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Supplemental Methods: including HUVEC fibrin gel bead assay, Production of anti-DLL4 F(ab')2, Mouse neonatal retina assay, Xenograft tumor models, Histological analysis of mouse liver, PK assay methods, and Anti-therapeutic antibody assay method. Table S1: PK parameters of anti-DLL4 IgG1 and F(ab')2 in mouse. Table S2: PK parameters for anti-DLL4 IgG1 and F(ab')2 in rat and money. Table S3: Summary of incidence of skin neoplasms in rats treated with anti-DLL4 IgG1 or anti-DLL4 F(ab')2. Figure S1: Generating and characterizing low affinity anti-DLL4 antibodies. Figure S2: Characterization of anti-DLL4 F(ab')2 using HUVEC bead sprouting assay. Figure S3: PK profile of anti-DLL4 IgG1 and F(ab')2 in rat toxicity studies. Figure S4. Mitigation of liver toxicity in mice with anti-DLL4 F(ab')2. Figure S5. Novel toxicities identified with anti-DLL4 F(ab')2 in rats.

Purpose: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody.Experimental Design: The F(ab′)2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab′)2) was generated and assessed in efficacy and toxicity studies.Results: Anti-DLL4 F(ab′)2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab′)2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition.Conclusions: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab′)2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans. Clin Cancer Res; 22(6); 1469–79. ©2015 AACR.

Mass spectrometry has recently emerged as a powerful analytical tool for the assessment of pharmacokinetics and biomarkers in drug development. Compared with ligand binding assays, a major advantage of mass spectrometry-based assays is that they are less dependent on high quality binding reagents, while a key limitation is the relatively lower sensitivity. To address the sensitivity issue, we have developed a generic reagent, ultratargeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method which combines commercially available protein A affinity capture, targeted analyte isolation by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). A targeted-2D-with-dilution configuration was designed to automate 2D-LC-MS/MS. This method was systematically evaluated using an anti-CD22 monoclonal antibody spiked into monkey and human serum, where lower limits of quantification (LLOQ) of 0.78 and 1.56 ng/mL were achieved, respectively. This represents an over 100-fold improvement in assay sensitivity compared to the conventional LC-MS/MS method. The performance of the method was further confirmed by analyzing another monoclonal antibody, bevacizumab, as well as a soluble antigen, circulating PD-L1. The results indicate that our method enables quantification of antibody therapeutics and antigen biomarkers in both clinical and nonclinical samples in the pg/mL to low ng/mL range. Protein A affinity capture was employed as a universal sample preparation procedure applicable to both full-length antibody therapeutics and antibody-antigen complexes. This novel method is also fully automated and proven to be highly robust for routine bioanalysis in drug development.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting. Graphical Abstract

Background The iMATRIX-atezolizumab study was a phase I/II, multicenter, open-label study designed to assess the safety and pharmacokinetics of atezolizumab in pediatric and young adult patients. We describe the pharmacokinetics (PK), exposure-safety, and immunogenicity of atezolizumab in pediatric and young adults with metastatic solid tumors or hematologic malignancies enrolled in this study. Methods Patients aged

Antibody therapeutics can be associated with unwanted immune responses resulting in the development of anti‐drug antibodies (ADA). Optimal methods to evaluate the potential effects of ADA on clinical outcomes in oncology are not well established. In this study, we assessed efficacy and safety, based on ADA status, in patients from over 10 clinical trials that evaluated the immune checkpoint inhibitor atezolizumab as a single agent or as combination therapy for several types of advanced cancers. ADA can only be observed post randomization, and imbalances in baseline prognostic factors can confound the interpretation of ADA impact. We applied methodology to account for the confounding effects of baseline clinical characteristics and survivorship bias on efficacy. Adjusted meta‐analyses revealed that despite numerical differences in overall survival and progression‐free survival between ADA‐positive and ADA‐negative patients from some studies, ADA‐positive patients from studies with an overall treatment effect derived benefit from atezolizumab, compared with their adjusted controls. Based on large, pooled populations from atezolizumab monotherapy or combination studies, unadjusted descriptive analyses did not identify a clear relationship between ADA status and frequency or severity of adverse events. Data also suggested that any ADA impact is not driven by neutralizing activity. Collectively, this exploratory analysis suggests that the potential for ADA development should not impact treatment decisions with atezolizumab.

