Synthetic integrin-binding immune stimulators target cancer cells and prevent tumor formation

Immuno-oncology approaches mainly utilize monoclonal antibodies or protein-based scaffolds that bind with high affinity to cancer cells and can generate an immune response. Peptides can also bind with high affinity to cancer cells and are intermediate in size between antibodies and small molecules. They are also synthetically accessible and therefore easily modified to optimize their stability, binding affinity and selectivity. Here we describe the design of immune system engagers (ISErs), a novel class of synthetic peptide-based compounds that bind specifically to cancer cells and stimulate the immune system. A prototype, Y9, targets integrin alpha(3), which is overexpressed on several cancer cells, and activates the immune system via a formyl methionine-containing effector peptide. Injection of Y9 leads to immune cell infiltration into tissue and prevents tumor formation in a guinea pig model. The antitumor activity and synthetic accessibility of Y9 illustrate that ISErs could be applied to a wide variety of targets and diseases

Activation of PAK by Rac1 and Rac1 S71E.

<p>The effect of Rac1 and Rac1 S71E on PAK phosphorylation was shown in HEp2 cells stably expressing either GTPase. A) A brief characterization of these stable transfected cell lines was done by scanning electron microscopy showing surface topology of the cells. B) Immunoblot analysis revealed comparable ectopic expression of HA-tagged Rac1 and Rac1 S71E and concomitant Ser-144 phosphorylation of PAK1 and Thr-402 phosphorylation of PAK2. C) densitometrical evaluation of three separate Immunoblots showing phosphorylation of PAK 1/2. Shown are mean values ± SD. D) Propidium iodide staining of stable cell lines indicates populations of cells with 2n (G1 phase) or 4n (G2/M phase) set of chromosomes. Shown are percentages of cells within different cell cycle phases (mean values of five separate experiments).</p

Activity status of Rac1 S71E and Cdc42 S71E.

<p>A) Cell lysates of <i>Rac1^fl/fl</i> and <i>rac1</i>^−/− mouse fibroblasts were analyzed for Ser-71 phosphorylation of Rac1 and Cdc42 after treatment with 100 ng/ml EGF for 2 h by immunoblot using anti-pRac1/pCdc42 (Ser71) antibody. Lack of specific signal in <i>rac1</i>^−/− cells strongly suggested specific phosphorylation of Rac1 and no detectable phosphorylation of Cdc42. Immunoblot is representative for three separate experiments B) GTP-binding of phosphomimetic (▴) Rac1 S71E and Cdc42 S71E in comparison with wild-type (○) Rac1 and Cdc42 was analyzed by a [γ-³²P]-GTP-binding assay. The diagrams show mean values ± SD of three separate experiments. C) Intracellular localization of pRac1/pCdc42 (Ser71). Phosphorylated Rac1/Cdc42 is exclusively present in the membrane fraction of cells. D) Pull down assay of pRac1/pCdc42 and Rac1 using PAK-PBD after overexpression of Rho-GDI. Total Rac, total pRac1/pCdc42, and expression of Rho GDI were checked by immunoblot of whole cell lysate (lower panel). E) Pull down assay in a recombinant system showed nucleotide dependent binding of wild-type Rac1, Rac1 S71E and Rac1 S71A to the PAK-p21 binding domain. E) The nucleotide-dependent binding of Rac1, Rac1 S71E and Rac1 S71A to full length PAK1 as determined by pull down experiments with HEp2 cell lysates using GTPases as bait.</p

GTPase-specific binding to their effectors.

<p>A) Coomassie-stained SDS-gel of precipitates of pull down experiments from HEp2 cell lysate. Arrows indicate GST-Rac1/−Rac1 S71E (48 kDa) used as bait and a coprecipitated 190 kDa protein that was identified as IQGAP1 by MALDI-TOF/TOF analysis. B) The interaction of active, GTP-bound Rac1/Cdc42 and their active forms (Q61L) with specific effectors was analyzed by immunoblot analyses of precipitates from pull down assays. Non-specific binding was tested by GST-loaded glutathione beads as control. C) Representative input control of pull down analyses using constitutively active (Q61L) mutants of Rac1 and Cdc42 and their S71E mutants. D) Representative immunoblots of pull down precipitates showing the interaction of constitutively active Rac1 and Cdc42 and constitutively active S71E mutants with their effector proteins. Rac1 Q61L/S71E and Cdc42 Q61L/S71E did hardly bind to their specific effectors Sra-1 and N-WASP, respectively and to their common effector protein PAK1. Both phosphomimetic GTPases, however bound to their common effectors IQGAP and MRCK alpha, although to a lesser extent. The bars show the arithmetic mean value ± SD of densitometrical evaluation of 3 independent experiments.</p

NF-κB is activated by phosphorylated Rac1.

<p>HEK 293 cells were co-transfected with NF-κB luciferase reporter plasmid as well as with Rac1 and Cdc42 mutants. Cells were lysed after 40 hours and analyzed for luciferase activity. Relative fold activity of mock-transfected cells is shown (arithmetic means±SD, n = 4). B) Expression of HA-tagged GTPases of quadruplicate samples from reporter gene assays was visualized by immunoblot using an anti-HA antibody.</p

Phosphomimetic Rac1 S71E induces filopodia formation.

<p>A) Treatment with 100 ng/ml EGF for 2 h induces pronounced formation of filopodia. Cells were stained for nuclei (DAPI, blue), actin cytoskeleton (rhodamin-phalloidin, red), and VASP (Alexa-488, green). B) HEp2 cells transfected with HA-tagged Rac1, Rac1 S71E, Cdc42, and Cdc42 S71E. Expression of GTPases was visualized by HA-staining, the actin cytoskeleton was stained with rhodamin-phalloidin. Only Rac1 S71E induced morphotype that is comparable with EGF-induced alterations. C) Phenotypes of HEp2 cells transfected with HA-tagged constitutive active mutants of Rac1 and Cdc42 as well as their phosphomimetic mutants S71E. Constitutively active (Q61L) Rac1 induced membrane ruffling whereas Rac1 S71E induced formation of filopodia. Filopodia formation is less pronounced in Cdc42 Q61L and Cdc42 Q61L/S71E transfected cells. Stained are nuclei (blue) and HA-tag (green); bar represents 10 µm. D) Active, GTP-bound form of Cdc42 was determined by G-LISA 24 h post transfection with constructs as indicated. Cdc42 Q61L was used for transfection experiments as positive control for experimental setup. Additionally, <i>C. difficile</i> toxin A (TcdA) was used as negative control for inactivation of Cdc42. The bar chart shows mean values ± SD of three (for TcdA) or four separate experiments.</p

Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation

Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac – although not essential – can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration