Patients whose tumors harbor oncogene amplification on extrachromosomal DNA (ecDNA) fail to respond to targeted or immune therapy and have poor prognosis. ecDNA are cancer-specific circular fragments of genomic DNA engendered with unique properties, including open chromatin architecture associated with hyper transcription and a predilection for structural variation. In addition, ecDNA+ tumors have tremendous copy number heterogeneity mediated through acentric, non-Mendelian segregation. These properties afford ecDNA+ tumors with unparalleled genomic plasticity permitting circumvention of therapeutic pressure. However, these features also confer heightened levels of DNA replication stress (RS), and we have found that ecDNA amplified tumor cells are hyper-reliant on CHK1, a master regulator of the cellular RS response. We identified CHK1 as an ecDNA essential target in a CRISPR genetic screen using a methotrexate-induced ecDNA amplification model in HeLa cancer cells. ecDNA-dependent cell fitness assessment of CHK1 was confirmed using a flow cytometry-based CRISPR competition assay. The target was further validated using a CHK1 inhibitor (CHK1i) tool compound in MYC-amplified COLO320 isogenic cell lines that demonstrated a 10-fold enhanced cytotoxicity in the ecDNA amplified setting.Based on these results, we developed a highly potent, selective, and orally bioavailable CHK1i optimized as an ecDNA-directed therapeutic (ecDTx). An advanced lead, BBI-cmpd1, robustly induced RS biomarkers (e.g., pRPA) in ecDNA+ COLO320 tumor cells as compared to matched chromosomally-amplified (ecDNA-) COLO320 cells, consistent with the increased reliance on CHK1 to manage elevated RS in the ecDNA amplified setting. BBI-cmpd1 demonstrated potent anti-proliferative activity against a panel of ecDNA+ oncogene amplified tumor lines as compared to non-amplified lines demonstrating oncogene and indication agnostic efficacy in ecDNA-based tumors. Oral administration of BBI-cmpd1 resulted in on-target activity against CHK1 and anti-tumor activity in an ecDNA oncogene amplified tumor model in vivo. These findings support the clinical utility of potent, selective, and oral CHK1i to address the significant unmet need driven by ecDNA oncogene amplified cancers. Citation Format: Sudhir Chowdhry, Snezana Milutinovic, Edison Tse, Salvador Garcia, Dean Perusse, Melissa Ritland, Juyeon Ko, Deepti Wilkinson, Kristen Turner, Auzon Steffy, Joshua Plum, Ben Norman, AnnMarie Pferdekamper, Todd Meyer, Debbie Liao, Rachelle Elsdon, Joshua Lange, Anthony Pinkerton, Ryan Hansen, Christian Hassig, Shailaja Kasibhatla. Tumors driven by oncogene amplified extrachromosomal DNA (ecDNA) demonstrate enhanced sensitivity to cell cycle checkpoint kinase 1 (CHK1) inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1626.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC 50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood–retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.

Triple negative (ER, PR and HER2/neu negative) breast cancers (TNBC) constitute 15-24% of breast cancers, but account for a disproportionate share of mortality. TNBC tumors are more aggressive, lack a targeted therapy, and are prone to relapse and metastasis after cytotoxic drug treatments, the only drug therapy currently available. Serum and glucocorticoid-regulated kinase 1 (SGK1) promotes the growth, invasiveness and chemo-resistance of cancer cells and is overexpressed in 48% of breast cancers, yet is not detected in normal breast tissue. In particular, SGK1 is overexpressed in TNBC cells and knockdown of SGK1 blocks the proliferation and invasiveness of TNBC cells. Thus, SGK1 is an attractive targeted therapy for TNBC. In the SGK1 inhibitors that have been reported previously, poor physicochemical properties have prevented these inhibitors from being active in vivo and advancing to the clinic. To discover drug-like inhibitors of SGK1, we used our technology platform (Leap-To-Lead™) and a fragment-based screening approach to find SGK1 inhibitor scaffolds. Low molecular weight inhibitors like ours are more readily optimized for potency while retaining drug-like properties. From this screen, we identified 11 scaffolds as novel inhibitors of SGK1. Four scaffold series had clear structure activity relationships (SAR). To illustrate the potential of these scaffolds, we carried out a limited synthetic expansion around one scaffold and improved potency more than 600-fold (SGK1 IC50 = 1 μM; LE = 0.4). Several compounds dose-dependently inhibited the proliferation of a TNBC cell line (MDA-MB-231) and inhibited the phosphorylation of the biomarker protein N-Myc downstream regulated 1 (NDRG-1). Unlike previously reported SGK1 inhibitors, our fragment lead compounds show excellent cellular penetration in Caco-2 assays. In summary, a fragment-based screening approach was used to identify four novel scaffolds with clear SAR trends. A single scaffold series was further optimized and shows anti-proliferative activity and biomarker modulation in a TNBC cell line. These SGK1 inhibitor series represent excellent starting points for further SAR studies and development of a novel preclinical candidate. Citation Format: James Zapf, Todd Meyer, Warren Wade, Laura Lingardo, Ayse Batova, Gordon Alton, Peter Pallai. Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2180.

