Teplizumab for treatment of type 1 diabetes (Protégé study): 1-year results from a randomised, placebo-controlled trial.

BACKGROUND: Findings of small studies have suggested that short treatments with anti-CD3 monoclonal antibodies that are mutated to reduce Fc receptor binding preserve ß-cell function and decrease insulin needs in patients with recent-onset type 1 diabetes. In this phase 3 trial, we assessed the safety and efficacy of one such antibody, teplizumab. METHODS: In this 2-year trial, patients aged 8-35 years who had been diagnosed with type 1 diabetes for 12 weeks or fewer were enrolled and treated at 83 clinical centres in North America, Europe, Israel, and India. Participants were allocated (2:1:1:1 ratio) by an interactive telephone system, according to computer-generated block randomisation, to receive one of three regimens of teplizumab infusions (14-day full dose, 14-day low dose, or 6-day full dose) or placebo at baseline and at 26 weeks. The Protégé study is still underway, and patients and study staff remain masked through to study closure. The primary composite outcome was the percentage of patients with insulin use of less than 0·5 U/kg per day and glycated haemoglobin A(1c) (HbA(1C)) of less than 6·5% at 1 year. Analyses included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00385697. FINDINGS: 763 patients were screened, of whom 516 were randomised to receive 14-day full-dose teplizumab (n=209), 14-day low-dose teplizumab (n=102), 6-day full-dose teplizumab (n=106), or placebo (n=99). Two patients in the 14-day full-dose group and one patient in the placebo group did not start treatment, so 513 patients were eligible for efficacy analyses. The primary outcome did not differ between groups at 1 year: 19·8% (41/207) in the 14-day full-dose group; 13·7% (14/102) in the 14-day low-dose group; 20·8% (22/106) in the 6-day full-dose group; and 20·4% (20/98) in the placebo group. 5% (19/415) of patients in the teplizumab groups were not taking insulin at 1 year, compared with no patients in the placebo group at 1 year (p=0·03). Across the four study groups, similar proportions of patients had adverse events (414/417 [99%] in the teplizumab groups vs 98/99 [99%] in the placebo group) and serious adverse events (42/417 [10%] vs 9/99 [9%]). The most common clinical adverse event in the teplizumab groups was rash (220/417 [53%] vs 20/99 [20%] in the placebo group). INTERPRETATION: Findings of exploratory analyses suggest that future studies of immunotherapeutic intervention with teplizumab might have increased success in prevention of a decline in ß-cell function (measured by C-peptide) and provision of glycaemic control at reduced doses of insulin if they target patients early after diagnosis of diabetes and children. FUNDING: MacroGenics, the Juvenile Diabetes Research Foundation, and Eli Lilly.

A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs.

Bioanalytical assessments of anti-drug antibodies (ADAs) provide an understanding of the immunogenicity of biological drug molecules. The potential to induce ADAs after treatment with biologics is a safety issue that has become an important consideration in the development of biologics and a critical aspect of regulatory filings. US and European regulatory agencies are recommending that sponsors study immunogenicity using a risk-based approach, encouraging sponsors to formulate and implement their own risk management plans and to conduct discussions with the agencies when necessary. It follows from this that the greater the safety risks of ADAs, the more diligently one should clarify the immunogenicity of the product. Here we propose a general strategy to broadly assign immunogenicity risk levels to biological drug products, and present risk level-based 'fit-for-purpose' bioanalytical schemes for the investigations of treatment-related ADAs in clinical and nonclinical studies.

Medication-Taking Practices of Patients on Antiretroviral HIV Therapy: Control, Power, and Intentionality.

Among people living with HIV (PLWH), adherence to antiretroviral therapy (ART) is crucial for health, but patients face numerous challenges achieving sustained lifetime adherence. We conducted six focus groups with 56 PLWH regarding ART adherence barriers and collected sociodemographics and ART histories. Participants were recruited through clinics and AIDS service organizations in North Carolina. Dedoose software was used to support thematic analysis. Participants were 59% male, 77% black, aged 23-67 years, and living with HIV 4-20 years. Discussions reflected the fluid, complex nature of ART adherence. Maintaining adherence required participants to indefinitely assert consistent control across multiple areas including: their HIV disease, their own bodies, health care providers, and social systems (e.g., criminal justice, hospitals, drug assistance programs). Participants described limited control over treatment options, ART's impact on their body, and inconsistent access to ART and subsequent inability to take ART as prescribed. When participants felt they had more decision-making power, intentionally choosing whether and how to take ART was not exclusively a decision about best treating HIV. Instead, through these decisions, participants tried to regain some amount of power and control in their lives. Supportive provider relationships assuaged these struggles, while perceived side-effects and multiple co-morbidities further complicated adherence. Adherence interventions need to better convey adherence as a continuous, changing process, not a fixed state. A perspective shift among care providers could also help address negative consequences of the perceived power struggles and pressures that may drive patients to exert control via intentional medication taking practices.

Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use.

A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers of chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, small-scale protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6 M urea, 6 M guanidine, 100 mM NaOH or 500 mM NaOH). Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG. Under all cleaning conditions tested, the log,10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles. In the two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by antibody (Ab) step yield and breakthrough. In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100 mM NaOH) or cycle 50 (500 mM NaOH). For media cycled 300+ times using 6 M urea or 6 M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident. In summary, our studies identify Ab step yield and breakthrough as performance attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled. Thus, we propose that virus removal validation studies should be performed on new media only and these attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media.

Teplizumab preserves C-peptide in recent-onset type 1 diabetes: two-year results from the randomized, placebo-controlled Protégé trial.

Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.

Nonclinical evaluation of GMA161--an antihuman CD16 (FcγRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.

Immunization with meningococcal polysaccharide-tetanus toxoid conjugate induces polysaccharide-reactive T cells in mice.

T cell clones were generated from mice immunized with a meningococcal group C (alpha2 --> 9-sialic acid) polysaccharide-tetanus toxoid (MCPS-TT) conjugate. Many clones were found to be specific for tetanus toxoid (TT), however, clones reactive with MCPS-TT and polysaccharide (PS) were isolated. Two clones were specific for MCPS and two cross-reacted with Escherichia coli K1-PS (alpha2 --> 8-sialic acid). Both TT and PS reactive clones were CD4+ and CD8-. TT and MCPS-TT-specific T cell clones were major histocompatibility complex (MHC) restricted, however, the PS-reactive clones were not. Both MHC-restricted TT clones and non-restricted PS clones, however, were dependent on contact with antigen presenting cells (APC) for maximal stimulation. The data suggest that multivalent repeating epitopes on PS antigen (Ag) can overcome the need for MHC restricted interactions, but not the requirement for cell-cell contact.

The Sr1 gene that controls diversity of the anti-inulin antibody response maps to mouse chromosome 14.

Previous studies demonstrated that the diversity of the antibody response of mice to the inulin (In) determinant of bacterial levan is regulated by the gene Spectrotype Regulation 1 ( Sr1). BALB/c mice produce a monoclonal anti-In response as shown by isoelectric focusing analysis. In contrast, the anti-In antibody response of (BALB/cxC57BL/6)F1 mice is significantly more heterogeneous. We performed a backcross and a genome-wide scan with microsatellite markers and found that Sr1 is tightly linked to D14Mit121 on chromosome (Chr) 14. This location for Sr1 was supported by analysis of CXB Recombinant Inbred strains. We further confirmed this by finding that the Chr 14 congenic mouse strain B6.C-H8 lacks the C57BL/6 allele of the Sr1 gene, indicating that Sr1 is located in the segment of Chr 14 replaced with BALB/c donor DNA. These data place Sr1 near to or coincident with the Tcra/Tcrd T-cell receptor gene complex and suggest a role for T cells in diversifying the anti-In response.

Surface plasmon resonance analysis of antipolysaccharide antibody specificity: responses to meningococcal group C conjugate vaccines and bacteria.

Antibody (Ab) responses to polysaccharides (PS), such as Neisseria meningitidis group C PS (MCPS), are characterized as being thymus independent and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled with tetanus toxoid or the diphtheria toxin derivative CRM197, elicit thymus-dependent responses. The present study developed a surface plasmon resonance approach to evaluate Ab responses to MCPS conjugate vaccines, including either O-acetylated (OAc+) or de-O-acetylated (OAc-) forms of the PS. The results were generally consistent with those obtained by enzyme-linked immunosorbent assay and showed that sera from mice immunized with conjugate vaccines contain Abs that bind more effectively to OAc+ and OAc- MCPS than sera from mice immunized with fixed bacteria. The data suggest a critical shared or overlapping epitope recognized by all the conjugate vaccine immune sera and strategies for assessing polyclonal Ab avidity.

Impact of cell culture process changes on endogenous retrovirus expression.

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.

Evaluation of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus in mAb cell-culture.

Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by evaluation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In a previous report, we demonstrated the utility of TaqMan fluorogenic 5'-nuclease product-enhanced reverse transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. In this report, we evaluate the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We find that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g. transmission electron microscopy, viral sequence specific TaqMan). Cell derived DNA polymerases appear to contribute only modestly to the assay background and RT activity in clarified cell culture harvests is contained largely in Type C particles. TM-PERT is linear and precise between 10(7)and 10(13) pU/ml, establishing the assay range. The assay is robust in that test article storage condition and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples.