Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names.

By combining and cross-checking data from the World Health Organization's lists of international nonproprietary names with three other databases, we assembled a dataset of amino acid sequences of 819 antibody-related therapeutics. This enabled the systematic tabulation of 57 different molecular formats and about 90 different constant-region variants (47 for silencing, 14 for enhancement, 8 for modifying binding to FcRn, 13 for heterodimerization, 4 for site-specific modification and 4 for stabilization), as well as the frequencies of different targets and immunoglobulin allotypes. The curated dataset provides a resource for researchers, giving insights into trends in antibody engineering and a guide to the most frequently tested designs.

Dry-compression packing of hydroxyapatite nanoparticles within a flat cuboid chromatography device and its use for fast protein separation.

We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out at quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously increased by increasing flow rate. The suitability of this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.

Fc-engineered antibodies with immune effector functions completely abolished.

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been described in the literature but none of them completely eliminates binding to all of the Fcγ receptors. Here we describe a set of novel variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses. Meanwhile, binding to FcRn, manufacturability, stability and potential for immunogenicity are unaffected. These variants have the potential to improve the safety and efficacy of therapeutic antibodies and Fc fusion proteins.

Ultrafast Separation and Analysis of Monoclonal Antibody Aggregates Using Membrane Chromatography.

We discuss a method for rapid and cost-effective analysis of monoclonal antibody (mAb) aggregates. Hydrophobic interaction membrane chromatography, which was previously shown to be highly suitable for such separation and analysis, was used in a recently developed format referred to as laterally fed membrane chromatography (or LFMC). A stack of rectangular polyvinylidene fluoride (or PVDF) membranes having 0.22 µm pores housed within a modified analytical-scale LFMC device was used for analyzing aggregate types and content in different monoclonal antibody samples. High-resolution separations could be achieved in less than 1.5 min, this being faster than other currently available techniques such as size exclusion ultraperformance liquid chromatography (SE-UPLC). Moreover, the operating pressure was less than 200 kPa, which eliminated the need for an expensive high-pressure pump and chromatography system. The resolution obtained using the LFMC was comparable to that obtained using SE-UPLC. The effect of design variations such as change in dead volume and pillar size within the lateral channels within the LFMC device was also examined.

A comparative study of reduced dose alemtuzumab in matched unrelated donor and related donor reduced intensity transplants.

In vivo T cell depletion with 100 mg alemtuzumab prevents graft-versus-host disease (GVHD) in reduced intensity conditioned transplants but is associated with delayed immune reconstitution, a higher risk of infection and relapse. De-escalation studies have shown that a reduced dose of 30 mg is as effective as 100 mg in preventing GVHD in matched related donor (MRD) transplants. Dose reduction in matched unrelated donor (MUD) transplants is feasible but the comparative efficacy of alemtuzumab in this setting is not known and opinions vary widely concerning the optimal level of GVHD prophylaxis that should be achieved. Through retrospective analysis we made an objective comparison of MUD transplants receiving an empirically reduced dose of 60 mg, with MRD transplants receiving a 30 mg dose. We observed proportionate levels of alemtuzumab according to dose but an inverse relationship with body surface area particularly in MRD transplants. MUD transplants experienced more acute and chronic GVHD, higher T cell chimerism, more sustained use of ciclosporin and less need for donor lymphocyte infusion than MRD transplants. Thus, doubling the dose of alemtuzumab to 60 mg did not provide equivalent prevention of GVHD after MUD transplant although there was no difference in non-relapse mortality or survival compared with MRD transplants.

Methods to measure the binding of therapeutic monoclonal antibodies to the human Fc receptor FcγRIII (CD16) using real time kinetic analysis and flow cytometry.

Two different methods have been developed to measure the binding of therapeutic antibodies to the low affinity human Fc receptor FcγRIII (CD16). The first measures binding of antibody to recombinant soluble receptor by surface plasmon resonance and the second uses flow cytometry to measure antibody binding to cells which express the receptor. Both methods have been formatted as parallel line assays and show high levels of accuracy, precision and linearity, making them suitable for comparability, potency and stability assays. They are both readily able to detect structural differences such as glycosylation, which affect Fc receptor binding. The same approaches can be used to measure the binding of any antibody to any Fc receptor. These assays show greater internal precision and long-term reproducibility than traditional cell-based assays such as antibody-dependent cell-mediated cytotoxicity. A combinational approach with a target binding might be appropriate for routine drug batch release as these assays are likely to be significantly more sensitive to small changes in drug structure or activity.

Human CD3 transgenic mice: preclinical testing of antibodies promoting immune tolerance.

