Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Characterization and root cause analysis of immunogenicity to pasotuxizumab (AMG 212), a prostate-specific membrane antigen-targeting bispecific T-cell engager therapy.

Introduction: In oncology, anti-drug antibody (ADA) development that significantly curtails response durability has not historically risen to a level of concern. The relevance and attention ascribed to ADAs in oncology clinical studies have therefore been limited, and the extant literature on this subject scarce. In recent years, T cell engagers have gained preeminence within the prolific field of cancer immunotherapy. These drugs whose mode of action is expected to potently stimulate anti-tumor immunity, may potentially induce ADAs as an unintended corollary due to an overall augmentation of the immune response. ADA formation is therefore emerging as an important determinant in the successful clinical development of such biologics. Methods: Here we describe the immunogenicity and its impact observed to pasotuxizumab (AMG 212), a prostate-specific membrane antigen (PSMA)-targeting bispecific T cell engager (BiTE®) molecule in NCT01723475, a first-in-human (FIH), multicenter, dose-escalation study in patients with metastatic castration-resistant prostate cancer (mCRPC). To explain the disparity in ADA incidence observed between the SC and CIV arms of the study, we interrogated other patient and product-specific factors that may have explained the difference beyond the route of administration. Results: Treatment-emergent ADAs (TE-ADA) developed in all subjects treated with at least 1 cycle of AMG 212 in the subcutaneous (SC) arm. These ADAs were neutralizing and resulted in profound exposure loss that was associated with contemporaneous reversal of initial Prostate Surface Antigen (PSA) responses, curtailing durability of PSA response in patients. Pivoting from SC to a continuous intravenous (CIV) administration route remarkably yielded no subjects developing ADA to AMG 212. Through a series of stepwise functional assays, our investigation revealed that alongside a more historically immunogenic route of administration, non-tolerant T cell epitopes within the AMG 212 amino acid sequence were likely driving the high-titer, sustained ADA response observed in the SC arm. Discussion: These mechanistic insights into the AMG 212 ADA response underscore the importance of performing preclinical immunogenicity risk evaluation as well as advocate for continuous iteration to better our biologics.

Immunogenicity assessment of bispecific antibody-based immunotherapy in oncology.

With increasing numbers of bispecific antibodies (BsAbs) and multispecific products entering the clinic, recent data highlight immunogenicity as an emerging challenge in the development of such novel biologics. This review focuses on the immunogenicity risk assessment (IgRA) of BsAb-based immunotherapies for cancer, highlighting several risk factors that need to be considered. These include the novel scaffolds consisting of bioengineered sequences, the potentially synergistic immunomodulating mechanisms of action (MOAs) from different domains of the BsAb, as well as several other product-related and patient-related factors. In addition, the clinical relevance of anti-drug antibodies (ADAs) against selected BsAbs developed as anticancer agents is reviewed and the advances in our knowledge of tools and strategies for immunogenicity prediction, monitoring, and mitigation are discussed. It is critical to implement a drug-specific IgRA during the early development stage to guide ADA monitoring and risk management strategies. This IgRA may include a combination of several assessment tools to identify drug-specific risks as well as a proactive risk mitigation approach for candidate or format selection during the preclinical stage. The IgRA is an on-going process throughout clinical development. IgRA during the clinical stage may bridge the gap between preclinical immunogenicity prediction and clinical immunogenicity, and retrospectively guide optimization efforts for next-generation BsAbs. This iterative process throughout development may improve the reliability of the IgRA and enable the implementation of effective risk mitigation strategies, laying the foundation for improved clinical success.

Immune Complex Formation Is Associated With Loss of Tolerance and an Antibody Response to Both Drug and Target.

AMG 966 is a bi-specific, heteroimmunoglobulin molecule that binds both tumor necrosis factor alpha (TNFα) and TNF-like ligand 1A (TL1A). In a first-in-human clinical study in healthy volunteers, AMG 966 elicited anti-drug antibodies (ADA) in 53 of 54 subjects (98.1%), despite a paucity of T cell epitopes observed in T cell assays. ADA were neutralizing and bound to all domains of AMG 966. Development of ADA correlated with loss of exposure. In vitro studies demonstrated that at certain drug-to-target ratios, AMG 966 forms large immune complexes with TNFα and TL1A, partially restoring the ability of the aglycosylated Fc domain to bind FcγRIa and FcγRIIa, leading to the formation of ADA. In addition to ADA against AMG 966, antibodies to endogenous TNFα were also detected in the sera of subjects dosed with AMG 966. This suggests that the formation of immune complexes between a therapeutic and target can cause loss of tolerance and elicit an antibody response against the target.

Assessment of romiplostim immunogenicity in pediatric patients in clinical trials and in a global postmarketing registry.

