The in-vitro effect of famotidine on sars-cov-2 proteases and virus replication.

The lack of coronavirus-specific antiviral drugs has instigated multiple drug repurposing studies to redirect previously approved medicines for the treatment of SARS-CoV-2, the coronavirus behind the ongoing COVID-19 pandemic. A recent, large-scale, retrospective clinical study showed that famotidine, when administered at a high dose to hospitalized COVID-19 patients, reduced the rates of intubation and mortality. A separate, patient-reported study associated famotidine use with improvements in mild to moderate symptoms such as cough and shortness of breath. While a prospective, multi-center clinical study is ongoing, two parallel in silico studies have proposed one of the two SARS-CoV-2 proteases, 3CLpro or PLpro, as potential molecular targets of famotidine activity; however, this remains to be experimentally validated. In this report, we systematically analyzed the effect of famotidine on viral proteases and virus replication. Leveraging a series of biophysical and enzymatic assays, we show that famotidine neither binds with nor inhibits the functions of 3CLpro and PLpro. Similarly, no direct antiviral activity of famotidine was observed at concentrations of up to 200 µM, when tested against SARS-CoV-2 in two different cell lines, including a human cell line originating from lungs, a primary target of COVID-19. These results rule out famotidine as a direct-acting inhibitor of SARS-CoV-2 replication and warrant further investigation of its molecular mechanism of action in the context of COVID-19.

PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models.

Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50-150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies.

Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBß, and IκBε. These NFκB family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NFκB dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NFκB dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NFκB dimer affinities and a strong preference of each IκB for a small subset of NFκB dimers. Once the preferred IκB is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NFκB transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.

Spectrum and Degree of CDK Drug Interactions Predicts Clinical Performance.

Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273-81. ©2016 AACR.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors.

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides a platform for structure-based drug design.

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.

Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction.

The heterodimer HIF-1α (hypoxia inducible factor)/HIF-ß (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics "hotspot identification" strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.

MIRG Survey 2011: snapshot of rapidly evolving label-free technologies used for characterizing molecular interactions.

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

ABRF-MIRG benchmark study: molecular interactions in a three-component system.

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-ß to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer.

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvß3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvß3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvß3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

A model-based approach to predicting the human pharmacokinetics of a monoclonal antibody exhibiting target-mediated drug disposition.

In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (k(on) and k(off)), internalization of the drug-target complex (k(int)), target degradation rate (k(deg)), and target abundance (R(0)), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone.

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K(d)=1.3 nM, K(i)=15 nM, and cellular IC(50)=0.5 µM).

Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

The RelA nuclear localization signal folds upon binding to IκBα.

The nuclear localization signal (NLS) polypeptide of RelA, the canonical nuclear factor-κB family member, is responsible for regulating the nuclear localization of RelA-containing nuclear factor-κB dimers. The RelA NLS polypeptide also plays a crucial role in mediating the high affinity and specificity of the interaction of RelA-containing dimers with the inhibitor IκBα, forming two helical motifs according to the published X-ray crystal structure. In order to define the nature of the interaction between the RelA NLS and IκBα under solution conditions, we conducted NMR and isothermal titration calorimetry studies using a truncated form of IκBα containing residues 67-206 and a peptide spanning residues 293-321 of RelA. The NLS peptide, although largely unfolded, has a weak tendency toward helical structure when free in solution. Upon addition of the labeled peptide to unlabeled IκBα, the resonance dispersion in the NMR spectrum is significantly greater, providing definitive evidence that the RelA NLS polypeptide folds upon binding IκBα. Isothermal titration calorimetry studies of single-point mutants reveal that residue F309, which is located in the middle of the more C-terminal of the two helices (helix 4) in the IκBα-bound RelA NLS polypeptide, is critical for the binding of the RelA NLS polypeptide to IκBα. These results help to explain the role of helix 4 in mediating the high affinity of RelA for IκBα.

Dual functional monoclonal antibody PF-04605412 targets integrin alpha5beta1 and elicits potent antibody-dependent cellular cytotoxicity.

Integrin α5ß1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5ß1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies.