Harbani Kaur Malik-Chaudhry, Ph.D

The use of recombinant interleukin‑2 (IL‑2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor‑regression in cancer patients. The adverse events and limited efficacy of IL‑2 treatment are due to the preferential binding of IL‑2 to cells that express the high‑affinity, trimeric receptor, IL‑2Rαβγ such as endothelial cells and T‑regulatory cells, respectively. Here, we describe a novel bispecific heavy‑chain only antibody… Show more

The use of recombinant interleukin‑2 (IL‑2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor‑regression in cancer patients. The adverse events and limited efficacy of IL‑2 treatment are due to the preferential binding of IL‑2 to cells that express the high‑affinity, trimeric receptor, IL‑2Rαβγ such as endothelial cells and T‑regulatory cells, respectively. Here, we describe a novel bispecific heavy‑chain only antibody which binds to and activates signaling through the heterodimeric IL‑2Rβγ receptor complex that is expressed on resting T‑cells and NK cells. By avoiding binding to IL‑2Rα, this molecule circumvents the preferential T‑reg activation of native IL‑2, while maintaining the robust stimulatory effects on T‑cells and NK‑cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL‑2R agonist that harnesses the benefits of the IL‑2 signaling pathway as a potential anti‑cancer therapy. Show less

The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies… Show more

The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy. Show less

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In… Show more

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode. Show less

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of… Show more

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of protein interactions with the engineered FRET pair, CyPet and YPet. The strong FRET signal from this pair made these developments possible. However, the direct link of fluorescent proteins with proteins of interests may interfere with the folding of some fusion proteins. Here, we report a new protein engineering strategy for improving FRET signals by adding a linker between the fluorescent protein and the targeted protein. This improvement allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. Three linkers, LAEAAAKEAA, TSGSPGLQEFGT, and LAAALAAA, which are alpha helix or random coil, all significantly improved the FRET signals. Our results show a general methodology for improving trans-FRET signals to effectively determine biochemical reaction intermediates. Show less

The ubiquitin-proteasome system (UPS) and UBL
pathways, such as SUMOylation and NEDDylation
are critical in protein homeostasis and activities
in vivo and are emerging as the target of new
strategies to treat many acute and chronic human
diseases, such as infections, inflammation and
cancers. Cytokines, including interferons (IFNs)
and Toll-like receptors, are the first line of
defense for host and have crucial roles in inducing
immune responses to the invading… Show more

The ubiquitin-proteasome system (UPS) and UBL
pathways, such as SUMOylation and NEDDylation
are critical in protein homeostasis and activities
in vivo and are emerging as the target of new
strategies to treat many acute and chronic human
diseases, such as infections, inflammation and
cancers. Cytokines, including interferons (IFNs)
and Toll-like receptors, are the first line of
defense for host and have crucial roles in inducing
immune responses to the invading pathogens.
SUMOylation inhibits the signaling pathways of
IFNs and Toll-like receptors, the JAK-STAT and
the NFκB pathways, respectively. NEDDylation
is required for activating the Cullin family
proteins, which mediate protein degradation as
part of apoptosis in many solid tumors. Although
only one proteasome inhibitor with a novel
mechanism has been approved for marketing so
far, targeting SUMOylation and other UBLs could
lead to a new paradigm for therapeutic agents for
a variety of pathological conditions. Recently,
the first NEDDylation inhibitor, MLN4924, was
shown to have great potential as a novel anti-tumor
drug and is in clinical development. A family of
other UBLs is emerging as different protein
modifiers for different biological processes and
may serve as potential drug targets in the future. Show less

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging… Show more

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples. Show less