Aqueous humour interleukin-6 and vision outcomes with anti-vascular endothelial growth factor therapy.

BACKGROUND: This analysis evaluated aqueous humour (AH) interleukin (IL)-6 concentrations and the association between AH IL-6 and visual outcomes in patients with neovascular age-related macular degeneration (nAMD) or diabetic macular oedema (DMO) receiving anti-vascular endothelial growth factor (VEGF) monotherapy. METHODS: Post hoc analysis of the multicentre, double-masked, randomised HARBOR (NCT00891735) and READ-3 (NCT01077401) trials. HARBOR enrolled treatment-naïve nAMD patients. READ-3 enrolled treatment-naïve/previously treated DMO patients. HARBOR patients received ranibizumab 0.5 or 2.0 mg monthly or as needed; AH samples were collected at month 2, after two previous intravitreal injections. READ-3 patients received ranibizumab 0.5 or 2.0 mg as needed; AH samples were collected at baseline and months 3, 6, 9, and 12. MAIN OUTCOME MEASURE: association between AH IL-6 concentrations and month 24 best-corrected visual acuity (BCVA). RESULTS: In both trials (HARBOR, N = 36; READ-3, N = 137), patients with higher AH IL-6 concentrations had worse visual outcomes. HARBOR patients with low AH IL-6 concentrations at month 2 had a mean (95% CI) BCVA change at month 24 of +2.9 (-2.6, 8.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of -9.0 (-22.7, 4.7) letters. READ-3 patients with low AH concentrations at baseline had a mean (95% CI) BCVA change at month 12 of +9.3 (7.4, 11.3) letters, whereas patients with high AH concentrations had a mean (95% CI) BCVA change of +5.6 (2.2, 9.1) letters. CONCLUSIONS: Higher IL-6 AH concentrations may predict suboptimal visual responses to anti-VEGF monotherapy in patients with nAMD/DMO.

Development of an anti-drug antibody assay to detect anti-drug antibodies to protein and PEG in a PEGylated molecule.

Background: PEGylation technology is one of long-acting delivery (LAD) platforms used to increase half-life of protein therapeutics. However, PEGylation of anti-Factor D Fab (PEG-aFD) poses challenges for detecting anti-drug antibody (ADA) to both Fab and polyethylene glycol (PEG) portions. Results: Although the bridging ELISA using traditional assay diluent containing Tween 20 is good for detecting ADA to Fab, it failed to detect ADA to PEG. Instead of only reducing Tween 20 in assay diluent, using a proprietary commercial buffer PEG50-1 as assay diluent successfully enabled the detection of ADA to both Fab and PEG with fit-for-purpose sensitivity and drug tolerance. Conclusion: Identification of appropriate assay diluent is critical for detection of ADA to both Fab and PEG in a PEGylated molecule.

Antibody-mediated activation of the FGFR1/Klothoß complex corrects metabolic dysfunction and alters food preference in obese humans.

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoß (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects.

Immunoaffinity LC-MS/MS is more suitable than ELISA to quantify a PEGylated molecule in cynomolgus monkey serum.

Background: Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. Results: An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80-120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC-MS/MS) assay was developed, achieving recovery within 80-120% for both free and conjugated Fab. Conclusion: Immunoaffinity LC-MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.

Immunoaffinity LC-MS/MS to quantify a PEGylated anti-Factor D Fab biotherapeutic in cynomolgus monkey serum.

Aim: To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Materials & methods: Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. Results & conclusion: The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.

Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions: Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

Bioanalytical challenges and unique considerations to support pharmacokinetic characterization of bispecific biotherapeutics.

Compared with conventional (monospecific) therapeutics, bispecific protein therapeutics present unique challenges for pharmacokinetic (PK) characterization - namely, the characterization of multiple functional domains as well as the consideration of biotransformation or interference by the formation of antitherapeutic antibodies against each functional domain. PK characterization is essential to the success of the overall drug development plan and for molecules with multiple binding domains; multiple bioanalytical methods may be needed to answer critical questions for each phase of drug development. The number of bispecific protein therapeutics entering drug development continues to increase, and therefore, a bioanalytical strategy for the PK characterization of bispecific molecules and study of their in vivo structure-function relationship is needed. This review presents case studies and a regulatory perspective.

