Eric Wakshull

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

Association of Endogenous Anti–Interferon-α Autoantibodies With Decreased Interferon-Pathway and Disease Activity in Patients With Systemic Lupus Erythematosus

OBJECTIVE Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE. METHODS Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays. RESULTS AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro. CONCLUSION Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.

The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.

Recommendations for the characterization of immunogenicity response to multiple domain biotherapeutics

Many biotherapeutics currently in development have complex mechanisms of action and contain more than one domain, each with a specific role or function. Examples include antibody-drug conjugates (ADC), PEGylated, fusion proteins and bi-specific antibodies. As with any biotherapeutic molecule, a multi-domain biotherapeutic (MDB) can elicit immune responses resulting in the production of specific anti-drug antibodies (ADA) when administered to patients. As it is beneficial to align industry standards for evaluating immunogenicity of MDBs, this paper highlights pertinent immunogenicity risk factors and describes steps involved in the design of a testing strategy to detect and characterize binding (non-neutralizing and neutralizing, NAb) ADAs. In a common tier based approach, samples identified as ADA screen positive are confirmed for the binding specificity of the antibodies to the drug molecule via a confirmatory assay. The confirmation of specificity is generally considered as a critical step of the tier based approach in overall ADA response evaluation. Further characterization of domain specificity of polyclonal anti-MDB ADA response may be required based on the analysis of molecule specific risk factors. A risk based approach in evaluating the presence of NAbs for MDB is discussed in this article. Analysis of domain-specific neutralizing antibody reactivity should be based on the risk assessment as well as the information learned during binding ADA evaluation. Situations where additional characterization of NAb specificity is possible and justified are discussed. Case studies demonstrating applicability of the risk factor based approach are presented. In general, the presence of a domain with high immunogenicity risk or presence of a domain with high endogenous protein homology may result in an overall high immunogenicity risk level for the entire MDB and can benefit from domain specificity characterization of immune response. For low immunogenicity risk MDBs, domain specificity characterization could be re-considered at later clinical phases based on the need to explain specific clinical observations. Inclusion of domain specificity characterization in early phase clinical studies for MDBs with limited clinical immunogenicity experience may be considered to help understand its value in later clinical development. It is beneficial and is recommended to have a well-defined plan for the characterization of ADA domain specificity and data analysis prior to the initiation of sample testing. Overall, best practices for immunogenicity evaluation of complex MDBs are discussed.

Monoclonal Antibodies to Skeletal Muscle Cell Surface

About half of the mass of a vertebrate’s body is muscle tissue, of which the principal cell is the muscle fiber: an extremely long, multinucleate cell which has a regionally specialized surface for interaction with tendons, for propagation of action potentials, and for reception of chemical signals from motor neurons. The intimate relation between motor neuron and muscle fiber has been of special importance to neurobiologists. Studies of the neuromuscular junction have provided fundamental information about synaptic communication. Furthermore, evidence of long-term trophic interactions between muscle fibers and motor neurons has been important for the development of ideas about the plasticity and stability of neuronal connections.

Randomized Phase II Trial of Parsatuzumab (Anti‐EGFL7) or Placebo in Combination with FOLFOX and Bevacizumab for First‐Line Metastatic Colorectal Cancer

Abstract Lessons Learned. These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses. Any further clinical development of anti‐EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents. Background. EGFL7 (epidermal growth factor‐like domain 7) is a tumor‐enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti‐EGFL7 IgG1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5‐fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC). Methods. One‐hundred twenty‐seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles. Results. The progression‐free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71–1.93; p = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46–2.1; p = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms. Conclusions. There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first‐line mCRC.

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG.

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.

Preexisting Antibodies to an F(ab′)2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment

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′)2 CDR using anti-DLL4 F(ab′)2 and 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.

Fit-for-purpose biomarker immunoassay qualification and validation: three case studies

AIM To improve on the efficiency of biomarker assay readiness, and for reliable biomarker data to support three drug programs, we implemented a fit-for-purpose approach, qualifying two biomarker assays and validating a third. Results/methodology: The qualification strategy and selection of experiments for two exploratory biomarkers (CXCL1, CCL19) was determined by the intended use of the biomarker data. The third biomarker, IL-6, was validated as the data would be used in monitoring patient safety during dose-escalation studies in a Phase I trial. All three assays passed a priori acceptance criteria. CONCLUSION These assays highlight strategies and methodologies for a fit-for-purpose approach. Minimum qualification, full qualification and validation were chosen and supported programs at different stages of drug development.

Proposal for a new protein therapeutic immunogenicity titer assay cutpoint

BACKGROUND Generally, immunogenicity assessment strategies follow this assay triage schema: screen→confirm→titer. Each requires the determination of a threshold value (cutpoint) for decision making. No guidance documents exist for the determination of a specific titration assay cutpoint. The default practice is to use the screening assay cutpoint, frequently leading to controls or samples not reaching this cutpoint. We propose a method for determination of a titration cutpoint based upon the variance of the negative-control sample. RESULTS Positive-control samples that did not cross a screening cutpoint did cross the titer cutpoint, albeit generating slightly lower titer values. CONCLUSION Our approach is consistent with the statistical methods currently recommended for the screening and confirmatory assay cutpoints and is operationally simple and efficient.