Development of first-generation thrombopoietins (TPOs) was halted due to antibodies that neutralized endogenous TPO, causing protracted thrombocytopenia in some patients. The second-generation TPO receptor agonist romiplostim, having no homology to TPO, was developed to circumvent potential immunogenicity. We examined the development of binding and neutralizing antibodies to romiplostim and TPO among pediatric patients with primary immune thrombocytopenia (ITP) in 5 clinical trials and a global postmarketing registry. In the trials, 25 of 280 (8.9%) patients developed anti-romiplostim binding antibodies. The first positive result was detected 67 weeks (median) after romiplostim treatment was initiated. The median romiplostim dose was 8 µg/kg, and the median platelet count was 87 × 109/L. Most patients who developed anti-romiplostim binding antibodies (18 of 25 [72%]) had ≥90% of platelet assessments showing a response. Anti-romiplostim neutralizing antibodies developed in 8 of 280 (2.9%) patients. The development of anti-romiplostim neutralizing antibodies was unrelated to the romiplostim dose, and most patients who developed the antibodies (7 of 8 [88%]) had platelet response. Nine of 279 (3.2%) patients developed anti-TPO binding antibodies, and 1 (0.4%) developed transient anti-TPO neutralizing antibodies. In 8 patients who developed anti-romiplostim neutralizing antibodies, no TPO cross-reactivity was observed. In the postmarketing registry, 3 of 19 (15.8%) patients developed anti-romiplostim binding antibodies; 1 (5.3%) patient developed anti-romiplostim neutralizing antibodies. These results suggest that immunogenicity to romiplostim occurs infrequently in pediatric patients with ITP and is generally not associated with loss of platelet response or other negative clinical sequelae.

Assessment of romiplostim immunogenicity in adult patients in clinical trials and in a global postmarketing registry.

Antibodies to first-generation recombinant thrombopoietin (TPO) neutralized endogenous TPO and caused thrombocytopenia in some healthy subjects and chemotherapy patients. The second-generation TPO receptor agonist romiplostim, having no sequence homology to TPO, was developed to avoid immunogenicity. This analysis examined development of binding and neutralising antibodies to romiplostim or TPO among adults with immune thrombocytopenia (ITP) in 13 clinical trials and a global postmarketing registry. 60/961 (6·2%) patients from clinical trials developed anti-romiplostim-binding antibodies post-baseline. The first positive binding antibody was detected 14 weeks (median) after starting romiplostim, at median romiplostim dose of 2 µg/kg and median platelet count of 29.5 × 109 /l; most subjects had ≥98·5% of platelet assessments showing response. Neutralising antibodies to romiplostim developed in 0·4% of patients, but were unrelated to romiplostim dose and did not affect platelet count. Thirty-three patients (3·4%) developed anti-TPO-binding antibodies; none developed anti-TPO-neutralising antibodies. In the global postmarketing registry, 9/184 (4·9%) patients with spontaneously submitted samples had binding antibodies. One patient with loss of response had anti-romiplostim-neutralising antibodies (negative at follow-up). Collectively, anti-romiplostim-binding antibodies developed infrequently. In the few patients who developed neutralising antibodies to romiplostim, there was no cross-reactivity with TPO and no associated loss of platelet response.

A randomized, double-blind, single-dose, three-arm, parallel group study to determine pharmacokinetic similarity of ABP 959 and eculizumab (Soliris® ) in healthy male subjects.

OBJECTIVES: ABP 959 is a proposed biosimilar to eculizumab, a monoclonal antibody targeting the human C5 complement protein. The objective of this randomized, double-blind, three-arm, study was to demonstrate pharmacokinetic (PK) and pharmacodynamic (PD) similarity of ABP 959 relative to the eculizumab reference product (RP) in healthy adult male subjects. METHODS: Eligible subjects aged 18-45 years were randomized to receive a 300-mg IV infusion of ABP 959, or FDA-licensed eculizumab (eculizumab US), or EU-authorized eculizumab (eculizumab EU). Primary PK endpoint was area under the total serum concentration-time curve from 0 to infinity (AUC0-∞ ); primary PD endpoint was area between the effect curve (ABEC) of CH50-time data. RESULTS: The geometric mean of PK and PD parameters were similar between ABP 959 versus eculizumab US and eculizumab EU; PK and PD similarity was established based on 90% confidence intervals of the geometric mean ratio being within prespecified equivalence margin of 0.8 and 1.25. The incidence of treatment-emergent adverse events was similar across groups. The incidence of binding anti-drug antibodies was similar across treatments; no subjects developed neutralizing antibodies. CONCLUSIONS: This study demonstrated PK and PD similarity of ABP 959 to eculizumab RP; safety and immunogenicity profiles were also similar.

Risk of pure red cell aplasia in patients with hepatitis C receiving antiviral therapy and an erythropoiesis-stimulating agent.

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin.

A detailed examination of the antibody prevalence and characteristics of anti-ESA antibodies.