INTRODUCTION:CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.METHODS:The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.RESULTS:CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.CONCLUSIONS:These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

The CD32a immunoglobulin G (IgG) receptor (Fcγ receptor IIa) is a potential therapeutic target for diseases in which IgG immune complexes (ICs) mediate inflammation, such as heparin-induced thrombocytopenia, rheumatoid arthritis, and systemic lupus erythematosus. Monoclonal antibodies (mAbs) are a promising strategy for treating such diseases. However, IV.3, perhaps the best characterized CD32a-blocking mAb, was recently shown to induce anaphylaxis in immunocompromised "3KO" mice. This anaphylactic reaction required a human CD32a transgene because mice lack an equivalent of this gene. The finding that IV.3 induces anaphylaxis in CD32a-transgenic mice was surprising because IV.3 had long been thought to lack the intrinsic capacity to trigger cellular activation via CD32a. Such an anaphylactic reaction would also limit potential therapeutic applications of IV.3. In the present study, we examine the molecular mechanisms by which IV.3 induces anaphylaxis. We now report that IV.3 induces anaphylaxis in immunocompetent CD32a-transgenic "FCGR2A" mice, along with the novel finding that IV.3 and 2 other well-characterized CD32a-blocking mAbs, AT-10 and MDE-8, also induce severe thrombocytopenia in FCGR2A mice. Using recombinant variants of these same mAbs, we show that IgG "Fc" effector function is necessary for the induction of anaphylaxis and thrombocytopenia in FCGR2A mice. Variants of these mAbs lacking the capacity to activate mouse IgG receptors not only failed to induce anaphylaxis or thrombocytopenia, but also very potently protected FCGR2A mice from near lethal doses of IgG ICs. Our findings show that effector-deficient IV.3, AT-10, and MDE-8 are promising candidates for developing therapeutic mAbs to treat CD32a-mediated diseases.

Preoperative evaluation of patients presenting with ovarian masses is challenging, partly due to shortcomings with the commonly used marker, CA-125. Ovarian cancer is associated with systemic coagulation activation. Measurement of D-dimer, serum tissue factor (TF), and the coagulation process as a whole are considered candidates for improving discrimination between benign and malignant ovarian masses. We therefore sought to identify possible benefits by analyzing preoperative coagulation status in conjunction with CA-125 in patients with ovarian masses. Preoperative blood from 95 patients with ovarian masses (75 benign, 20 malignant) and 30 controls was analyzed, prospectively. Thromboelastography served for global hemostatic assessment. Plasma TF antigen and D-dimer were measured by ELISA and microparticle-associated TF activity by thrombin generation assay. TF+ microparticles were enumerated by flow cytometry. Time to clot formation by thromboelastography was similar between patients having either benign or malignant ovarian tumors. Clot formation rate, clot strength, and coagulation index were significantly increased in patients having malignant versus benign tumors, indicating that thromboelastography differentiated malignant from benign tumors. D-dimer alone differentiated malignant from benign ovarian tumors and also improved differentiation when combined with CA-125. Circulating TF antigen, activity, and TF+ microparticle numbers, however, failed to differentiate benign from malignant tumors. Significant coagulation activation occurs in women with ovarian malignancies. Plasma D-dimer may help discriminate between patients with benign and malignant tumors. Thromboelastography may also contribute meaningfully when combined with CA-125 in the preoperative evaluation of ovarian masses. Larger studies are needed to assess these possibilities.

Abstract Abstract 106 Antibodies targeting β2-glycoprotein 1 (β2-GP1; β2-Abs) are of primary importance in antiphospholipid syndrome (APS), a thrombotic autoimmune disorder. The predominance of the IgG antibody isotype in APS is conspicuously associated with increased risk of thrombosis, raising the question whether the platelet IgG receptor, FcγRIIa, may play a role in thrombosis in APS, as is the case in heparin-induced thrombocytopenia (HIT). The hypothesis that platelet FcγRIIa may contribute to thrombosis in APS has received little attention, with research emphasis instead placed on several proposed alternative mechanisms of action. We have shown that, like HIT antibodies, antibodies targeting VEGF or CD154 are also potently thrombotic in mice transgenic for human FcγRIIa but have no activity in mice lacking FcγRIIa (i.e., wild type mice). We therefore asked whether antiphospholipid antibodies can activate platelets and cause thrombosis via FcγRIIa. To this end, we tested mouse monoclonal (mAb) and goat polyclonal anti-human β2-Abs alone or complexed to human β2-glycoprotein 1 (i.e., to form immune complexes, or ICs) by the serotonin release assay (SRA) and platelet aggregation methods using washed human platelets. We found that two of three commercially available polyclonal anti-β2-Abs (pAb1 and pAb3), both alone or in IC form, induced platelet granule release and aggregation, and that this activity was abolished by anti-FcγRIIa mAb, IV.3. pAb2 and mAb were inactive. Activity analysis (SRA) of preformed ICs using constant pAb1 concentration with varied β2-GP1 stoichiometries revealed a zone-of-equivalence pattern, with maximal activity near balanced stoichiometry (1:1). Because pAb2 did not activate platelets, we sought to determine its capacity to form higher order ICs (which are known to be required for FcγRIIa activation) by size exclusion chromatography (SEC). All antibodies tested in isolation were shown by HPLC-SEC to be free of aggregates or degradation products. Antibody-antigen complex size analysis revealed that, as expected, mAb+β2-GP1 in 1:1 stoichiometry failed to form higher order ICs (i.e., complexes having ≥2 IgGs/complex). pAb2 (inactive) did form higher order ICs at 1:1 and 4:1 (IgG:Ag) stoichiometries, but less extensively than pAb1 (active), which efficiently formed higher order ICs at both 1:1 and 4:1 stoichiometries. pAb1 alone (i.e., not in IC form) caused aggregation, while pAb2 did not. Furthermore, preincubation of washed platelets with pAb2 prevented pAb1-induced aggregation, suggesting that pAb1 activity is β2-GP1-specific. A single intravenous injection of anti-β2-GP1 ICs (20 μg β2-GP1 plus 120 μg pAb1, a 1:2 molar stoichiometry) induced severe thrombocytopenia (>90%) and caused thrombotic shock in FcγRIIa-transgenic mice but not in wild type mice. Symptoms of shock occurred within 10 minutes. Pervasive occlusive thrombi were observed in the lungs of all FcγRIIa-transgenic but not in any wild type mice (H&E microscopy). Injection of pAb2 ICs produced none of these effects in transgenic or wild type mice. Finally, injection of pAb1 or pAb2 alone (neither of which bind mouse β2-GP1) had no effects in transgenic mice. In summary, these findings confirm previous in vitro studies that β2-Abs can directly activate platelets in a manner wholly dependent on the platelet IgG receptor, FcγRIIa. Additionally, we have shown for the first time in vivo that β2-Abs can also cause FcγRIIa-dependent thrombosis. This mechanism may contribute to thrombosis in APS, suggesting that further studies on the importance of FcγRIIa in APS are warranted. Disclosures: No relevant conflicts of interest to declare.

In a recent issue of Swiss Medical Weekly [1], Fehr and colleagues report findings from a study designed to address the clinical relevance of a molecular mechanism we suggested may link bevacizumab (bev) to thrombosis [2]. However, we would respectfully point out that the data presented by Fehr et al. do not support their conclusion that our proposed mechanism cannot be “a major contributing factor” to bev-associated thrombosis. In our study, we showed that when bev and VEGF165 were combined in balanced stoichiometry, the resulting higher order immune complexes (ICs) activated the IgG receptor, FcγRIIa, both in human platelets and in mice transgenic for human FcγRIIa [2]. We identified heparin as a vital component of this mechanism, as VEGF165 is a heparin-binding protein [3]. Although the authors state their study was not designed to assess the impact of heparin in bev-treated patients, the study objective (to test the clinical relevance of our proposed mechanism) should have led the authors to design their experiments with attention to the heparin-binding capacity of VEGF165. However, Fehr et al. used a type of VEGF predicted to lack heparin binding activity. Santa Cruz Biotechnology (SCBT) “VEGF (hBA-165)” (sc-4570) has an amino acid sequence “deletion at 141-208” [4], which removes the heparin-binding domain [3]. It is doubtful this type of VEGF could support the heparin-dependent activity we originally described [2]. This matter aside, the authors tested bev and VEGF alone and in combination, but not in the presence of heparin. We have already shown that under these conditions, bev typically lacks platelet activating activity [2]. Fehr and colleagues combined 500 μg/milliliter bev with 500 μg/liter VEGF, resulting in a molar stoichiometric imbalance far outside the zone of equivalence (i.e. >100-fold less VEGF than bev). These conditions do not favour the formation of higher order ICs, which are required for FcγRIIa signaling. Indeed, we have previously determined that stoichiometric imbalances of this magnitude render bev+VEGF inactive, even in the presence of heparin (unpublished data). Fehr et al. used the PFA-100 instrument to test the effect of bev+VEGF on platelet function. Citing several reports, the authors contend that, although the PFA-100 was specifically designed to detect platelet dysfunction, “it is also capable of detecting an increased adhesive and aggregatory platelet function.” Such enhanced platelet function would be reflected in shortened PFA “closure time” (CT) values, which were not observed with bev±VEGF. However, the “question of whether shortened PFA-100 CTs per se might also reflect a prothrombotic tendency” remains a topic of ongoing investigation [5]. The PFA-100 has not been clinically validated for such use, and its design principle suggests it would not be a sensitive measure of thrombotic risk, since blood coagulation is blocked in the system by citrate, and since CT is controlled by collagen plus epinephrine or ADP. Indeed, preincubation of blood with bev plus little or no VEGF and no heparin can hardly be expected to add significantly to the potent activation stimuli of collagen plus epinephrine or ADP, which together rank among the most potent of platelet agonists. If bev-induced platelet activation contributes to thrombosis in cancer patients, we suggested the process would not be rapid, due to limited VEGF availability [2]. A physiologically plausible way that our proposed mechanism could contribute to thrombosis would be through the gradual accumulation of bev+VEGF complexes at sites of vascular disturbance, leading to micro-thromboembolism [6]. In this view, bev+VEGF-activated platelets would be removed from circulation, much as in the case of heparin-induced thrombocytopenia (HIT). Because of this, we would not expect to find any profound changes in systemic markers of primary or secondary haemostasis due to bev therapy, beyond those already linked to cancer and chemotherapy. Interestingly, this was the case in a study by Gerber and colleagues, who subjected mice lacking FcγRIIa to prolonged high dose bev therapy and were unable to demonstrate systemic haemostatic dysfunction [7]. In an effort to identify systemic platelet activation in bev recipients, Fehr et al. measured serum soluble P-selectin (sPsel) levels. However, measurement of sPsel in serum rather than plasma merely reflects complete release of the molecule from all platelets, which are activated during the production of serum [8]. Therefore, the significance of the sPsel values obtained is unclear. For these reasons, we believe the work of Fehr et al. fails to exclude the possibility that bev-associated platelet activ-

Abstract Anticalins are high affinity scaffold proteins based on human lipocalins and represent a new class of biologics. Although lipocalins share limited sequence identity among each other they are all made up of a single conserved beta-barrel. The four loops protruding from the opening of the calyx contribute to the observed various binding functions of different lipocalins. The loop residues of human tear lipocalin were randomized to generate a combinatorial library and successive rounds of panning and screening were employed to generate an Anticalin with high affinity and specificity for VEGF-A, Angiocal. Angiocal was found to antagonize VEGF-A function by preventing its interaction with cognate membrane receptors. Following the initial characterization in cell-based and in vivo models as communicated previously (AACR 2008 #4077, AACR 2009 #2318) we sought to estimate the human pharmacokinetics of Angiocal by interspecies scaling. Therefore, PK profiles in mice, rats and cynomolgus monkeys were obtained and will be presented. Therapeutic antibodies such as bevacizumab are usually well tolerated while significant toxicities occur in a subset of patients, for example leading to an enhanced risk for thromboembolic events. It has been demonstrated that bevacizumab forms multimeric immune complexes in vivo which may be a cause for these effects. Furthermore, immune complex deposition in glomeruli of the kidney may cause glomerulosclerosis. With respect to thromboembolic complications of bevacizumab use, these were mirrored in human FcgammaIIa receptor transgenic mice where administration of complexes between heparin, Bevacizumab and heparin-binding isoforms of its VEGF-A target lead to platelet aggregation and thrombosis (Meyer, T. et al. J. Thromb. Haemost. 2009, 7: 171-181). Angiocal was tested in the same model and does not cause any of these adverse effects on platelets in vivo. GLP toxicology studies with Angiocal were performed in two relevant species to determine the NOAEL. In addition, we did not observe effects on inflammatory cytokines or complement factors in these studies while exaggerated pharmacodynamic effects consistent with potent VEGF neutralization were seen. Angiocal is currently undergoing phase Ib clinical testing in cancer patients to establish Anticalins as novel and safe class of therapeutic molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2011-3632