Monoclonal antibodies have proven to be potent agents to promote immunological tolerance in animal models of autoimmune disease and transplantation. However, optimal clinical application and pharmaceutical development have been limited by the species specificity of therapeutic antibodies, as well exemplified in the case of anti-CD3 antibodies. Compelling evidence in the nonobese diabetic (NOD) mouse, recently translated to clinical autoimmune insulin-dependent diabetes, demonstrates that a short CD3 antibody treatment effectively and durably controls disease progression. We established transgenic mice expressing the human ε chain of the CD3 complex bred onto the NOD background. These mice developed a high incidence of spontaneous autoimmune diabetes and harbored T cells sensitive both in vitro and in vivo to anti-human CD3 antibodies. Treatment of diabetic transgenic mice with otelixizumab, an anti-human CD3 antibody that has proven effective in the clinic, resulted in durable disease remission dependent on transferable T cell-mediated tolerance. This model should enable the evaluation of anti-human CD3 antibodies to determine their potential clinical utility.

Impact of in vivo alemtuzumab dose before reduced intensity conditioning and HLA-identical sibling stem cell transplantation: pharmacokinetics, GVHD, and immune reconstitution.

In vivo alemtuzumab reduces the risk of graft-versus-host disease (GVHD) and nonrelapse mortality after reduced intensity allogeneic transplantation. However, it also delays immune reconstitution, leading to frequent infections and potential loss of graft-versus-tumor responses. Here, we tested the feasibility of alemtuzumab dose deescalation in the context of fludarabine-melphalan conditioning and human leukocyte antigen (HLA)-identical sibling transplantation. Alemtuzumab was given 1-2 days before graft infusion, and dose reduced from 60 mg to 20 mg in 4 sequential cohorts (total n = 106). Pharmacokinetic studies were fitted to a linear, 2-compartment model in which dose reduction led to incomplete saturation of CD52 binding sites and greater antibody clearance. Increased elimination was particularly evident in the 20-mg group in patients who had CD52-expressing tumors at time of transplantation. The 20-mg dose was also associated with greater risk of severe GVHD (acute grade III-IV or chronic extensive) compared with > 20 mg (hazard ratio, 6.7; 95% CI, 2.5-18.3). In contrast, dose reduction to 30 mg on day -1 was associated with equivalent clinical outcomes to higher doses but better lymphocyte recovery at 12 months. In conclusion, alemtuzumab dose reduction to 30 mg is safe in the context of reduced intensity conditioning and HLA-identical sibling transplantation. This trial was registered at http://www.ncrn.org.uk as UKCRN study 1415.

A novel strategy to reduce the immunogenicity of biological therapies.

Biological therapies, even humanized mAbs, may induce antiglobulin responses that impair efficacy. We tested a novel strategy to induce tolerance to a therapeutic mAb. Twenty patients with relapsing-remitting multiple sclerosis received an initial cycle of alemtuzumab (Campath-1H), up to 120 mg over 5 d, preceded by 500 mg SM3. This Ab differs from alemtuzumab by a single point mutation and is designed not to bind to cells. Twelve months later, they received a second cycle of alemtuzumab, up to 72 mg over 3 d. One month after that, 4 of 19 (21%) patients had detectable serum anti-alemtuzumab Abs compared with 145 of 197 (74%) patients who received two cycles of alemtuzumab without SM3 in the phase 2 CAMMS223 trial (p < 0.001). The efficacy and safety profile of alemtuzumab was unaffected by SM3 pretreatment. Long-lasting "high-zone" tolerance to a biological therapy may be induced by pretreatment with a high i.v. dose of a drug variant, altered to reduce target-binding.

Pharmacokinetics and antibody responses to the CD3 antibody otelixizumab used in the treatment of type 1 diabetes.

Otelixizumab is a chimeric CD3 antibody that has been genetically engineered to remove the glycosylation site in the Fc domain. This limits its ability to bind to complement or Fc receptors and reduces the risk of adverse clinical reactions due to cytokine release. In a trial for treatment of type 1 diabetes, a short treatment with otelixizumab resulted in a reduced requirement for insulin lasting at least 18 months. In the course of this trial, the blood concentrations of the antibody were measured by flow cytometry to determine its pharmacokinetic profile. Dose-dependent accumulation of otelixizumab was demonstrated and modeling of the data indicated that the terminal half-life was approximately 1.5 days. Antibody responses to otelixizumab were measured by 2 methods: a bridging enzyme-linked immunosorbent assay and surface plasmon resonance. The surface plasmon resonance method had a greater sensitivity and was able to detect responses in all patients, starting at 8 days after the commencement of therapy. Neutralizing antibodies were detected in a significant proportion of patients by days 22 to 29. Although no adverse clinical effects were associated with these antibody responses and they did not appear to affect the clearance of the drug, they might have important implications for possible retreatment of the patients.

Transient Epstein-Barr virus reactivation in CD3 monoclonal antibody-treated patients.

Here we report a unique situation in which an early and synchronized Epstein-Barr virus (EBV) reactivation was induced by a 6-day course of treatment with a humanized CD3-specific monoclonal antibody in patients with recent onset of type 1 diabetes. The virologic and immunologic analysis demonstrated that this reactivation was transient, self-limited, and isolated, associated with the rapid advent of an EBV-specific T-cell response. The anti-CD3 antibody administration induced short-lasting immunosuppression and minor yet clear-cut signs of T-cell activation that preceded viral reactivation. Early posttransplant monitoring of renal and islet allograft recipients showed that no comparable phenomenon was observed after the administration of full-dose immunosuppressive therapy. This EBV reactivation remains of no apparent clinical concern over the long term and should not preclude further development of therapeutic anti-CD3 antibodies. This phenomenon may also direct new research avenues to understand the still ill-defined nature of stimuli triggering EBV reactivation in vivo.

Haemopoietic stem-cell transplantation with antibody-based minimal-intensity conditioning: a phase 1/2 study.

BACKGROUND: Stem-cell transplantation can cure primary immunodeficiencies. However, in patients with pre-existing organ toxicity, patients younger than 1 year, and those with DNA or telomere repair disorders, chemotherapy-based conditioning is poorly tolerated and results in major morbidity and mortality. We tested a novel antibody-based minimal-intensity conditioning (MIC) regimen to assess whether this approach allowed curative donor stem-cell engraftment without non-haemopoietic toxicity. METHODS: 16 high-risk patients underwent stem-cell transplantation for primary immunodeficiencies with an MIC regimen consisting of two rat anti-CD45 monoclonal antibodies YTH 24.5 and YTH 54.12 for myelosuppression, and alemtuzumab (anti-CD52) and fludarabine, and low dose cyclophosphamide for immunosuppression. Donors were matched siblings (n=5), and matched (9) and mismatched (2) unrelated donors. FINDINGS: Antibody-based conditioning was well tolerated, with only two cases of grade 3 and no grade 4 toxicity. Rates of clinically significant acute (n=6, 36%) and chronic graft-versus-host disease (GVHD) (n=5, 31%) were acceptable. 15 of 16 patients (94%) engrafted, of whom 11 (69%) achieved full or high-level mixed chimerism in both lymphoid and myeloid lineages, and three achieved engraftment in the T-lymphoid lineage only. One patient needed retransplantation. At a median of 40 months post-transplant, 13 of 16 patients (81%) in this high-risk cohort were alive and cured from their underlying disease. INTERPRETATION: Monoclonal antibody-based conditioning seems well tolerated and can achieve curative engraftment even in patients with severe organ toxicity or DNA repair defects, or both. This novel approach represents a shift from the paradigm that intensive chemotherapy or radiotherapy, or both, is needed for donor stem-cell engraftment. This antibody-based conditioning regimen may reduce toxicity and late effects and enable SCT in virtually any primary immunodeficiency patient with a matched donor. FUNDING: None.

Preclinical development of monoclonal antibodies: considerations for the use of non-human primates.

The development of mAbs remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies. Often, the only relevant species for preclinical safety assessment of mAbs are non-human primates (NHPs), and this raises important scientific, ethical and economic issues. To investigate evidence-based opportunities to minimize the use of NHPs, an expert working group with representatives from leading pharmaceutical and biotechnology companies, contract research organizations and institutes from Europe and the USA, has shared and analyzed data on mAbs for a range of therapeutic areas. This information has been applied to hypothetical examples to recommend scientifically appropriate development pathways and study designs for a variety of potential mAbs. The addendum of ICHS6 provides a timely opportunity for the scientific and regulatory community to embrace strategies which minimize primate use and increase efficiency of mAb development.

Morbidity and mortality in rheumatoid arthritis patients with prolonged therapy-induced lymphopenia: twelve-year outcomes.

OBJECTIVE: To assess immunologically relevant outcomes in a cohort of rheumatoid arthritis (RA) patients with prolonged therapy-induced lymphopenia. METHODS: Morbidity (infection or malignancy) and mortality were assessed in 53 RA patients who were treated with the lymphocytotoxic monoclonal antibody alemtuzumab between 1991 and 1994. Data were obtained by interview, medical record review, and Office for National Statistics mortality monitoring. Lymphocyte subsets were enumerated by flow cytometry. A retrospective, matched-cohort study of mortality was performed with 102 control subjects selected from the European League Against Rheumatism database of patients with rheumatic disorders. RESULTS: Lymphopenia persisted in the patients: median CD3+CD4+, CD3+CD8+, CD19+, and CD56+ lymphocyte counts measured at a median followup of 11.8 years from the first administration of alemtuzumab were 0.50 x 10(9)/liter, 0.26 x 10(9)/liter, 0.11 x 10(9)/liter, and 0.09 x 10(9)/liter, respectively. Twenty-seven of 51 cases and 46 of 101 controls with available data had died, yielding a mortality rate ratio of 1.20 (95% confidence interval 0.72-1.98). Causes of death were similar to those that would be expected in a hospital-based RA cohort. No opportunistic infections were noted, and only 3 infections were documented following 36 elective orthopedic procedures. CONCLUSION: Despite continued lymphopenia 11.8 years after therapy, our patient cohort did not exhibit excess mortality or unusual infection-related morbidity, and surgery was well tolerated. These data should be reassuring for clinicians and patients who are considering lymphocytotoxic or other immunomodulatory therapy for RA.

