Developing a dual VEGF/PDL1 inhibitor based on high-affinity scFv heterodimers as an anti-cancer therapeutic strategy

Abstract Cancer progression is enhanced by the interaction of programmed death-ligand 1 (PDL1), which is associated with inhibition of the immune response against tumors, and vascular endothelial growth factor (VEGF), which inhibits immune cell activity while inducing angiogenesis and proliferation of cancer cells. Dual inhibition of PDL1 and VEGF may therefore confer a synergistic anti-cancer therapeutic effect. We present a novel strategy for developing a therapeutic that simultaneously binds and inhibits both PDL1 and VEGF. We generated a bi-specific protein, designated DuRan-Bis, comprising a single chain variable fragment (scFv)-based inhibitor of PDL1 fused to an scFv-based inhibitor of VEGF, with the latter being attached to an Fc fragment. We found that DuRan-Bis binds to both PDL1 and VEGF with high affinity. Compared to treatments with mono-specific proteins, alone or in combination, the DuRan-Bis chimera showed superior inhibition of the proliferation of glioblastoma cells. In comparison to treatment with immune cells alone, a combination of immune cells with DuRan-Bis decreased the viability of head and neck cancer cells. To the best of our knowledge, this study is the first to use a single polypeptide chain scFv-scFv-Fc scaffold for engineering a high-affinity bi-specific inhibitor of PDL1 and VEGF

The Contribution of the Minimal Promoter Element to the Activity of Synthetic Promoters Mediating CAR Expression in the Tumor Microenvironment

Harnessing immune effector cells to benefit cancer patients is becoming more and more prevalent in recent years. However, the increasing number of different therapeutic approaches, such as chimeric antigen receptors and armored chimeric antigen receptors, requires constant adjustments of the transgene expression levels. We have previously demonstrated it is possible to achieve spatial and temporal control of transgene expression as well as tailoring the inducing agents using the Chimeric Antigen Receptor Tumor Induced Vector (CARTIV) platform. Here we describe the next level of customization in our promoter platform. We have tested the functionality of three different minimal promoters, representing three different promoters’ strengths, leading to varying levels of CAR expression and primary T cell function. This strategy shows yet another level of CARTIV gene regulation that can be easily integrated into existing CAR T systems

Poly Q aggregates lead to depletion of histone ubiquitination.

<p>U2OS cells were transiently transfected with Htt-Q91-Cherry and fixed for immunofluorescence with an antibody that identifies ubiquitinated H2B <b>(A)</b> and ubiquitinated H2AX The staining of the IB with the Ubi-H2B and Ubi-H2AX antibodies is likely due to cross reaction of these antibodies with the high concentrations of ubiquitin on the aggregates <b>(B)</b>. Cells were either imaged under the microscope <b>(A,B)</b> or analyzed in bulk by flow cytometry. The red bars indicate cells that lack aggregates and the blue bars cells with aggregates. The presented experiment is a representative of three repeats <b>(C)</b>.</p

Firefly luciferase aggregates are associated with histone de-ubiquitination and a compromised DNA damage response.

<p>U2OS Cells were transiently transfected with FLUC-DM-YFP and fixed for immunofluorescence with an Fk2, an antibody that identifies ubiquitinated proteins <b>(A)</b> and ubiquitinated H2AX <b>(B)</b>. U2OS cells were transiently transfected with FLUC-DM-YFP, treated with the DNA damaging agent neocarzinostatin and fixed at indicated time points <b>(C,E)</b>. Cells were either stained with an antibody against phosphor-gamma-H2AX and analyzed by flow cytometry <b>(C)</b> The presented experiment is a representative of three repeats. Alternatively, cells were stained or against the 53BP1<b>(D)</b>, Cells from three experiments were counted and quantified <b>(E)</b>. Error bars represent SE, three repeats.</p

Poly Q aggregates accumulate ubiquitin and lead to depletion of nuclear ubiquitin.

<p>Cells stably expressing YFP-Ubi were transiently transfected with Htt-Q91-Cherry and followed by live cell imaging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169054#pone.0169054.s006" target="_blank">S3 Mov</a>). Shortly after the Htt-Q91-Cherry aggregates it starts to accumulate ubiquitin. T = 0 was arbitrarily set to the time protein aggregation started in this particular cell. Strikingly YFP-Ubi staining in the nucleus is depleted <b>(A)</b>. U2OS Cells were transiently transfected with Htt-Q91-Cherry and fixed for immunofluorescence with an antibody that identifies ubiquitinated proteins (Fk2). The experiment was repeated in N2A and PC12 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169054#pone.0169054.s002" target="_blank">S2 Fig</a>). In each of these lines the cells with the IB lack nuclear ubiquitin staining compared to the non-expressing cells. <b>(B)</b>. U2OS Cells were transiently transfected with untagged Htt-Q91 and fixed for immunofluorescence with Fk2. Ubiquitin accumulated on the untagged IB and was depleted from the nucleus like with the Htt-Q91-Cherry <b>(C)</b>. U2OS cells were co-transfected with Ubi[9]-mRFP[1] and YFP-Htt-Q91. Overexpression of ubiquitin reduces depletion of nuclear ubiquitin by Htt-Q91 in spite of the accumulation of ubiquitin on the IB <b>(D)</b>.</p

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response

<div><p>Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington’s disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.</p></div

Inducible expression of Poly Q aggregates leads to depletion of histone ubiquitination.

<p>U2OS cells expressing an inducible Htt-Q91-Cherry vector were induced with dox. Cells were fixed and stained with Fk2 antibodies that identify ubiquitinated proteins B, antibodies against ubiquitinated H2B <b>(B)</b> and ubiquitinated H2AX <b>(C)</b>. Cells were imaged under the microscope <b>(B,C)</b>. Several hundred cells in multiple fields were photographed and quantified by ImageJ. The red bars indicate cells that lack aggregates (normalized to 1) and the blue bars cells with aggregates, error bars are SE <b>(D)</b>.</p

Cells with Poly Q aggregates have an impaired DNA damage response, which can be partially rescued by ubiquitin over-expression.

<p>U2OS cells were transiently transfected with Htt-Q91-Cherry, treated with DNA damaging agent neocarzinostatin and fixed at indicated time points. Cells were stained with an antibody against the early marker for DDR phosphor-gamma-H2AX. The red line indicates cells that lack aggregates and the blue line cells with aggregates. The presented experiment is a representative of three repeats <b>(A, B).</b> U2OS cells were co-transfected with Ubi[9]-mRFP[1] (red) and Htt-Q91-Venus treated with neocarzinostatin and fixed after one hour <b>(C)</b>. Cells transiently transfected with Htt-Q91-Cherry, and treated with neocarzinostatin were fixed at indicated time points and stained with antibodies against the late DDR marker 53BP1 <b>(D, E)</b>. Cells were either imaged by microscope <b>(A, C, D, E)</b> or analyzed by flow cytometry. The red bar indicates cells that lack aggregates and the blue bar cells with aggregates. Number of foci of 53BP1 were counted manually, error bars represent SE, three repeats <b>(E)</b>.</p