared to decrease the severity of CRS grade (mean grade reduction 0.54) compared to pts that received a one-step LID during cycle 1. In addition, lower median peak cytokine levels are observed with two-step LID during W1 and after achieving W2 target dose. Fourteen pts treated at the threshold 500 ng/kg/day dose cohort and beyond (700ng/kg/day dose cohort) have completed at least one cycle of treatment and had a post-treatment bone marrow (BM) biopsy. Anti-leukemic activity was documented in 57% (8/14) pts, 6/14 reached IWG criteria (3 CR, 1 CRi, 1 MLF, 1 PR) for an overall response rate (ORR) of 43%, and 2 pts had stable disease and bone marrow (BM) blast reduction of 20 and 25% from baseline, respectively. Blast reduction occurred rapidly, often within one cycle of therapy, and extended beyond flotetuzumab discontinuation. Multispectral immunohistochemistry analysis of BM samples showed flotetuzumab in situ with a significant increase (in CD-8 T cells (1.58-fold increase, p=0.0013). Consistent with T-cell activation, CD-25, CD-69 and PD-1 upregulation on both CD-4 and CD-8 T-cells was also observed in peripheral blood samples. Conclusions: Flotetuzumab in R/R AML and MDS demonstrated evidence of anti-leukemic activity (ORR 43%) with a manageable safety profile. This program advances an immune-activating agent in treating AML and continues to enroll patients in cohort expansion (24 AML and 24 MDS patients at the MTDS) in the US and Europe. clinicaltrials.gov NCT02152956. Disclosures Uy: Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport. Foster: Macrogenics: Research Funding; Shire: Honoraria; Pfizer: Research Funding; Amgen: Honoraria; Incyte: Honoraria; Celgene: Research Funding; Celator: Research Funding. Arellano: Cephalon Oncology: Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Topp: Roche: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Research Funding; Macrogenics: Consultancy, Research Funding; Celgene: Other: Travel; Regeneron: Consultancy, Honoraria, Research Funding. Martinelli: Incyte, Pfizer, MSD, Abbvie, J&J, Seattle Genetics, Jazz Pharmaceuticals, Astellas: Consultancy, Other: Advisory Board, Speakers Bureau. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Lelièvre: Institut de recherches international Servier: Employment. La Motte-Mohs: Sunnybrook Health Sciences Centre: Patents & Royalties; MacroGenics: Employment, Equity Ownership, Patents & Royalties. Sun: Macrogenics Inc: Employment, Equity Ownership. Baughman: MacroGenics, Inc.: Employment. Shannon: MacroGenics, Inc.: Employment. Fox: Bristol Myers-Squibb: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy, Research Funding; PerkinElmer: Consultancy, Research Funding; Janssen/Johnson and Johnson: Consultancy, Research Funding; Argos: Consultancy; Bayer: Consultancy; Definiens: Consultancy; OncoSec: Consultancy, Research Funding; PrimeVax: Consultancy, Equity Ownership; Peregrine: Consultancy; UbiVac: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and managing Member of LLC; Ventana/Roche: Consultancy; Viralytics: Consultancy, Research Funding. Bonvini: MacroGenics, Inc.: Employment, Equity Ownership, Research Funding. Wigginton: MacroGenics: Employment, Equity Ownership. Davidson-Moncada: MacroGenics: Employment, Equity Ownership.

among species and populations can be understood as adaptations. But at some stages of evolution certain characters effectively "click in" and remain fixed in the descendent group of species (5-7). For instance, And the impact of repair machinery on the quentially during clonal expansion, and the H, clarke J, Exp, Med, 161, 687 (1985).immune response needs clarification. Indeed, influence of selection is seen by the distribu-6. D. J. McKean eta/., J. k p . Med. 161,687 (1985). there is a discrepancy between the observed tion of mutations leading to amino acid sub- 7.mutation frequency and that expected in the stitutions, known as replacement (R) muta-85, 107 (1996); J, F, McBlane 83, 387 absence of Pms2 activity. Loss of Pms2 functions. V genes expressed in expanded clones (1995).

Mouse CD1.1 is an MHC class I-like, non-MHC-encoded, surface glycoprotein that can be recognized by T cells, in particular NK1.1+ T cells, a subset of αβ T cells with semiinvariant TCRs that promptly releases potent cytokines such as IL-4 and IFN-γ upon stimulation. To gain insight into the function of CD1.1, a panel of nine mAbs was generated and used to biochemically characterize and monitor the surface expression of CD1.1 on different cell types. CD1.1 is a heavily glycosylated, β2-microglobulin-associated surface protein. Its recognition by a panel of 12 Vα14-positive and -negative CD1-specific αβ T cell hybridomas was blocked by two groups of mAbs that bound to adjacent clusters of epitopes, indicating that different αβ TCRs bind to the same region of CD1.1, presumably above the groove. Remarkably, CD1.1 was mainly expressed by dendritic cells, B cells, and macrophages, suggesting a function in Ag presentation to Th cells. Furthermore, the cell type that expressed the highest levels of CD1.1 was the splenic marginal zone B cell, a distinct subset of B cells that also expresses CD21 (the C3d receptor) and may be involved in natural responses to bacterial Ags. Altogether, the results support the idea that CD1.1 may function in recruiting a form of innate help from specialized cytokine producer αβ T cells to APCs, a role that might be important at the preadaptive phase of immune responses to some microbial pathogens.