Baseline patient characteristics and prognostic factors are important considerations in oncology when evaluating the impact of immunogenicity on pharmacokinetics (PK) and efficacy. Here, we assessed the impact of anti‐drug antibodies (ADA) on the PK of the immune checkpoint inhibitor atezolizumab (an anti–PD‐L1 monoclonal antibody). We evaluated data from ≈ 4500 patients from 12 clinical trials across different tumor types, treatment settings, and dosing regimens. In our dataset, ~ 30% of patients (range, 13–54%) developed treatment‐emergent ADA, and in vitro neutralizing antibodies (NAb) were seen in ~ 50% of ADA‐positive (+) patients. Pooled time course data showed a trend toward lower atezolizumab exposure in ADA+ patients, which was more pronounced in ADA+/NAb+ patients. However, the atezolizumab concentration distributions overlapped, and drug concentrations exceeded 6 µg/ml, the target concentration required for receptor saturation, in greater than 95% of patients. Patients had sufficient exposure regardless of ADA status. The dose selected to allow for dosing over effects from ADA resulted in a flat exposure‐response relationship. Analysis of study results by ADA titer showed that exposure and overall survival were not affected in a clinically meaningful way. High tumor burden, low albumin, and high CRP at baseline showed the greatest association with ADA development but not with subsequent NAb development. These imbalanced factors at baseline can confound analysis of ADA impact. ADA increases atezolizumab clearance minimally (9%), and its impact on exposure based on the totality of the clinical pharmacology assessment does not appear to be clinically meaningful.

Significance Using mouse models that recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis, we report that chemically induced colitis promoted development of colon tumors that were largely resistant to anti-VEGF antibody treatment. Serum G-CSF levels were markedly elevated after induction of colitis. Inhibition of G-CSF or Bv8/PROK2 increased the efficacy of anti-VEGF antibody and prevented onset of resistance. To verify the potential clinical relevance of these findings, we examined a series of CRC specimens and found that tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. These findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents., We tested cis-ApcΔ716/Smad4+/− and cis-ApcΔ716/Smad4+/− KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/− KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/− and cis-ApcΔ716/Smad4+/− KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 μL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab′)2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′)2CDR using anti-DLL4 F(ab′)2and a control F(ab′)2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

Atezolizumab, a humanized immunoglobulin G1 (IgG1) monoclonal antibody targeting human programmed death-ligand 1 (PD-L1), is US Food and Drug Administration (FDA) approved in metastatic urothelial carcinoma (MUC) and is being investigated in various malignancies. This analysis based upon 906 patients from two phase I and one phase II MUC studies, is the first report of the clinical pharmacokinetics (PK) and pharmacodynamics (PD) of atezolizumab. Atezolizumab exhibited linear PK over a dose range of 1-20 mg/kg, including the labeled 1,200 mg dose. The clearance, volume of distribution, and terminal half-life estimates from population pharmacokinetic (PopPK) analysis of 0.200 L/day, 6.91 L, and 27 days, respectively, were as expected for an IgG1. Exposure-response analyses did not identify statistically significant relationships with either objective response rate or adverse events of grades 3-5 or of special interest. None of the statistically significant covariates from PopPK (body weight, gender, antitherapeutic antibody, albumin, and tumor burden) would require dose adjustment.

Background: The standard safety evaluation of biotherapeutics includes assessment of immunogenicity. Anti-therapeutic antibodies (ATA) can be detected in serum using immunoassays with a bridging format. However, these assays can be subject to interference. Results: In the bridging ATA assay for 3A5 TDC, an antibody–drug conjugate that binds to the multimeric extracellular domain of MUC16 (CA125), soluble CA125 in the serum caused false-positive results by binding to the ATA assay reagents. This interaction was blocked by wheat germ agglutinin lectin as it binds to the glycans in CA125; thus, the specificity of the assay improved. Conclusion: The assay development and validation results showed that the addition of wheat germ agglutinin eliminates the interference from circulating CA125 without impacting the ability to detect ATA.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Purpose: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. Experimental Design: The F(ab′)2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab′)2) was generated and assessed in efficacy and toxicity studies. Results: Anti-DLL4 F(ab′)2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab′)2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. Conclusions: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab′)2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans. Clin Cancer Res; 22(6); 1469–79. ©2015 AACR.

Intravenous administration of a humanized monoclonal antibody of IgE (E25) attenuates the early and late phase response to inhaled allergen in allergic asthmatic subjects. To test whether direct delivery of E25 to the airway might have the same effect, we conducted a randomized, double-blind, three group study in 33 subjects with mild allergic asthma (20 to 46 yr of age, 21 men, FEV(1)70% predicted). The airway responses to aerosolized allergen were determined at baseline, after 2 and 8 wk of once daily treatment with aerosolized placebo (n = 11), aerosolized E25 1 mg (n = 12), or aerosolized E25 10 mg (n = 10), and after 4 wk of treatment withdrawal. We found that E25 was detectable in the serum during aerosol treatment, although serum IgE did not change significantly in any of the three groups during treatment. In addition, both doses of E25 were no more effective than placebo in attenuating the early phase responses to allergen at both times during treatment. Although aerosolized E25 was generally well tolerated, one subject receiving aerosolized E25 10 mg daily was found to have serum IgG and IgA antibodies to E25. We conclude that aerosol administration of an anti-IgE monoclonal antibody does not inhibit the airway responses to inhaled allergen in allergic asthmatic subjects. We speculate that the observed lack of efficacy may be due to the inability of aerosol route of delivery to result in high enough concentrations of E25 in the tissue compartments surrounding IgE effector cells to neutralize IgE arising from local airway and pulmonary sources and IgE arising from the vascular space. Additionally, the aerosol route of delivery of monoclonal antibodies may be more immunogenic than the parenteral route.

Purpose. Interaction of human IgE with its high affinity receptor (FceRI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and FceRI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE FceRI α chain, the extracellular portion of IgE high affinity receptor (sFceRIα) was compared with the binding of IgE and IgE immunoadhesin (FceRIα-IgG).

Background: OX40 is a co-stimulatory receptor that is transiently expressed by T cells upon antigen recognition. In murine models, OX40 engagement by an agonist anti-OX40 antibody can promote durable tumor regression associated with co-stimulation of effector T cells and reduction of regulatory T cells. MOXR0916 is a humanized effector-competent agonist IgG1 monoclonal antibody that targets OX40. Methods: A Phase I, open-label, multicenter study was conducted to evaluate the safety and pharmacokinetics (PK) of MOXR0916 in patients (pts) with locally advanced or metastatic refractory solid tumors that have progressed after available standard therapy. MOXR0916 was administered at fixed doses every 3 weeks (q3w), and treatment beyond RECIST progression was permitted in the absence of clinical deterioration. A 3+3 dose-escalation was conducted in immunotherapy-naïve pts with a 21-day window to evaluate dose-limiting toxicity (DLT). A dedicated expansion cohort enrolled pts who consented to serial tumor biopsies, enabling immune profiling by immunohistochemistry and gene expression methods. In the biopsy cohort, prior immunotherapy with adequate washout was permitted, provided there was no history of Grade (G) ?3 immune-mediated adverse events (AEs). Results: Enrollment in the dose-finding phase of the trial is complete, with 34 pts treated across 10 dose escalation cohorts (dose levels 0.2-1200 mg) and 36 pts treated in the serial biopsy cohort (dose levels 3.2-600 mg). While NSCLC (n = 8), clear cell RCC (n = 6), melanoma (n = 2), and bladder (n = 2) were represented, less immunogenic tumor types predominated. The median number of prior regimens for metastatic disease was 2 (range 0-9); 4 pts had received prior checkpoint inhibitors. As of 11 Jan 2016, no DLTs, G4/5 AEs attributed to study treatment, or AEs leading to treatment discontinuation were reported. The majority of treatment-related AEs were G1 in severity; 4 related G3 events (autoimmune hepatitis responsive to steroids, worsening dyspnea in a pt with malignant pleural effusions, hypertension, and fatigue) were reported. At doses ?40 mg q3w, PK was linear and sustained peripheral blood OX40 receptor saturation was achieved. Tumor pharmacodynamic (PD) biomarker modulation supportive of the mechanism of action was observed in a subset of pts. Eleven of 70 pts (16%) have been treated with MOXR0916 for > 6 months (?9 cycles) with a best response of stable disease per RECIST v1.1. Updated clinical data will be presented. Conclusions: In a heterogeneous, refractory population, MOXR0916 was well-tolerated at all doses evaluated. The recommended dose and schedule based on PK and OX40 receptor saturation is 300 mg q3w. Tumor PD modulation and evidence of prolonged stable disease support the ongoing expansion phase to evaluate anti-tumor activity in select indications and Phase Ib trial in combination with atezolizumab (anti-PD-L1). Citation Format: Aaron R. Hansen, Jeffrey R. Infante, Grant McArthur, Michael S. Gordon, Alexander M. Lesokhin, Ann-Lee Stayner, Todd M. Bauer, Shahneen Sandhu, Frank Tsai, Alexandra Snyder, Deepa S. Subramaniam, Jeong Kim, Eric Stefanich, Chi-Chung Li, Jane Ruppel, Maria Anderson, Houston Gilbert, Bruce McCall, Mahrukh A. Huseni, Ina Rhee, Michael Pishvaian. A first-in-human phase I dose escalation study of the OX40 agonist MOXR0916 in patients with refractory solid tumors. [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 CT097.

PD-L1 expressed in the tumor microenvironment regulates Th1 immune responses and mediates cancer immune evasion through interactions with PD-1 or B7.1 receptors on activated T cells. MPDL3280A, an engineered human monoclonal antibody, targets PD-L1 and inhibits its function. To identify immunologic predictive and pharmacodynamic biomarkers of MPDL3280A treatment, we performed a comprehensive analysis of tumors and blood samples collected at baseline and/or on treatment from ≈280 patients with locally advanced or metastatic solid tumors, including NSCLC, RCC, melanoma and bladder cancer. Regardless of tumor type, clinical responses were characterized by PD-L1 expression, the presence of markers of T cell activation (Th1 gene signature and CTLA4), and the absence of fractalkine at baseline in the tumor microenvironment. Elevated baseline expression of IFN-γ and IFN-γ-inducible genes (e.g., IDO1 and CXCL9) was associated with MPDL3280A response in melanoma but not NSCLC or RCC. On treatment, responding tumors showed increased infiltration of Th1-dominant immune infiltrate and evidence of adaptive PD-L1 up-regulation. In contrast, progressing tumors displayed the following patterns of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression: (1) few/no TILs and absent PD-L1 expression (immunologic ignorance), (2) TILs present with minimal/no PD-L1 expression (non-functional immune responses), or (3) TILs residing solely around the tumor cell mass outer edge (excluded infiltrate), suggesting that resistance to MPDL3280A may be associated with impaired T cell trafficking and/or function. Profiling of ≈180 circulating biomarkers revealed that plasma concentrations of IL-18 and interferon-inducible T cell alpha chemoattractant (ITAC) increased in all patients following MPDL3280A treatment, representing a pharmacodynamic measurement of PD-L1 inhibition. In addition, analysis of PBMC showed an increase in T cell activation, as measured by IFN-γ and granzymes A and B gene expression in responders following MPDL3280A treatment, consistent with the observations in responding tumors. Baseline soluble PD-L1 was not associated with response. Some indication-specific biomarkers, such as plasma VEGF, decreased in responders with RCC but not with other indications. In NSCLC, a decrease in tumor burden markers, CA-125 and CEA, was associated with response. Similarly, IL-6 and IL-8 were differentially expressed on treatment in responders vs non-responders. Additionally, responders exhibited a decrease in circulating tumor DNA (ctDNA) in plasma, suggesting that ctDNA may be used to monitor MPDL3280A clinical activity in NSCLC. In conclusion, these data provide general and indication-specific mechanistic insights into immune checkpoint inhibition, potential mechanisms of response and resistance, as well as identification of potential predictive and pharmacodynamic biomarkers of anti-PD-L1/PD-1 clinical activity across multiple tumor types.