Jennifer L. Stevenson

Differential Metastasis Inhibition by Clinically Relevant Levels of Heparins—Correlation with Selectin Inhibition, Not Antithrombotic Activity

Purpose: Unfractionated heparin reduces metastasis in many murine models. Multiple mechanisms are proposed, particularly anticoagulation and/or inhibition of P-selectin and L-selectin. However, the doses used are not clinically tolerable and other heparins are now commonly used. We studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. Experimental Design: Five clinically approved heparins were evaluated for inhibition of P-selectin and L-selectin binding to carcinoma cells. Pharmacokinetic studies determined optimal dosing for clinically relevant anticoagulant levels in mice. Experimental metastasis assays using carcinoma and melanoma cells investigated effects of a single injection of various heparins. Heparins were compared for structural relationships to selectin inhibition. Results: One (Tinzaparin) of three low molecular weight heparins showed increased selectin inhibitory activity, and the synthetic pentasaccharide, Fondaparinux, showed none when normalized to anticoagulant activity. Experimental metastasis models showed attenuation with unfractionated heparin and Tinzaparin, but not Fondaparinux, at clinically relevant anticoagulation levels. Tinzaparin has a small population of high molecular weight fragments not present in other low molecular weight heparins, enriched for selectin inhibitory activity. Conclusions: Heparin can attenuate metastasis at clinically relevant doses, likely by inhibiting selectins. Equivalent anticoagulation alone with Fondaparinux is ineffective. Clinically approved heparins have differing abilities to inhibit selectins, likely explained by size distribution. It should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. Caution is also raised about the current preference for smaller heparins. Despite equivalent anticoagulation, hitherto unsuspected benefits of selectin inhibition in various clinical circumstances may be unwittingly discarded.

L-Selectin Facilitation of Metastasis Involves Temporal Induction of Fut7-Dependent Ligands at Sites of Tumor Cell Arrest

Hematogenous carcinoma metastasis is supported by aggregated platelets and leukocytes, forming tumor cell emboli. Early tumor cell-platelet interactions can be mediated by P-selectin binding to tumor cell surface ligands and this process is blocked by heparin. We previously showed that L-selectin deficiency also attenuates experimental metastasis. However, the mechanisms and timing of L-selectin action remained unknown. Here, we study how L-selectin facilitates establishment of pulmonary metastatic foci in syngeneic mice by using experimental metastasis to time events following entry of tumor cells into the bloodstream. Although L-selectin deficiency did not affect platelet aggregation or initial tumor cell embolization, the association of leukocytes with tumor cells was reduced and tumor cell survival was diminished 24 hours later. Temporal inhibition of L-selectin by a function-blocking antibody reduced metastasis. Moreover, although selectin blockade by heparin 6 to 18 hours after tumor cell injection was synergistic with P-selectin deficiency in reducing metastasis, there was no further effect in L-selectin-deficient animals. Thus, heparin apparently works at these time points primarily by blocking L-selectin. Endogenous L-selectin ligands were concomitantly induced adjacent to established intravascular tumor cell emboli in a similar time window when leukocytes were also present. Metastasis was attenuated in mice missing these induced endogenous L-selectin ligands due to fucosyltransferase-7 deficiency. Thus, L-selectin facilitation of metastasis progression involves leukocyte-endothelial interactions at sites of intravascular arrest supported by local induction of L-selectin ligands via fucosyltransferase-7. These data provide the first explanation for how leukocyte L-selectin facilitates tumor metastasis.

Response of a concentrated monoclonal antibody formulation to high shear

There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin‐G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates between 20,000 and 250,000 s−1 for between 5 min and 30 ms using a parallel‐plate and capillary rheometer, respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s−1 and 0.06 pN, respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel‐plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat‐induced aggregates of a mAb. We calculate that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air–water interface or the 20–150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases, air‐bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production. Biotechnol. Bioeng. 2009;103: 936–943.

Monoclonal Antibody Interactions With Micro- and Nanoparticles: Adsorption, Aggregation, and Accelerated Stress Studies

Therapeutic proteins are exposed to various wetted surfaces that could shed subvisible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose, and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel, or Fe(2)O(3) microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe(2)O(3) adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage.

Heparin attenuates metastasis mainly due to inhibition of P- and L-selectin, but non-anticoagulant heparins can have additional effects

Heparin and low molecular weight heparin (LMWH) are widely used for treatment of cancer patients with thrombosis, a common complication of malignant disease. Several recent prospective clinical studies indicate that heparin might improve outcomes of human cancer. Meanwhile, experimental evidence from mouse models consistently demonstrates that heparin efficiently inhibits metastasis. We have previously shown that P- and L-selectin play independent roles in supporting the initial stages of hematogeneous metastasis. Heparin is a known potent inhibitor of such selectin-mediated interactions. Here we provide evidence that the absence of both P- and L-selectin (PL -/- mice) dramatically improved survival in an experimental metastasis model. The use of clinically acceptable amounts of heparin did not further aff5ct metastasis rates in such mice. However, a non-anticoagulant derivative of heparin with P- and L-selectin inhibitory properties reduced metastasis to similar levels as observed in PL -/- mice. The virtual elimination of metastasis by a single treatment with a modified heparin without anticoagulant activity strongly suggests that heparin primarily reduces metastatic disease by inhibiting P- and L-selectin interactions. However, such heparins could have further effects at higher doses.

Production of particles of therapeutic proteins at the air–water interface during compression/dilation cycles

Particles in protein therapeutics are undesirable because they may have the potential for causing adverse immunogenicity in patients. Agitation-induced exposure to the air–water interface during manufacturing, shipping, and administration can cause particle formation in therapeutic protein products. We systematically studied how application of surface pressure during periodic interfacial compressions caused a model monoclonal antibody to form particles. Above a critical interfacial compression ratio of 5 we observed a dramatic increase in the rate of protein particle formation. During continuous interfacial compression/dilation cycles, particle numbers increased but the particle size distribution remained unchanged. When cyclic compressions were halted, particles did not nucleate additional particles or grow further in bulk solution suggesting that they are formed only at the air–water interface. In fact, we found that particles in the bulk slowly decreased in number upon standing. The rate of particle formation was only weakly dependent on both the bulk protein concentration and the period of cyclical interfacial compressions. These observations are consistent with the interfacial aggregation of proteins during periods of high surface pressure, followed by collapse of the adsorbed layer and detachment of protein particles from the interface into the bulk.