BACKGROUND: The antibody characteristics in erythropoiesis-stimulating agent (ESA)-treated patients who develop antibody-mediated pure red cell aplasia (PRCA; amPRCA) can be described as high-affinity, neutralizing anti-ESA antibodies with a mixed immunoglobulin G (IgG) subclass. The characteristics of an early-onset anti-ESA antibody response are not well documented, especially in the months prior to the development of amPRCA. Therefore, a detailed characterization of anti-ESA antibodies was performed in patients in both clinical studies and in a post-market setting. Both baseline and post-dose samples were tested and antibody-positive samples were characterized. Antibody characteristics such as concentration, isotype and specificity were evaluated in subjects with non-neutralizing anti-ESA antibodies and subjects that developed neutralizing anti-ESA antibodies associated with amPRCA. METHODS: Serum samples were analyzed for the presence of anti-ESA antibodies, using a validated surface plasmon resonance (SPR)-based immunoassay or SPRIA. RESULTS: Among the clinical studies, pre-existing non-neutralizing anti-ESA antibodies were found in 6% of the subjects from clinical studies in nephrology, oncology and congestive heart failure (CHF). After ESA treatment, 2.3% of the subjects developed binding, non-neutralizing antibodies with 0.1% confirmed as having an IgG isotype and were specific to the ESA protein. IgM antibodies were detected at baseline and post-ESA treatment and reported to be specific to the glycosylation of the ESA. No clinical study subjects progressed to amPRCA. In contrast, anti-ESA antibody-positive subjects from the post-market setting with a confirmed IgG subclass were specific to the ESA protein. Subjects that had progressed to amPRCA were noted to have high antibody concentrations with neutralizing activity and a diverse IgG subtype. CONCLUSIONS: A low prevalence of non-neutralizing anti-ESA IgM specific to glycosylation on the ESA and IgG1 antibodies specific to the ESA protein was detected across all clinical patient populations. Patients with amPRCA were noted to have high IgG antibody concentrations, neutralizing antibodies and the presence of anti-ESA IgG4 antibodies.

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing.

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance. Further, this human anti-ESA antibody panel may help set the limits of each assay platform in terms of the full repertoire of the anti-ESA antibodies, and may facilitate standardization of ESA immunogenicity reporting across assay platforms.

Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison.

BACKGROUND: The immunological methods for detecting antibodies to erythropoiesis-stimulating agents (ESAs) differ in assay sensitivity. However, this parameter, routinely determined in clinical assays using a high-affinity non-human polyclonal antibody, gives a one-dimensional assessment of antibody detection. We compare three widely used immunological methods and evaluate the ability of each to detect mature human antibodies and human antibodies characteristic of an early immune response. METHODS: The detection of anti-ESA antibodies was compared between a radioimmunoprecipitation (RIP) assay, an electrochemiluminescence (ECL) bridging enzyme-linked immunosorbent assay and a surface plasmon resonance (SPR)-based immunoassay. All three methods were validated for sensitivity, specificity and precision. Specimens from clinical studies or post market testing were categorized as pure red cell aplasia (PRCA) or non-PRCA and then analyzed in each method. RESULTS: Among the antibody-mediated PRCA samples, which contain high affinity neutralizing antibodies, there was strong correlation between all methods. The results from non-PRCA sample analysis show high correlation between RIP and ECL methods; however, differences between the SPR immunoassay and the ECL and RIP were demonstrated. The samples that scored positive in the SPR immunoassay and negative by RIP and ECL were characterized to be of low antibody concentration, contained a high percentage of rapidly dissociating antibodies, or were antibodies of the IgM isotype. CONCLUSIONS: All three immunological methods are appropriate for detection of antibodies associated with antibody-mediated PRCA. However, the SPR immunoassay platform detected an early, low affinity IgG and IgM antibody response as well as detected and characterized a pathogenic antibody response associated with antibody-mediated PRCA.

The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.

A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.

Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of panitumumab.

Evaluation of the immunogenicity of panitumumab, a fully human anti-epidermal growth factor receptor mAb approved for use in colorectal cancer patients, led to the development of two separate immunoassays for the detection of anti-panitumumab Abs. The first immunoassay used a bridging ELISA capable of detecting 10 ng/ml positive control anti-panitumumab Ab. The ELISA incorporated an acid dissociation step to reduce drug interference and tolerated the presence of approximately 100-fold molar excess of drug. During eight clinical trials, the ELISA detected developing Ab responses in 2 of 612 (0.3%) subjects. In one of the ELISA positive subjects, neutralizing Abs were detected using an epidermal growth factor receptor phosphorylation bioassay. The second immunoassay used a Biacore biosensor immunoassay format capable of detecting 1 mug/ml positive control Ab while tolerating the presence of equal molar amounts of drug. Although less sensitive and less tolerant to competing drug in the assay, the Biacore assay detected developing Ab responses in 25 of the 604 (4.1%) subjects. Additionally, the Biacore assay identified eight subjects who developed neutralizing Abs. Mouse mAbs with affinities ranging from 1.1 x 10(-6) to 8.4 x 10(-10) M were used to characterize both assay types. The ELISA was more sensitive for the detection of higher affinity mAbs and detected high-affinity mAbs in the presence of higher molar ratio of drug to mAb. The Biacore assay was more sensitive for detection of lower affinity mAbs and detected low affinity Abs in the presence of higher molar ratios of drug to mAb.