Controlling CAR-T cell activity and specificity with synthetic SparX adapters.

While conventional chimeric antigen-receptor (CAR)-T therapies have shown remarkable clinical activity in some settings, they can induce severe toxicities and are rarely curative. To address these challenges, we developed a controllable cell therapy where synthetic D-domain-containing proteins (soluble protein antigen-receptor X-linker [SparX]) bind one or more tumor antigens and mark those cells for elimination by genetically modified T cells (antigen-receptor complex [ARC]-T). The chimeric antigen receptor was engineered with a D-domain that specifically binds to the SparX protein via a unique TAG, derived from human alpha-fetoprotein. The interaction is mediated through an epitope on the TAG that is occluded in the native alpha-fetoprotein molecule. In vitro and in vivo data demonstrate that the activation and cytolytic activity of ARC-T cells is dependent on the dose of SparX protein and only occurs when ARC-T cells are engaged with SparX proteins bound to antigen-positive cells. ARC-T cell specificity was also redirected in vivo by changing SparX proteins that recognized different tumor antigens to combat inherent or acquired tumor heterogeneity. The ARC-SparX platform is designed to expand patient and physician access to cell therapy by controlling potential toxicities through SparX dosing regimens and enhancing tumor elimination through sequential or simultaneous administration of SparX proteins engineered to bind different tumor antigens.

Effect of Center of Mass Immobilization on Center of Pressure Displacement in Single and Dual-Task.

Recent research showed that artificially immobilizing the center of mass (COM) of participants in a standing position increased the center of pressure (COP) variability. This increase has been interpreted as an exploratory behavior. The objectives of this study are to investigate if this exploratory behavior is (1) reflected in other COP variables and (2) automatically controlled using a dual-task paradigm. Sixteen young adults were strapped to an apparatus which allowed them to sway freely ("unlocked") or to be immobilized ("locked") without their knowledge. Participants undertook the two phases (unlocked and locked) in a single-task and dual-task condition. Results suggested the presence of an exploratory behavior through different COP variables. Results also suggested this exploratory behavior to be automatic by nature.

The impact of eye movement on postural control depends on the type of oculomotor behavior and the visual task.

Recent evidence suggests that performing a task inducing saccades will improve stability when compared to static fixation. However, they assume the linearity of postural control by only interpreting the area of displacement and/or the velocity of sway. Conversely, non-linear measures could bring a complementary understanding of postural control. The aim is to examine the effect of eye movements on different linear and non-linear measures of stability. 21 healthy adults (24.0 ± 3.3 years) were asked to stand on a force platform with their feet together and look at the monitor in front of them. Five conditions were tested: eyes closed, random looking, fixed static point, saccade, and visual pursuit (gaze shift angle of 15°). Five 60-second trials per condition were performed. An ANOVA with repeated measures was completed for each postural control variables in each direction: antero-posterior (AP) and medio-lateral (ML). The absence of vision had a negative impact on sway, as seen by an increase in sway area and variability as well as reduced contributions from the ultra-low band. The saccade led to greater stability than the random looking, as evidenced by a smaller area. However, the visual pursuit led to decreased stability compared to random looking, as evidenced by a larger area, as well as increased variability. Of note, the energy contained in the very-low band, which indicates the contribution of the vestibular system, was highest in the visual pursuit compared to the fixed static point. The findings support that the visual system is an important, but complex contributor to stability as different eye movements result in distinct postural responses with saccade and visual pursuit causing a decrease and an increase in sway, respectively.

Preclinical Efficacy of BCMA-Directed CAR T Cells Incorporating a Novel D Domain Antigen Recognition Domain.

Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.

Modification of pharmacokinetic and abuse-related effects of cocaine by human-derived cocaine hydrolase in monkeys.

Although substantial research effort has focused on developing pharmacological treatments for cocaine abuse, no effective medications have been developed. Recent studies show that enzymes that metabolize cocaine in the periphery, forestalling its entry into the brain, can prevent cocaine toxicity and its behavioral effects in rodents. Here we report on effects of one such enzyme (Albu-CocH) on the pharmacokinetic and behavioral effects of cocaine in squirrel monkeys. Albu-CocH was developed from successive mutations of human butyrylcholinesterase (BChE) and has 1000-fold greater catalytic activity against cocaine than naturally occurring BChE. Pharmacokinetic studies showed that Albu-CocH (5 mg/kg) had a half-life of 56.6 hours in squirrel monkeys. In these studies, plasma levels of cocaine following i.v. 1 mg/kg cocaine were reduced 2 hours after administration of Albu-CocH, whereas plasma levels of the cocaine metabolite ecgonine methyl ester were increased. These effects were still evident 72 hours following Albu-CocH administration. In behavioral experiments in monkeys, pre-treatment with 5 mg/kg Albu-CocH dramatically decreased self-administration of a reinforcing dose of i.v. cocaine (30 µg/kg/injection) for over 24 hours. Pre-treatment with 5 mg/kg Albu-CocH also attenuated the reinstatement of extinguished cocaine self-administration by an i.v. priming injection of cocaine (0.1 or 0.3 mg/kg) and, in separate studies, attenuated the discriminative-stimulus effects of cocaine. The ability of Albu-CocH to attenuate the abuse-related effects of cocaine in squirrel monkeys indicates that further investigation of BChE mutants as potential treatment for cocaine abuse and toxicity is warranted.

