Development of CDX-1140, an agonist CD40 antibody for cancer immunotherapy.

Limitations of immunotherapy include poorly functioning events early in the immune response cycle, such as efficient antigen presentation and T cell priming. CD40 signaling in dendritic cells leads to upregulation of cell surface costimulatory and MHC molecules and the generation of cytokines, which promotes effective priming of CD8+ effector T cells while minimizing T cell anergy and the generation of regulatory T cells. This naturally occurs through interaction with CD40 ligand (CD40L) expressed on CD4+ T-helper cells. CD40 signaling can also be achieved using specific antibodies, leading to several agonist CD40 antibodies entering clinical development. Our approach to select a CD40 agonist antibody was to define a balanced profile between sufficiently strong immune stimulation and the untoward effects of systemic immune activation. CDX-1140 is a human IgG2 antibody that activates DCs and B cells and drives NFkB stimulation in a CD40-expressing reporter cell line. These activities are Fc-independent and are maintained using an F(ab')2 fragment of the antibody. CDX-1140 binds outside of the CD40L binding site, and addition of recombinant CD40L greatly enhances DC and B activation by CDX-1140, suggesting that CDX-1140 may act synergistically with naturally expressed CD40L. CDX-1140 also has both direct and immune-mediated anti-tumor activity in xenograft models. CDX-1140 does not promote cytokine production in whole blood assays and has good pharmacodynamic and safety profiles in cynomolgus macaques. These data support the potential of CDX-1140 as part of a cancer therapy regimen, and a phase 1 trial has recently commenced.

Excessive peptidergic sensory innervation of cutaneous arteriole-venule shunts (AVS) in the palmar glabrous skin of fibromyalgia patients: implications for widespread deep tissue pain and fatigue.

OBJECTIVE: To determine if peripheral neuropathology exists among the innervation of cutaneous arterioles and arteriole-venule shunts (AVS) in fibromyalgia (FM) patients. SETTING: Cutaneous arterioles and AVS receive a convergence of vasoconstrictive sympathetic innervation, and vasodilatory small-fiber sensory innervation. Given our previous findings of peripheral pathologies in chronic pain conditions, we hypothesized that this vascular location may be a potential site of pathology and/or serotonergic and norepinephrine reuptake inhibitors (SNRI) drug action. SUBJECTS: Twenty-four female FM patients and nine female healthy control subjects were enrolled for study, with 14 additional female control subjects included from previous studies. AVS were identified in hypothenar skin biopsies from 18/24 FM patient and 14/23 control subjects. METHODS: Multimolecular immunocytochemistry to assess different types of cutaneous innervation in 3 mm skin biopsies from glabrous hypothenar and trapezius regions. RESULTS: AVS had significantly increased innervation among FM patients. The excessive innervation consisted of a greater proportion of vasodilatory sensory fibers, compared with vasoconstrictive sympathetic fibers. In contrast, sensory and sympathetic innervation to arterioles remained normal. Importantly, the sensory fibers express α2C receptors, indicating that the sympathetic innervation exerts an inhibitory modulation of sensory activity. CONCLUSIONS: The excessive sensory innervation to the glabrous skin AVS is a likely source of severe pain and tenderness in the hands of FM patients. Importantly, glabrous AVS regulate blood flow to the skin in humans for thermoregulation and to other tissues such as skeletal muscle during periods of increased metabolic demand. Therefore, blood flow dysregulation as a result of excessive innervation to AVS would likely contribute to the widespread deep pain and fatigue of FM. SNRI compounds may provide partial therapeutic benefit by enhancing the impact of sympathetically mediated inhibitory modulation of the excess sensory innervation.

The evolution and multi-molecular properties of NF1 cutaneous neurofibromas originating from C-fiber sensory endings and terminal Schwann cells at normal sites of sensory terminations in the skin.

In addition to large plexiform neurofibromas (pNF), NF1 patients are frequently disfigured by cutaneous neurofibromas (cNF) and are often afflicted with chronic pain and itch even from seemingly normal skin areas. Both pNFs and cNF consist primarily of benign hyperproliferating nonmyelinating Schwann cells (nSC). While pNF clearly arise within deep nerves and plexuses, the role of cutaneous innervation in the origin of cNF and in chronic itch and pain is unknown. First, we conducted a comprehensive, multi-molecular, immunofluorescence (IF) analyses on 3mm punch biopsies from three separate locations in normal appearing, cNF-free skin in 19 NF1 patients and skin of 16 normal subjects. At least one biopsy in 17 NF1 patients had previously undescribed micro-lesions consisting of a small, dense cluster of nonpeptidergic C-fiber endings and the affiliated nSC consistently adjoining adnexal structures-dermal papillae, hair follicles, sweat glands, sweat ducts, and arterioles-where C-fiber endings normally terminate. Similar micro-lesions were detected in hind paw skin of mice with conditionally-induced SC Nf1-/- mutations. Hypothesizing that these microlesions were pre-cNF origins of cNF, we subsequently analyzed numerous overt, small cNF (s-cNF, 3-6 mm) and discovered that each had an adnexal structure at the epicenter of vastly increased nonpeptidergic C-fiber terminals, accompanied by excessive nSC. The IF and functional genomics assays indicated that neurturin (NTRN) and artemin (ARTN) signaling through cRET kinase and GFRα2 and GFRα3 co-receptors on the aberrant C-fiber endings and nSC may mutually promote the onset of pre-cNF and their evolution to s-cNF. Moreover, TrpA1 and TrpV1 receptors may, respectively, mediate symptoms of chronic itch and pain. These newly discovered molecular characteristics might be targeted to suppress the development of cNF and to treat chronic itch and pain symptoms in NF1 patients.

Development of a Novel Antibody-Drug Conjugate for the Potential Treatment of Ovarian, Lung, and Renal Cell Carcinoma Expressing TIM-1.

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody-drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1-expressing cell lines, but not on TIM-1-negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1-expressing tumors. Mol Cancer Ther; 15(12); 2946-54. ©2016 AACR.

Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes.

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.