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Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

The advent of large molecule therapeutics has revolutionized treatment options for previously unmet medical needs. This advent has also led to an increased impact of immunogenicity on drug efficacy and safety. In order to maximize the potential of large molecule therapeutics, immunogenicity-related liabilities must be identified as early in development as possible, using an integrated risk assessment that takes into account the various cell types and processes involved. Here, we describe the development of an ex vivo B-cell immunogenicity assay, to capture a key component of the immune response that has been missing from previously published ex vivo immunogenicity assays. Plasmablasts/plasma cells were preferentially expanded in this assay, a subset of which were drug-specific and presented drug-specific peptides on MHC Class II. This assay represents an important tool in the immunogenicity risk assessment toolkit, to allow liabilities to be identified and mitigated early in the drug development process.

Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.

Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

A critical step in the immunogenicity cascade is attributed to human leukocyte antigen (HLA) II presentation triggering T cell immune responses. The liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based major histocompatibility complex (MHC) II-associated peptide proteomics (MAPPs) assay is implemented during preclinical risk assessments to identify biotherapeutic-derived T cell epitopes. Although studies indicate that HLA-DP and HLA-DQ alleles are linked to immunogenicity, most MAPPs studies are restricted to using HLA-DR as the dominant HLA II genotype due to the lack of well-characterized immunoprecipitating antibodies. Here, we address this issue by testing various commercially available clones of MHC-II pan (CR3/43, WR18, and Tü39), HLA-DP (B7/21), and HLA-DQ (SPV-L3 and 1a3) antibodies in the MAPPs assay, and characterizing identified peptides according to binding specificity. Our results reveal that HLA II receptor-precipitating reagents with similar reported specificities differ based on clonality and that MHC-II pan antibodies do not entirely exhibit pan-specific tendencies. Since no individual antibody clone is able to recover the complete HLA II peptide repertoire, we recommend a mixed strategy of clones L243, WR18, and SPV-L3 in a single immunoprecipitation step for more robust compound-specific peptide detection. Ultimately, our optimized MAPPs strategy improves the predictability and additional identification of T cell epitopes in immunogenicity risk assessments.

ObjectiveRandomised trials of type I anti-CD20 antibodies rituximab and ocrelizumab failed to show benefit in proliferative lupus nephritis (LN). We compared obinutuzumab, a humanised type II anti-CD20 monoclonal antibody that induces potent B-cell depletion, with placebo for the treatment of LN in combination with standard therapies.MethodsPatients with LN receiving mycophenolate and corticosteroids were randomised to obinutuzumab 1000 mg or placebo on day 1 and weeks 2, 24 and 26, and followed through week 104. The primary endpoint was complete renal response (CRR) at week 52. Exploratory analyses through week 104 were conducted. The prespecified alpha level was 0.2.ResultsA total of 125 patients were randomised and received blinded infusions. Achievement of CRR was greater with obinutuzumab at week 52 (primary endpoint, 22 (35%) vs 14 (23%) with placebo; percentage difference, 12% (95% CI −3.4% to 28%), p=0.115) and at week 104 (26 (41%) vs 14 (23%); percentage difference, 19% (95% CI 2.7% to 35%), p=0.026). Improvements in other renal response measures, serologies, estimated glomerular filtration rate and proteinuria were greater with obinutuzumab. Obinutuzumab was not associated with increases in serious adverse events, serious infections or deaths. Non-serious infusion-related reactions occurred more frequently with obinutuzumab.ConclusionsImproved renal responses through week 104 were observed in patients with LN who received obinutuzumab plus standard therapies compared with standard therapies alone. Obinutuzumab was well tolerated and no new safety signals were identified.Trial registration numberNCT02550652.

