Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus.

The essential role of the Delta-like ligand 4 (Dll4)-Notch signaling pathway in T-lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post-natal development leads to a deregulation in T-cell differentiation. However, whether ongoing Dll4-Notch signaling is required for T-cell development in the adult thymus is unknown. The use of anti-Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4-Notch signaling blockade in T-cell development in adult mice. We found that anti-Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T-cell maturation at the pro-T-cell double negative 1 (CD4(-) CD8(-) c-kit(+) CD44(+) CD25(-) ) stage, leading to decreased numbers of immature double-positive (CD4(+) CD8(+) ) T cells without affecting the frequency of mature single positive CD4(+) and CD8(+) thymocytes, while promoting alternative thymic B-cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4-Notch signaling during T-cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4-Notch inhibitors.

VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade.

VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.

Direct correlation between adsorption-induced changes in protein structure and platelet adhesion.

It is widely recognized that adsorbed proteins on biomaterial surfaces tend to initiate thrombus formation, although the specific mechanisms involved are still not well understood. In attempts to decrease the conformational change of adsorbed proteins, surface treatments that reduce surface hydrophobicity have been considered, such as the sulfonation of low-density polyethylene and isotactic polypropylene. The objectives of this present research were to study how changes in surface chemistry influence the degree of conformational change of adsorbing proteins and to investigate the correlation between the change in adsorbed protein structure and platelet response. Adsorbed porcine serum albumin and porcine fibrinogen were used as the model proteins for determining the effects of sulfonation on protein conformational change. Circular dichroism spectroscopy studies showed that the proteins were less altered structurally on the sulfonated surfaces. Platelet adhesion studies were used to correlate the number of adhered platelets with the amount of conformational change in adsorbed proteins on the polymer surface. The results of these studies show a linear correlation between platelet adhesion and the degree of adsorption-induced protein conformational change. These findings suggest that the degree of protein conformational change after adsorption is a dominant mechanism governing platelet interactions with biomaterial surfaces.

Endometrial angiopoietin expression and modulation by thrombin and steroid hormones: a mechanism for abnormal angiogenesis following long-term progestin-only contraception.

The angiopoietins (Ang) are endothelial cell-related factors necessary for the development and maintenance of all vessels. Altering the expression of these proteins would be expected to result in aberrant angiogenesis. Indeed the fragile endometrial vasculature and bleeding observed in women treated with long-term progestin-only contraceptives has been associated with changes in the expression of Ang-1 and Ang-2. Since bleeding would result in thrombin formation, we have assessed the effects of thrombin on the expression of the Angs in human endometrial cells. This study shows that thrombin significantly reduces the expression of Ang-1 protein and mRNA expression in human endometrial stromal cells (HESCs) and minimally decreases the production of Ang-2 protein in human endometrial endothelial cells (HEECs). Hence the presence of thrombin due to aberrant bleeding could affect the angiogenic potential of the endometrium, creating a feed forward loop resulting in more thrombin, weak vasculature, and more bleeding. In addition, since the exact localization of Ang in the human endometrium remains a subject of controversy, we have addressed this issue in an in vivo system by analyzing the expression of Angs by microdissection of HESCs, HEECs, and human endometrial glandular epithelial cells followed by real time, quantitative RT-PCR.

VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier.

Vascular endothelial growth factor (VEGF) plays a central role in the development of retinal neovascularization and diabetic macular edema. There is also evidence suggesting that VEGF is an important stimulator for choroidal neovascularization. In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF-TRAP(R1R2), in models for these disease processes. VEGF-TRAP(R1R2) is a fusion protein, which combines ligand binding elements taken from the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous injections or a single intravitreous injection of VEGF-TRAP(R1R2) strongly suppressed choroidal neovascularization in mice with laser-induced rupture of Bruch's membrane. Subcutaneous injection of VEGF-TRAP(R1R2) also significantly inhibited subretinal neovascularization in transgenic mice that express VEGF in photoreceptors. In two models of VEGF-induced breakdown of the blood-retinal barrier (BRB), one in which recombinant VEGF is injected into the vitreous cavity and one in which VEGF expression is induced in the retina in transgenic mice, VEGF-TRAP(R1R2) significantly reduced breakdown of the BRB. These data confirm that VEGF is a critical stimulus for the development of choroidal neovascularization and indicate that VEGF-TRAP(R1R2) may provide a new agent for consideration for treatment of patients with choroidal neovascularization and diabetic macular edema.

Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis.

Gene therapy approaches involving vascular endothelial growth factor (VEGF) to promote therapeutic angiogenesis are under consideration for conditions ranging from ischemic heart disease to nonhealing skin ulcers. Here we make the surprising observation that the transgenic delivery of VEGF to the skin results in a profound inflammatory skin condition with many of the cellular and molecular features of psoriasis, including the characteristic vascular changes, epidermal alterations, and inflammatory infiltrates. Even longstanding psoriatic disease remains dependent on the transgenic VEGF in this model because it can be effectively reversed by the addition of VEGF Trap, a potent VEGF antagonist. Previous attempts to faithfully replicate the psoriatic phenotype through the transgenic delivery of epidermal keratinocyte growth factors or inflammatory mediators generated phenotypes with only partial resemblance to human psoriasis, leaving unanswered questions about the etiology of this disease. The ability of transgenic VEGF to induce a psoriasiform phenotype suggests a new etiology and treatment approach for this disease and further substantiates emerging concerns about possible proinflammatory adverse effects that might be associated with therapeutic attempts to deliver VEGF.

VEGF-Trap: a VEGF blocker with potent antitumor effects.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.