The multifunctional cytokine, TWEAK (TNF-like weak inducer of apoptosis), is a member of the TNF alpha superfamily. TWEAK is found in a broad range of cell types and has been linked to cell growth and survival, angiogenesis and other inflammatory processes. These functions and their importance in inflammatory diseases have made TWEAK an attractive pharmaceutical target, particularly for immunotherapy with monoclonal antibodies (mAbs). Immunotherapy targeting another TNF alpha family member, CD154, was associated with thrombosis in clinical trials. Subsequent studies identified platelets, which contain CD154, as a possible contributing factor to thrombosis in these trials. Since clinical trials with anti-TWEAK mAbs have already begun, we considered it important to determine whether platelets contain TWEAK. Using a variety of immunologic methods we found that, upon activation, human platelets expose TWEAK antigen and release it in soluble form (sTWEAK). By flow cytometry we determined that human platelets activated by TRAP (Thrombin Receptor Agonist Peptide) and other agonists expose TWEAK antigen (22% median positivity) and release TWEAK positive microparticles. The presence of TWEAK on platelets was confirmed by confocal microscopy. By ELISA, we found that sTWEAK is released by activated platelets. Finally, western blot analysis revealed TWEAK protein (similar to 34 kDa) in washed platelet lysates. The finding that human platelets contain TWEAK raises important questions about its possible functions in normal physiology, as well as in inflammatory diseases and their treatment.

It is well established that the blood coagulation system is activated in cancer. In addition, there is considerable evidence to suggest that clotting activation plays an important role in the biology of malignant tumors, including the process of blood-borne metastasis. For many years our laboratory has used experimental models of lung metastasis to study the events that follow the introduction of procoagulant-bearing tumor cells into circulating blood. This chapter focuses on the basic methods involved in assessing the anti-metastatic effects of anticoagulants and anti-platelet agents using rodent models of experimental metastasis. In addition, it summarizes our experience with these models, which collectively suggests that intravascular coagulation and platelet activation are a necessary prelude to lung tumor formation and that interruption of coagulation pathways or platelet aggregation may be an effective anti-metastatic strategy.