Phase I study of the anti-α5ß1 monoclonal antibody MINT1526A with or without bevacizumab in patients with advanced solid tumors.

PURPOSE: MINT1526A is a monoclonal antibody that blocks the interaction of integrin alpha 5 beta 1 (α5ß1) with its extracellular matrix ligands. This phase I study evaluated the safety and pharmacokinetics of MINT1526A with or without bevacizumab in patients with advanced solid tumors. METHODS: MINT1526A was administered every 3 weeks (Q3W) as monotherapy (arm 1) or in combination with bevacizumab 15 mg/kg, Q3W (arm 2). Each arm included a 3 + 3 dose-escalation stage and a dose-expansion stage. RESULTS: Twenty-four patients were enrolled in arm 1 (dose range 2-30 mg/kg) and 30 patients were enrolled in arm 2 (dose range 3-15 mg/kg). Monocyte α5ß1 receptor occupancy was saturated at a dose of 15 mg/kg. No dose-limiting toxicities were observed, and the maximum tolerated dose was not reached in either arm. The most common adverse events, regardless of causality, included abdominal pain (25%), diarrhea (25%), nausea (21%), vomiting (21%), and fatigue (21%) in arm 1 and nausea (40%), fatigue (33%), vomiting (30%), dehydration (30%), headache (30%), and hypertension (30%) in arm 2. No grade ≥ 3 bleeding events were observed in either arm. No confirmed partial responses (PR) were observed in arm 1. In arm 2, one patient with thymic carcinoma experienced a confirmed PR and two patients with hepatocellular carcinoma (HCC) experienced durable minor radiographic responses. CONCLUSIONS: MINT1526A, with or without bevacizumab, was well-tolerated. Preliminary evidence of combination efficacy, including in patients with HCC, was observed, but cannot be distinguished from bevacizumab monotherapy in this phase I study.

Development and validation of a novel semi-homogenous clinical assay for quantitation of Ranibizumab in human serum.

Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and efficacy. For drugs administered intravitreally, systemic drug levels during the course of clinical studies are typically 100 to 1000-fold lower than those of similar therapeutics dosed intravenously, posing a significant bioanalytical challenge for PK measurements. Thus, the development of a highly-sensitive assay for measuring pg/mL levels of ranibizumab in patients' sera after intravitreal administration was needed to support clinical studies. In this report, we describe the development of a novel method that utilizes a high-affinity murine monoclonal anti-ranibizumab-VEGF-complexes antibody (MARA) reagent to measure ranibizumab in human serum. The assay format utilizes a semi-homogeneous solution phase step using a monoclonal antibody (the MARA) that binds specifically to the ranibizumab-VEGF complex, but not to either alone. This unique reagent exhibited low non-specific binding and high selectivity, increasing signal-to-noise readouts and maximizing assay sensitivity. The resulting MARA enzyme-linked immunosorbent assay (ELISA) has a lower limit of quantification of 15 pg/mL in human serum. In the assay, serum samples are incubated overnight with a mixture containing biotinylated-VEGF and MARA, which form a three-molecule complex with ranibizumab in the sample. These complexes are then captured onto streptavidin-coated wells, followed by enzymatic detection using a horseradish peroxidase-labeled-anti-murine antibody reagent and a colorimetric reaction. The assay conditions were optimized to allow for quantitative detection of "total" ranibizumab levels in serum. The assay was fully validated, establishing its high tolerance to sample matrix, as well as its suitable specificity, accuracy, dilution linearity, as well as intra- and inter-assay precision. The MARA ELISA's novel and unique approach has resulted in a considerably more sensitive ranibizumab PK assay compared to earlier versions of this assay. The MARA ELISA has been used for PK measurements in multiple ranibizumab studies, supporting this drug's life-cycle management and related preclinical and clinical-development studies.

Randomized Phase II Trial of Parsatuzumab (Anti-EGFL7) or Placebo in Combination with Carboplatin, Paclitaxel, and Bevacizumab for First-Line Nonsquamous Non-Small Cell Lung Cancer.

