Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16a/NKG2D Immune-Modulating TriNKET® CC-96191.

Increasing efforts are focusing on natural killer (NK) cell immunotherapies for AML. Here, we characterized CC-96191, a novel CD33/CD16a/NKG2D immune-modulating TriNKET®. CC-96191 simultaneously binds CD33, NKG2D, and CD16a, with NKG2D and CD16a co-engagement increasing the avidity for, and activation of, NK cells. CC-96191 was broadly active against human leukemia cells in a strictly CD33-dependent manner, with maximal efficacy requiring the co-engagement of CD16a and NKG2D. A frequent CD33 single nucleotide polymorphism, R69G, reduced CC-96191 potency but not maximal activity, likely because of reduced CD33 binding. Similarly, the potency, but not the maximal activity, of CC-96191 was reduced by high concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact activity. In the presence of CD33+ AML cells, CC-96191 activated NK cells but not T cells; while maximum anti-AML efficacy was similar, soluble cytokine levels were 10- to >100-fold lower than with a CD33/CD3 bispecific antibody. While CC-96191-mediated cytolysis was not affected by ABC transporter proteins, it was reduced by anti-apoptotic BCL-2 family proteins. Finally, in patient marrow specimens, CC-96191 eliminated AML cells but not normal monocytes, suggesting selectivity of TriNKET-induced cytotoxicity toward neoplastic cells. Together, these findings support the clinical exploration of CC-96191 as in NCT04789655.

Effect of claw horn lesion type and severity at the time of treatment on outcome of lameness in dairy cows.

Prompt diagnosis and treatment of claw horn lesions in cattle affects the likelihood of recovery; however, it is unknown if the type of lesion influences the likelihood of recovery. The aim of this study was to investigate whether the type, severity and frequency of claw horn lesions in newly lame cows (lame for no more than 2 weeks) at the time of corrective foot trimming affects the probability of recovery from lameness after treatment. The images of 112 feet (224 claws) from newly lame cows (n=112; lame in only one hind foot), which were treated with a standardised therapeutic hoof trim only, were used to score claw horn lesions (sole ulcer, sole haemorrhage, white line haemorrhage or white line separation). Most cows (n=107/112; 95.5%) were classified as mildly lame at the time of treatment. The proportion of cows that recovered 2 weeks after therapeutic hoof trimming was 88/112 (78.6%). Results of a multilevel logistic regression model indicated that severely lame cows were less likely to recover than those that were mildly lame (odds ratio, OR, 0.16; P=0.04). White line haemorrhage had a significant negative impact on the likelihood of recovery from lameness (OR 0.14; P>0.01); however, recovery of cows with white line haemorrhage was positively associated with the length of the lesion (OR 1.05; P=0.03). This latter finding may be associated with the severity of the lesion, since mild claw horn lesions affected a significantly larger area of the claw than more severe lesions. The length and type of claw horn lesion were associated with recovery from lameness.

Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein.

Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant ∼12kDa fragment was generated from ∼24kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.

Expression of invariant chain can cause an allele-dependent increase in the surface expression of MHC class I molecules.

Invariant chain (Ii) has been shown to play a significant part in the assembly of MHC class II molecules. Ii also binds to MHC class I, although it is not known when this first occurs or whether it can affect class I assembly. Our examination of lysates of L(d)-transfected T2 cells showed that Ii bound intracellularly to folded, but not to open, forms of MHC class I. Furthermore, addition of peptides to the lysates dissociated Ii from the Ii-folded MHC class I complex. Thus, unlike other known chaperones, Ii associates only with folded, peptide-free class I molecules. To determine whether Ii can affect MHC class I transport and surface expression, we used both wild-type Ii and a mutant Ii that lacked the endosomal targeting sequence. Neither Ii nor Ii(Delta 20) increased the rate of MHC class I migration; however, Ii and (to a greater extent) Ii(Delta 20) increased cell surface expression of MHC class I. In HeLa cells, this effect was allele-specific, affecting HLA-A28 more than -B75. Ii also increased the surface expression of K(b) more than D(b) on Panc02 pancreatic adenocarcinoma cells. Neither form of Ii was detectable at the cell surface with MHC class I, indicating that Ii had exercised its effect on class I intracellularly. In total, these data suggest that Ii can bind peptide-free folded class I/beta(2)m heterodimers, but not open MHC class I heavy chains, in the endoplasmic reticulum, and that Ii can facilitate the surface expression of the MHC class I molecule.