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis.

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.

An albumin-butyrylcholinesterase for cocaine toxicity and addiction: catalytic and pharmacokinetic properties.

Butyrylcholinesterase (BChE, EC 3.1.1.8) is important in human cocaine metabolism despite its limited ability to hydrolyze this drug. Efforts to improve the catalytic efficiency of this enzyme have led to a quadruple mutant cocaine hydrolase, "CocH", that in animal models of addiction appears promising for treatment of overdose and relapse. We incorporated the CocH mutations into a BChE-albumin fusion protein, "Albu-CocH", and evaluated the pharmacokinetics of the enzyme after i.v. injection in rats. As assessed from the time course of cocaine hydrolyzing activity in plasma, Albu-CocH redistributed into extracellular fluid (16% of estimated total body water) with a t(1/2) of 0.66h and it underwent elimination with a t(1/2) of 8h. These results indicate that the enzyme has ample stability for short-term applications and may be suitable for longer-term treatment as well. Present data also confirm the markedly enhanced power of Albu-CocH for cocaine hydrolysis and they support the view that Albu-CocH might prove valuable in treating phenomena associated with cocaine abuse.

A cocaine hydrolase engineered from human butyrylcholinesterase selectively blocks cocaine toxicity and reinstatement of drug seeking in rats.

Successive rational mutations of human butyrylcholinesterase (BChE) followed by fusion to human serum albumin have yielded an efficient hydrolase that offers realistic options for therapy of cocaine overdose and abuse. This albumin-BChE prevented seizures in rats given a normally lethal cocaine injection (100 mg/kg, i.p.), lowered brain cocaine levels even when administered after the drug, and provided rescue after convulsions commenced. Moreover, it selectively blocked cocaine-induced reinstatement of drug seeking in rats that had previously self-administered cocaine. The enzyme treatment was well tolerated and may be worth exploring for clinical application in humans.

Analysis of IFN-kappa expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.

Interferon-kappa (IFN-kappa) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells (DCs). In human keratinocytes, it is produced in response to double-stranded RNA (dsRNA) and other IFNs and protects from viral infections. In monocytes and DCs, IFN-kappa induces tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) and inhibits lipopolysaccharide (LPS)-induced IL-12. In this study, we evaluated IFN-kappa expression in skin lesions of patients with common immune-mediated inflammatory disorders using immunohistochemical techniques. IFN-kappa was not detectable in healthy skin but was strongly expressed in allergic contact dermatitis and lichen planus-affected skin. IFN-kappa was localized mainly in basal and suprabasal keratinocytes and in some leukocytes infiltrating the dermis. In contrast, IFN-kappa expression in psoriatic or atopic dermatitis (AD) pidermis was weak and detectable in only 2 of 5 patients examined. Consistently, cultured keratinocytes and monocytes obtained from psoriatic and AD patients expressed null or low levels of IFN-kappa in response to IFN-gamma, which strongly upregulates IFN-kappa in normal keratinocytes. IFN-kappa accumulated in keratinocyte cytoplasm and plasma membrane, and only limited amounts were released extracellularly. Soluble IFN-kappa did not influence keratinocyte proliferation or chemokine and membrane molecule expression, and only its membrane-associated form activated IFN-stimulated response element (ISRE) signaling. Given the difference in IFN-kappa expression levels in the skin disorders examined, IFN-kappa presence or deficiency might have different pathogenetic consequences depending also on other disease-specific intrinsic alterations.

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.

Regulatory effect of IFN-kappa, a novel type I IFN, on cytokine production by cells of the innate immune system.

IFN-kappa is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-kappa on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-kappa or two recombinant IFN subtypes, IFN-beta and IFN-alpha2a. Although IFN-alpha2a failed to stimulate monocyte cytokine secretion, IFN-kappa, like IFN-beta, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-kappa was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-beta, IFN-kappa did not induce release of IFN-gamma by PBL. Expression of the IFN-kappa mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-gamma stimulation in monocytes, while IFN-beta mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-kappa was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-kappa, like IFN-beta, binds strongly to heparin (K(d): 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-kappa in monocytes, coupled with the unique induction of IFN-kappa mRNA by IFN-gamma, indicates a potential role for IFN-kappa in the regulation of immune cell functions.