ObjectiveTo investigate changes in peripheral B cells, B-cell subsets and biomarkers in NOBILITY.MethodsIn the randomized Phase II NOBILITY trial (NCT02550652), patients with lupus nephritis received standard care (MMF) plus obinutuzumab 1000 mg or placebo on Weeks 0, 2, 24 and 26. We analyzed autoantibodies, complement C3/C4, total CD19+ B cells and B-cell subsets using high-sensitivity flow cytometry at Weeks 4, 12, 24, 52, 76 and 104. Sustained B-cell depletion was defined as total B cells below the lower limit of quantitation (LLOQ; 0.4 cells/μL) at Weeks 24 and 52. Detectable B cells were defined as total B cells above LLOQ.ResultsBaseline biomarker levels were similar between treatment groups. By Week 4, 89.3% (50/56) of obinutuzumab-treated patients had total and all B-cell subsets below LLOQ; by Week 104, 91.8% (45/49) had total B cells above LLOQ. At Week 104, mean memory B cells remained low compared with baseline in obinutuzumab-treated patients (3.5 vs 65 cells/µL), whereas mean naïve B cells were repleted (127 vs 229 cells/µL; figure 1). Of 52 obinutuzumab-treated patients with B-cell measurements at Weeks 24 and 52, 61.5% had sustained B-cell depletion. A trend towards higher renal response rates at Week 76 were observed in obinutuzumab-treated patients with sustained B-cell depletion and those with detectable B cells compared with placebo-treated patients (figure 2). In patients positive for anti-dsDNA antibodies at baseline (>30 IU/mL), median anti-dsDNA antibodies at Week 104 were reduced by 84% and 39% in the obinutuzumab and placebo groups, respectively; anti-C1q antibodies were reduced by 61% and 7%, respectively. Among patients who were hypocomplementemic at baseline (C3 <90 mg/dL or C4 <16 mg/dL), more patients who received obinutuzumab than placebo had normal C3 (75% vs 38%) and C4 (82% vs 31%) levels at Week 52.ConclusionIn patients with lupus nephritis, obinutuzumab treatment resulted in robust and sustained reductions in B cells, greater renal response in patients with sustained B-cell depletion and greater improvements in autoantibodies and C3/C4 compared with placebo. These data support the superiority of obinutuzumab plus MMF at inducing renal response.AcknowledgementsFunded by F. Hoffmann-La Roche Ltd. Editorial assistance was provided by Health Interactions, Inc., and funded by F. Hoffmann-La Roche Ltd.DisclosuresR.A. Furie has received research support and consulting fees from Genentech, Inc.S.W. Tas has received research support from F. Hoffmann-La Roche Ltd/Genentech, Inc.A. Malvar has received consulting fees from Genentech, Inc., and F. Hoffmann-La Roche Ltd.C.M. Looney, E. Martins, A. Mao and T. Schindler are employees and shareholders of F. Hoffmann-La Roche Ltd.H. Raghu, V.G. Anania and J.A. Ross Terres are employees of Genentech, Inc., and shareholders of F. Hoffmann-LaRoche Ltd.E.M. Vital has received consulting fees from F. Hoffmann-La Roche Ltd/Genentech, Inc.Abstract O33 Figure 1Depletion of B cells and B-cell subsets over time in patients with lupus nephritis in NOBILITYCD, cluster of differentiation; MMF, mycophenolate mofetil.Abstract O33 Figure 2Renal response at week 76 by B-cell depletion status at weeks 24 and 52. HPF, high-powered field; RBC, red blood cell; LLOQ, lower limit of quantitation; ULN, upper limit of normal; UPCR, urine protein-to-creatinine ratio. †Total B cells below LLOQ (0.4 cells/μL). ‡Total B cells above LLOQ (0.4 cells/μL). §A composite measure requiring UPCR of <0.5, normal renal function (serum creatinine ≤ ULN) without worsening of baseline serum creatinine by >15% and inactive urinary sediment (<10 RBCs/HPF without RBC casts). A composite measure requiring serum creatinine of ≤15% above baseline value, no urinary RBC casts and either RBCs/HPF of ≤50% above baseline or <10 RBCs/HPF, 50% UPCR improvement, with 1 of the following conditions met (1) if the baseline UPCR is ≤3.0, then UPCR of <1.0 or (2) if the baseline UPCR is >3.0, then UPCR of <3.0. * P<0.2 vs placebo group. ** P<0.05 vs placebo group. *** P<0.001 vs placebo group.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified . By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.