Abstract Abstract 3200 Anti-angiogenic therapy with the monoclonal antibody (mAb) bevacizumab (bev; Genentech) is associated with venous and arterial thrombosis in cancer patients. We recently showed that bev immune complexes (ICs) activate platelets in vitro and cause thrombotic thrombocytopenia in mice transgenic for the human platelet IgG receptor, FcγRIIa (FCGR2A mice; Jax Labs). Previous studies conducted in other labs using wild type mice injected with bevacizumab failed to identify thrombotic side effects; however, wild type mice do not have platelet FcγRIIa IgG receptors. Platelets from human donors or from FCGR2A mice are directly activated when FcγRIIa is clustered by the Fc domains of mAbs that have been assembled into higher order ICs by multivalent antigens, such as was shown for bev (Meyer, et al, J Thromb Haemost, 2009;7:171) and for anti-CD154 ICs (Robles-Carrillo, et al, J Immunol, 2010;185:1577). Aflibercept (aflib, Regeneron/sanofi-aventis, also known as VEGF Trap) is a soluble decoy receptor protein that binds all isoforms of VEGF-A including VEGF165 and inhibits VEGF165 activity. Aflib also binds the related angiogenic molecule PlGF, and like bev has anti-angiogenic activity. The aflib molecule contains the Fc domain of the IgG isotype antibody; however, although VEGF165 efficiently clusters bev into higher order ICs, VEGF165 does not cluster aflib into higher order complexes (Rudge JS, et al, PNAS, 2007;104:18363). This suggests that aflib+VEGF165 should not be able to trigger platelet FcγRIIa activation (because FcγRIIa requires clustering for activation). We therefore hypothesized that aflib+VEGF165 complexes would fail to activate platelet FcγRIIa in vitro or in FCGR2A transgenic mice. For in vitro studies, we chose the washed platelet aggregation method as a measure of IC-induced platelet activation. To assess platelet activation in vivo, we measured IC-induced reductions in the number of circulating platelets (thrombocytopenia) following intravenous injection of ICs into FCGR2A mice. Aflib+VEGF165 or bev+VEGF165 complexes were pre-formed at room temperature in the presence or absence of heparin. (Heparin can enhance bev IC-induced platelet activation. Many cancer patients are routinely exposed to heparin to maintain patency of chemotherapy ports.) In 5 donors, bev+VEGF165 complexes (500nM) rapidly induced full aggregation of human platelets in an FcγRIIa-dependent manner, whereas 500nM aflib+VEGF165 failed to induce aggregation under all conditions tested. Within 5 minutes following intravenous injection of bev+VEGF165, FCGR2A mice (n=10) experienced signs of distress consistent with thrombotic shock, including rapid shallow breathing, hunched posture, and decreased or dysfunctional locomotor activity. Animals receiving aflib complexes (n=10) did not exhibit these symptoms. Platelet counts 10 minutes following IC injection revealed thrombocytopenia in animals receiving bev+VEGF165 but not aflib+VEGF165 complexes. Specifically, animals injected with PBS (vehicle; n=5) had baseline platelet counts of (1173±179;mean±SD), whereas animals receiving bev+VEGF165 had mean platelet counts of 331±217 (with heparin; P < 0.001) and 725±303 (without heparin; P < 0.003). Animals receiving aflib+VEGF165 had mean platelet counts of 966±168 (with heparin) and 878±250 (without heparin), which were not statistically different from baseline animal counts. These results indicate that aflibercept lacks that platelet activating potential observed with bevacizumab, and also suggest that anti-angiogenic therapy with aflibercept may lack the capacity to induce the platelet associated thrombotic side effects observed in patients receiving bevacizumab. Disclosures: Meyer: Regeneron Pharmaceuticals, Inc.: Florida Hospital received research funding from Regeneron Pharmaceuticals in support of some of this work.

Anti-CD40L immunotherapy in systemic lupus erythematosus patients was associated with thromboembolism of unknown cause. We previously showed that monoclonal anti-CD40L immune complexes (ICs) activated platelets in vitro via the IgG receptor (FcγRIIa). In this study, we examined the prothrombotic effects of anti-CD40L ICs in vivo. Because mouse platelets lack FcγRIIa, we used FCGR2A transgenic mice. FCGR2A mice were injected i.v. with preformed ICs consisting of either anti-human CD40L mAb (M90) plus human CD40L, or a chimerized anti-mouse CD40L mAb (hMR1) plus mouse CD40L. ICs containing an aglycosylated form of hMR1, which does not bind FcγRIIa, were also injected. M90 IC caused shock and thrombocytopenia in FCGR2A but not in wild-type mice. Animals injected with hMR1 IC also experienced these effects, whereas those injected with aglycosylated-hMR1 IC did not, demonstrating that anti-CD40L IC-induced platelet activation in vivo is FcγRIIa-dependent. Sequential injections of individual IC components caused similar effects, suggesting that ICs were able to assemble in circulation. Analysis of IC-injected mice revealed pulmonary thrombi consisting of platelet aggregates and fibrin. Mice pretreated with a thrombin inhibitor became moderately thrombocytopenic in response to anti-CD40L ICs and had pulmonary platelet-thrombi devoid of fibrin. In conclusion, we have shown for the first time that anti-CD40L IC-induced thrombosis can be replicated in mice transgenic for FcγRIIa. This molecular mechanism may be important for understanding thrombosis associated with CD40L immunotherapy. The FCGR2A mouse model may also be useful for assessing the hemostatic safety of other therapeutic Abs.

