Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.

• Bispecific antibodies binding CD3 and CLL-1 deplete CLL-1 1 target cells in animal models.• An appropriately engineered CLL-1/CD3 bispecific antibody could be effective in treating AML.Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the highand low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. (Blood. 2017;129(5):609-618)

DNA microarrays enable users to obtain information on differences in transcript abundance on a massively parallel scale. Recently, however, data analyses have revealed potential pitfalls related to image acquisition, variability and misclassifications in replicate measurements, cross-hybridization and sensitivity limitations. We have generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. Together, we have used these tools to optimize performance in an expression profiling study. We demonstrate three significant advantages of the Motorola CodeLink platform: sensitivity of one copy per cell, coefficients of variation of 10% in the hybridization signals across slides and across target preparations, and specificity in distinguishing highly homologous sequences. Slides where oligonucleotide probes are spotted in 6-fold redundancy were used to demonstrate the effect of replication on data quality. Lastly, the differential expression ratios obtained with the CodeLink expression platform were validated against those obtained with quantitative reverse transcription-PCR assays for 54 genes.

An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linkedread de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those~20 million years divergent.