LESSONS LEARNED: The lack of efficacy associated with anti-EGFL7 combined with standard bevacizumab and chemotherapy in this phase II trial in non-small cell lung carcinoma is consistent with the lack of benefit observed in colorectal carcinoma, highlighting the challenge of enhancing the efficacy of VEGF inhibition in unselected populations.Future efforts with agents like anti-EGFL7 should be guided by advances in pharmacodynamic and predictive biomarker development for antiangiogenic agents. BACKGROUND: Epidermal growth factor-like domain 7 (EGFL7) is an extracellular matrix-associated protein that is upregulated during angiogenesis and supports endothelial cell survival. This phase II trial evaluated the efficacy of the anti-EGFL7 antibody, parsatuzumab, in combination with bevacizumab plus platinum-based therapy for advanced or recurrent nonsquamous non-small cell lung cancer (NS-NSCLC). METHODS: Patients (n = 104) were randomized to either placebo or parsatuzumab (600 mg) in combination with bevacizumab (15 mg/kg) and carboplatin/paclitaxel, administered on day 1 of each 21-day cycle. Carboplatin and paclitaxel were administered for up to six cycles. Bevacizumab and parsatuzumab/placebo were administered for a maximum of 24 months. RESULTS: The progression-free survival (PFS) hazard ratio (HR) was 1.7 (95% confidence interval [CI], 1.0-2.8; p = .047). The median PFS was 6.7 months for the parsatuzumab arm versus 8.1 months for the placebo arm. The hazard ratio for overall survival (OS) was 1.1 (95% CI, 0.5-2.2; p = .847). The objective response rate (ORR) was 29% in the parsatuzumab arm and 56% in the placebo arm. Overall safety and tolerability were consistent with the established toxicity profile of bevacizumab. CONCLUSION: There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first-line NS-NSCLC.

Advancements in Understanding Immunogenicity of Biotherapeutics in the Intraocular Space.

Therapeutic breakthroughs in a number of retinal degenerative diseases have come about through the development of biotherapeutics administered directly into the eye. As a consequence of their use, we have gained more insight into the immune privileged status of the eye and the various considerations that development, manufacturing, and use of these drugs require. It has been observed that therapeutic proteins injected into the vitreous can elicit an immune response resulting in the production of anti-drug antibodies (ADAs) which can have clinical consequences. This review includes discussion of the anatomy, physiology, and specific area of the eye that are targeted for drug administration. The various immunologic mechanisms involved in the immune responses to intraocularly administered protein are discussed. This review entails discussion on chemistry, manufacturing, and control (CMC) and formulation-related issues that may influence the risk of immunogenicity. Based on the available immunogenicity profile of the marketed intraocular drugs and their reported adverse events, the animal models and the translational gap from animals to human are discussed. Thus, the objective of this review article is to assess the factors that influence immunogenicity in relation to intraocular administration and the steps taken for mitigating immunogenicity risks.

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

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. The Oncologist 2017;22:375-e30.

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.

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.

Workshop Report: AAPS Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment.

A novel format was introduced at the recent AAPS NBC Workshop on Method Development, Validation and Troubleshooting in San Diego on 18th May 2014. The workshop format was initiated by Binodh De Silva; Marie Rock and Sherri Dudal joined the initiative to develop and chair the workshop. Questions were solicited by a variety of avenues, including a Linked-In Discussion Group. Once collated and clarified, the topics covered assay development, validation, and analysis of PK, Immunogenicity, and Biomarkers with an additional topic on alternative bioanalytical technologies. A panel of experts (workshop report co-authors) was assigned to each topic to bring forward thought-provoking aspects of each topic. The format of the workshop was developed to target the needs of bioanalytical scientists with intermediate to advanced experience in the field ranging to enable robust discussion and to delve deeper into the current bioanalytical hot topics. While the new format allowed for an interactive session with the topical discussion driven by the audience members, it did not foster equal discussion time for all of the proposed topics, especially Biomarkers and alternative LBA technologies.

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.

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.