A decade ago the world of monoclonal antibody (mAb) therapeutics was surprised when clinical trials in patients with systemic lupus erythematosus (SLE) using anti-CD154 immunotherapy were halted due to fatal thrombosis [ [1] Boumpas D.T. Furie R. Manzi S. Illei G.G. Wallace D.J. Balow J.E. et al. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum. Mar 2003; 48: 719-727 Crossref PubMed Scopus (551) Google Scholar ]. Questions focused on how thrombosis could result from antibodies targeting the T-lymphocyte antigen, CD154. As lymphocytes are not substantively involved in blood coagulation, speculation arose that anti-CD154 mAbs might produce fibrin clots by activating coagulation indirectly (e.g., via the vascular endothelial cell inflammatory response) [ [2] Kawai T. Andrews D. Colvin R.B. Sachs D.H. Cosimi A.B. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med. Feb 2000; 6: 114 Crossref Scopus (557) Google Scholar ]. To date there is no compelling evidence this process occurs in vivo, and this problem has delayed widespread use of anti-CD154 immunotherapy. Safe and effective anti-CD154 immunotherapy is urgently needed for diseases such as SLE.

Abstract Abstract 5128 Objectives: Hu5c8, a whole IgG1 anti-CD40L antibody, is known to cause thrombotic side effects in patients with Systemic Lupus Erythematosus (SLE). Anti-CD40L-mediated activation of platelets in vitro was shown to be FcγRIIa-dependent1, suggesting that the adverse effects of hu5c8 could be due to this mechanism, and that anti-CD40L antibodies lacking a functional Fc region would not activate platelets. CDP7657 is a monovalent Fab' PEG anti-CD40L antibody being developed for the treatment of SLE. The aim of the present work was to study a range of anti-CD40L antibody formats in isolated platelet assays and to evaluate the role of the Fc region and of valency. Methods: Isolated human platelets from healthy donors were used in the platelet aggregometry and serotonin release assay systems. Antibodies were added either alone, or pre-complexed with soluble CD40L trimer (sCD40L) at optimal stoichiometry. Antibody IV.3 was used to block FcγRIIa. The antibodies and antibody fragments tested included hu5c8 in IgG1, aglycosyl IgG, Fab′, & (Fab′)2 formats, CDP7657, and the CDP7657 variable regions in di-Fab' PEG, tri-Fab' PEG and aglycosyl formats. Results: Hu5c8 in complex with sCD40L caused strong aggregation (in 2–3 minutes) and >85% serotonin release, both alone and in complex with sCD40L. This activation was completely abrogated by blocking FcγRIIa. In contrast, platelet activation did not occur with CDP7657 or other antibody formats, in any assay, regardless of the valency or presence of sCD40L. Conclusions: The data support the hypothesis that platelet activation by anti-CD40L antibodies is dependent on the presence of an active Fc moiety, via interactions with FcγRIIa, and indicate that CDP7657 has the potential to deliver the therapeutic effect of anti-CD40L blockade in SLE without thrombotic toxicity. References:(1) Langer F et al. 2005; Thromb Haemost 93: 1137. Disclosures: Amirkhosravi: UCB: Research Funding.

It is established that experimental metastasis requires platelet activity. CD154 expressed on and released from activated platelets induces an inflammatory response in endothelial cells and monocytes, including tissue factor production. CD154 has also been shown to activate platelets in vitro and promote thrombus stability in vivo. These CD154 effects may be mediated, at least in part, by CD40 signaling on platelets and vascular endothelial cells. We have previously demonstrated prolonged bleeding and PFA-100 closure times in mice deficient for Cd154 or its receptor Cd40. In the present study, we hypothesized that Cd40 and Cd154 promote lung tumor formation in experimental metastasis in mice. We created mice doubly deficient in Cd40 and Cd154 (Dbl KO) and found them to be both fertile and viable. Injected tumor cells seeded poorly in mice deficient in Cd40 or Cd154, as well as Dbl KO, compared to wild-type mice. We sought to determine whether blood-borne Cd40 versus endothelial Cd40 contribute differentially to reduced experimental lung metastasis, as observed in Cd40 deficient mice. By bone marrow transplantation, we created mice deficient for Cd40 either in the blood compartment but not in the endothelium, or vice versa. We found that mice deficient in blood compartment Cd40 had fewer lung nodules compared to wild-type mice and mice deficient in endothelial Cd40. Our findings suggest an important contribution of the Cd40-Cd154 pathway to experimental lung metastasis. Furthermore, the data points to a selective role for peripheral blood cell Cd40 in this process.