33 research outputs found
A DNA Self-Assembled Monolayer for the Specific Attachment of Unmodified Double- or Single-Stranded DNA
No full textAbstractA novel method for DNA surface immobilization and a paradigm for the attachment of unmodified DNA of any length or sequence are described herein. The development of a DNA self-assembled monolayer (DNA-SAM) that incorporates a DNA-thiol into a monolayer of inert alkane thiolates is reported. This DNA-SAM specifically hybridized complementary oligonucleotides while resisting the nonspecific adsorption of noncomplementary DNA and irrelevant proteins. Duplex DNA, having a single-stranded “capture tail,” specifically bound to the DNA-SAM if the sequence of the “tail” was complementary to DNA presented in the SAM. The sense strand of the hybridized duplex DNA could be covalently attached to the surface by an enzymatic ligation reaction (leaving the anti-sense strand dissociable). DNA-binding proteins specifically bound to these surfaces only if their cognate sites were present in the duplex DNA
The Use of Variable Density Self-Assembled Monolayers to Probe the Structure of a Target Molecule
No full textAbstractVP16, a protein encoded by herpes simplex virus, has a well-characterized 78 amino acid acidic activation domain. When tethered to DNA, tandem repeats of an eight amino acid motif taken from this region stimulate the transcription of a nearby gene. This work addresses how these minimal activation motifs interact with a putative target, the general transcription factor TATA box binding protein (TBP), and the biological relevance of this mechanism of action. I developed novel biophysical techniques to discriminate among three possible mechanistic models that describe how reiterated peptide motifs could synergistically effect transcription: 1) the peptide motifs simultaneously bind to quasi-identical sites on TBP, producing a high-affinity bivalent interaction that holds the general transcription factor near the start site of transcription; 2) the binding of one recognition motif causes an allosteric effect that enhances the subsequent binding of additional peptide motifs; or 3) a high-affinity interaction between the peptide repeats and TBP does occur, but rather than being the result of a “bivalent” interaction, it results from the summation of multiple interactions between the target protein and the entire length of the peptide. I generated self-assembled monolayers (SAMs) that presented different densities of the activation motif peptide in a two-dimensional array to test for avidity effects. Surface plasmon resonance (SPR) was used to measure the amount of target (TBP) binding as a function of the peptide density; a marked increase in avidity above a characteristic, critical peptide surface density was found. Competitive inhibition experiments were performed to compare the avidity of peptide motifs, tandemly repeated two or four times, and single motifs separated by a flexible linker. Four iterations of the motif, preincubated with TBP, inhibited its binding to high-density peptide surfaces ∼250-fold better than two iterations. Single peptide motifs joined by a flexible amino acid linker inhibited TBP binding to surface peptide nearly as well as four tandem repeats. The results favor mechanistic model 1: reiterated activation motifs interact with TBP through a high-affinity interaction that is the result of the cooperative effect of single motifs simultaneously binding to separate sites on TBP. This finding is consistent with the idea that DNA-bound activation domains trigger the transcription of a nearby gene by tethering the general transcription factor, TBP, near the start site of transcription
Abstract 3330: MUC1* targeting CAR T
No full textAbstract
Purpose: Over 75% of all solid tumor cancers are characterized by aberrant expression of MUC1. Yet to date, no effective MUC1 targeting therapeutic has succeeded. The purpose of this body of work was to develop an antibody therapeutic that would effectively treat MUC1 positive solid tumor cancers, without damaging healthy MUC1 positive tissues.
Experimental Procedures: MUC1 is a heavily glycosylated transmembrane protein whose expression is restricted to the apical border of healthy epithelial tissues but uniformly expressed over cancerous tissues. We discovered that the cancer-associated form of MUC1 is transmembrane cleavage product that remains after cleavage and release of the bulk of the extracellular domain. We named cleaved MUC1, MUC1* (muk 1 star), and showed that it is a growth factor receptor that is activated by ligand-induced dimerization of its small extracellular domain. We also showed that 100% of pluripotent human stem cells express MUC1* but cleavage stops with the onset of differentiation. By studying stem cells and cancer cells in parallel, we sought to develop antibodies that would only recognize MUC1* as it is expressed on cancerous tissues but would not recognize MUC1* as it exists on some healthy cells and tissues.