Outcomes after myeloablative unrelated donor stem cell transplantation using both in vitro and in vivo T-cell depletion with alemtuzumab.

HLA-matched unrelated donor (MUD) stem cell transplantation (MUD) is complicated by a high incidence of graft-versus-host-disease (GVHD) resulting in significant morbidity and mortality. To circumvent this problem we included alemtuzumab for in vivo and in vitro T-cell depletion in a myeloablative MUD-SCT regimen. After SCT, no severe acute GVHD was observed in the 30 transplanted patients. Donor lymphocyte infusion administered at a later time point resulted in sustained anti-tumor responses in most patients with chronic myeloid leukemia. After donor lymphocyte infusion three patients developed severe acute GVHD. Due to good responsiveness to immunosuppressive therapy only two patients developed persistent chronic GVHD. The main advantage of the transplantation regimen including alemtuzumab is that not only mortality due to GVHD is limited but also extensive chronic GVHD, which potentially leads to chronic morbidity and diminished quality of life, is hardly observed.

CD52 expression patterns in myeloma and the applicability of alemtuzumab therapy.

Alemtuzumab has been proposed as a therapeutic agent in myeloma. CD52 was detected on plasma cells in 46/106 patients but levels were 30-fold lower than on alemtuzumab-responsive cells (n=138) and 8-fold lower than on alemtuzumab-resistant cells (n=57). The data suggest that myeloma plasma cells are unlikely to be depleted by alemtuzumab in most patients.

Non-size-based membrane chromatographic separation and analysis of monoclonal antibody aggregates.

Humanized monoclonal antibody produced by mammalian cell culture may contain significant amounts of antibody dimers and smaller amounts of higher order aggregates. These are undesirable in therapeutic formulations, and their content should be lower than specific allowable limits. Quantitative analysis of aggregate content is usually carried out by size exclusion chromatography (SEC), which is slow and often gives poorly resolved peaks. We describe a novel hydrophobic interaction membrane chromatography-based technique for rapid, non-size-based separation and analysis of the aggregate content in monoclonal antibody samples. The typical sample analysis time using this technique is less than 3 min, this being significantly faster than SEC. The technique gives excellent resolution of the antibody, its dimer, and higher order aggregates and could potentially be scaled up for large-scale manufacture of aggregate-free monoclonal antibody. This work also clearly shows that monoclonal antibody aggregates are more hydrophobic than the monomer form, a fact that could have significant theoretical and practical implications.

A mouse monoclonal antibody against Epstein-Barr virus envelope glycoprotein 350 prevents infection both in vitro and in vivo.

A mouse monoclonal antibody (MAb) against Epstein-Barr virus (EBV) envelope glycoprotein 350, 72A1, inhibited EBV infection of B lymphocytes in vitro. When severe combined immunodeficient mice were injected with EBV-seronegative donors' peripheral-blood mononuclear cells and challenged with EBV, 72A1 MAb prevented development of EBV-positive tumors: none of the test mice (0/12) developed EBV-positive tumors. In contrast, 67% (8/12) of control mice developed EBV-positive tumors (P=.001). Purified 72A1 MAb was infused into 1 healthy adult and 4 EBV-seronegative children after liver transplant. No adverse reactions were seen in the adult or in 3 of the transplant recipients. The remaining patient developed a hypersensitivity reaction, thus underlining the need to humanize the MAb.

Therapeutic antibodies--delivering the promise?

For more than a century, therapeutic antibodies held the promise of providing specific cures for a wide range of diseases. It was not till the monoclonal era that the difficulties with purity and reproducibility were surmounted. But many obstacles still remained, and it has been a complex process to identify the best specificities, optimise effector functions and avoid unwanted immunogenicity. The academic community made substantial contributions, but higher regulatory hurdles will make this less significant in the future. Optimal delivery to the site of action remains one of the most important issues to be addressed. Monoclonal antibodies are already a significant part of the pharmaceutical market but there is a considerable potential still to be tapped.