Summary New Data: We previously showed that we had developed antibodies that recognized the cleaved, growth factor receptor form, MUC1* but not full length MUC1. However, MUC1 is cleaved to a healthy form of MUC1* on, for example, pluripotent stem cells, hematopoietic stem cells and stem cells of intestinal crypts. We now report that we have developed monoclonal antibodies that only recognize the cancerous form of MUC1* and do not recognize these healthy forms of MUC1*. To demonstrate that we have succeeded in deciphering the differences between cancerous MUC1* and healthy MUC1*, we also developed a set of monoclonal antibodies that only recognize healthy MUC1*. Thousands of human cancerous versus normal tissue specimens attest to the specificity of these antibodies. Cancer-specific anti-MUC1* antibodies (Fabs) alone blocked the growth of MUC1* positive breast and hormone refractory prostate cancers in animals, with no detectable adverse effects. On the basis of tissue safety studies and efficacy studies, a clinical candidate antibody MNC2 has been identified, humanized and incorporated into more than 20 CAR constructs. huMNC2-scFv-CAR T cells display typical cytokine release only when co-cultured with MUC1* positive cancer cells. huMNC2-scFv-CAR T cells selectively kill MUC1* positive cancers, while stimulating T cell expansion.
Conclusions: The relevant target for anti-cancer drugs is the extracellular domain of MUC1*, not full length MUC1. MUC1* is a growth factor receptor that is activated by NME family growth factors. Subtle differences between MUC1* as expressed on healthy stem-like cells and MUC1* expressed on cancerous cells has allowed the development of cancer-specific MUC1* antibodies, especially suited for cancer immunotherapies.
Citation Format: Cynthia Carol Bamdad, Andrew K. Stewart, Benoit J. Smagghe, Luke T. Deary, Victoria L. Kohler, Jared L. Dietz. MUC1* targeting CAR T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2017-3330</jats:p
A Multiclass Simulation-Based Dynamic Traffic Assignment Model for Mixed Traffic Flow of Connected and Autonomous Vehicles and Human-Driven Vehicles
No full textOne of the potential capabilities of Connected and Autonomous Vehicles (CAVs)
is that they can have different route choice behavior and driving behavior
compared to human Driven Vehicles (HDVs). This will lead to mixed traffic flow
with multiple classes of route choice behavior. Therefore, it is crucial to
solve the multiclass Traffic Assignment Problem (TAP) in mixed traffic of CAVs
and HDVs. Few studies have tried to solve this problem; however, most used
analytical solutions, which are challenging to implement in real and large
networks (especially in dynamic cases). Also, studies in implementing
simulation-based methods have not considered all of CAVs' potential
capabilities. On the other hand, several different (conflicting) assumptions
are made about the CAV's route choice behavior in these studies. So, providing
a tool that can solve the multiclass TAP of mixed traffic under different
assumptions can help researchers to understand the impacts of CAVs better. To
fill these gaps, this study provides an open-source solution framework of the
multiclass simulation-based traffic assignment problem for mixed traffic of
CAVs and HDVs. This model assumes that CAVs follow system optimal principles
with rerouting capability, while HDVs follow user equilibrium principles.
Moreover, this model can capture the impacts of CAVs on road capacity by
considering distinct driving behavioral models in both micro and meso scales
traffic simulation. This proposed model is tested in two case studies which
shows that as the penetration rate of CAVs increases, the total travel time of
all vehicles decreases
A minimal fragment of MUC1 mediates growth of cancer cells.
No full textThe MUC1 protein is aberrantly expressed on many solid tumor cancers. In contrast to its apical clustering on healthy epithelial cells, it is uniformly distributed over cancer cells. However, a mechanistic link between aberrant expression and cancer has remained elusive. Herein, we report that a membrane-bound MUC1 cleavage product, that we call MUC1*, is the predominant form of the protein on cultured cancer cells and on cancerous tissues. Further, we demonstrate that transfection of a minimal fragment of MUC1, MUC1*(1110), containing a mere forty-five (45) amino acids of the extracellular domain, is sufficient to confer the oncogenic activities that were previously attributed to the full-length protein. By comparison of molecular weight and function, it appears that MUC1* and MUC1*(1110) are approximately equivalent. Evidence is presented that strongly supports a mechanism whereby dimerization of the extracellular domain of MUC1* activates the MAP kinase signaling cascade and stimulates cell growth. These findings suggest methods to manipulate this growth mechanism for therapeutic interventions in cancer treatments
Abstract CT232: Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*)
No full textMUC1* mediates the growth of human pluripotent stem cells.
No full textThe MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions
