Modulation of drug-linker design to enhance in vivo potency of homogeneous antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising class of anticancer agents which have undergone substantial development over the past decade and are now achieving clinical success. The development of novel site-specific conjugation technologies enables the systematic study of architectural features within the antibody conjugated drug linker that may affect overall therapeutic indices. Here we describe the results of a systematic study investigating the impact of drug-linker design on the in vivo properties of a series of homogeneous ADCs with a conserved site of conjugation, a monodisperse drug loading, a lysosomal release functionality and monomethyl auristatin E as a cytotoxic payload. The ADCs, which differed only in the relative position of certain drug-linker elements within the reagent, were first evaluated in vitro using anti-proliferation assays and in vivo using mouse pharmacokinetics (PK). Regardless of the position of a discrete polymer unit, the ADCs showed comparable in vitro potencies, but the in vivo PK properties varied widely. The best performing drug-linker design was further used to prepare ADCs with different drug loadings of 4, 6 and 8 drugs per antibody and compared to Adcetris® in a Karpas-299 mouse xenograft model. The most efficacious ADC showed complete tumor regression and 10/10 tumor free survivors at a single 0.5mg/kg dose. This study revealed drug-linker design as a critical parameter in ADC development, with the potential to enhance ADC in vivo potency for producing more efficacious ADCs.

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead.

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

A comparison of the ability of the human IgG1 allotypes G1m3 and G1m1,17 to stimulate T-cell responses from allotype matched and mismatched donors.

The immunogenicity of clinically administered antibodies has clinical implications for the patients receiving them, ranging from mild consequences, such as increased clearance of the drug from the circulation, to life-threatening effects. The emergence of methods to engineer variable regions resulting in the generation of humanised and fully human antibodies as therapeutics has reduced the potential for adverse immunogenicity. However, due to differences in sequence referred to as allotypic variation, antibody constant regions are not homogeneous within the human population, even within sub-classes of the same immunoglobulin isotype. For therapeutically administered antibodies, the potential exists for an immune response from the patient to the antibody if the allotype of patient and antibody do not match. Allotypic distribution in the human population varies within and across ethnic groups making the choice of allotype for a therapeutic antibody difficult. This study investigated the potential of human IgG1 allotypes to stimulate responses in human CD4(+) T cells from donors matched for homologous and heterologous IgG1 allotypes. Allotypic variants of the therapeutic monoclonal antibody trastuzumab were administered to genetically defined allotypic matched and mismatched donor T cells. No significant responses were observed in the mismatched T cells. To investigate the lack of T-cell responses in relation to mismatched allotypes, HLA-DR agretopes were identified via MHC associated peptide proteomics (MAPPs). As expected, many HLA-DR restricted peptides were presented. However, there were no peptides presented from the sequence regions containing the allotypic variations. Taken together, the results from the T-cell assay and MAPPs assay indicate that the allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity.

The INNs and outs of antibody nonproprietary names.

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity.

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.

In Vitro and In Vivo Evaluation of Cysteine Rebridged Trastuzumab-MMAE Antibody Drug Conjugates with Defined Drug-to-Antibody Ratios.

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bis-alkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug-to-antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro potency, and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to trastuzumab-DM1 (T-DM1, Kadcyla), as evaluated in a low HER2 expressing JIMT-1 xenograft model.

Managing unwanted immunogenicity of biologicals.

All protein drugs (biologicals) have an immunogenic potential and we are armed with multiple guidelines, regulatory documents and white papers to assist us in assessing the level of risk for unwanted immunogenicity of new biologicals. However, for certain biologicals, significant immunogenicity becomes only apparent after their use in patients. Causes of immunogenicity are multifactorial but not yet fully understood. Within the pharmaceutical industry there are only a few opportunities to openly discuss the causes and consequences of immunogenicity with regard to the development of new biologicals. The annual Open Scientific Symposium of the European Immunogenicity Platform (EIP) is one such meeting that brings together scientists and clinicians from academia and industry to build know-how and expertise in the field of immunogenicity. The critical topics discussed at the last EIP meeting (February 2014) will be reviewed here. The current opinion of this expert group is that the assessment of unwanted immunogenicity can be improved by using prediction tools, optimizing the performance of immunogenicity assays and learning from the clinical impact of other biologicals that have already been administered to patients. A multidisciplinary approach is warranted to better understand and minimize drug immunogenicity and its clinical consequences.

Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

PURPOSE: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. METHODS: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. RESULTS: Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. CONCLUSIONS: These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1ß, IL-6, IL-10, MCP-1, MIP-1α, MIP-1ß, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 µm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

Pre-clinical considerations in the assessment of immunogenicity for protein therapeutics.

Protein therapeutics offer distinct advantages over other classes of drugs largely due to the high level of target specificity and generally low toxicity. Problems have, however, been encountered with some protein therapeutics inducing undesirable immune responses in patients. This immunogenicity can produce pleiotropic effects including the development of a high affinity B cell-mediated humoral response that is often directed against the therapeutic. Opinions are divided as to the principal causes of clinical immunogenicity and, as a result, this area has been the subject of much research. One thing that has emerged as a result of this intense activity is the development of pre-clinical models that can provide a level of prediction of the immunogenic potential of novel protein therapeutics before administration in man.

Prediction of immunogenicity of therapeutic proteins: validity of computational tools.

Most protein therapeutics have the potential to induce undesirable immune responses in patients. Many patients develop anti-therapeutic antibodies, which can affect the safety and efficacy of the therapeutic protein, particularly if the response is neutralizing. There are a variety of factors that influence the immunogenicity of protein therapeutics and, in particular, the presence of B- and T-cell epitopes is considered to be of importance. In silico tools to identify the location of both B- and T-cell epitopes and to assess the potential for immunogenicity have been developed, and such tools provide an alternative to more complex in vitro or in vivo immunogenicity assays. This article reviews computational epitope prediction methods and also the use of manually curated databases containing experimentally derived epitope data. However, due to the complexities of the molecular interactions involved in epitope recognition by the immune system, the heterogeneity of key proteins in human populations and the adaptive nature of the immune response, in silico methods have not yet achieved a level of accuracy that enables them to be used as stand-alone tools for predicting clinical immunogenicity. Computational methods, particularly with regard to T-cell epitopes, only consider a limited number of events in the process of epitope formation and therefore routinely over-predict the number of epitopes within a molecule. Epitope databases such as the Immune Epitope Database (IEDB) and the proprietary T Cell Epitope Database (TCED) have reached a size and level of organization that increases their utility; however, they are not exhaustive. These methods have greatest utility as an adjunct to in vitro assays where they can be used either to reduce the amount and complexity of the in vitro screening, or they can be used as tools to analyze the sequence of the identified epitope in order to locate amino acids critical for its properties.

Immunogenicity of protein therapeutics: The key causes, consequences and challenges.

The immunogenicity of protein therapeutics has so far proven to be difficult to predict in patients, with many biologics inducing undesirable immune responses directed towards the therapeutic resulting in reduced efficacy, anaphylaxis and occasionally life threatening autoimmunity. The most common effect of administrating an immunogenic protein therapeutic is the development of a high affinity anti-therapeutic antibody response. Furthermore, it is clear from clinical studies that protein therapeutics derived from endogenous human proteins are capable of stimulating undesirable immune responses in patients, and as a consequence, the prediction and reduction of immunogenicity has been the focus of intense research. This review will outline the principle causes of the immunogenicity in protein therapeutics, and describe the development of pre-clinical models that can be used to aid in the prediction of the immunogenic potential of novel protein therapeutics prior to administration in man.

Circumventing immunogenicity in the development of therapeutic antibodies.

The development of anti-therapeutic antibody immune responses can limit efficacy and reduce the safety of antibody treatments. Despite significant advances to minimize such immune responses, these responses still occur, even against fully human antibodies. Thus, the ability to assess the immunogenic potential of a therapeutic antibody can provide significant benefits during the development cycle of a therapeutic protein, and may lead to the future development of therapeutic antibodies that possess minimal immunogenicity. In particular, in vitro, in silico and ex vivo methods have been developed for the prediction of T-cell epitopes that can lead to immunogenicity. Ultimately, immunogenicity may be completely avoided by the generation of humanized or fully human non-immunogenic therapeutic antibodies using techniques such as deimmunization and composite human antibodies.

Deimmunization of monoclonal antibodies.

Immunogenicity is a major limitation to therapy with certain monoclonal antibodies and proteins. A major driver for immunogenicity is the presence of human T-cell epitopes within the protein sequence which can activate helper T-cells resulting in the sustained production of antibodies and neutralization of the therapeutic effect. Deimmunization is a new technology for location and removal of T-cell epitopes through the combined use of immunological and molecular biology techniques. In the case of deimmunization of antibodies, mutations to remove T-cell epitopes can generally be introduced without significantly reducing the binding affinity of the antibody. Typically, "deimmunized" antibodies are created with human constant regions and by expression of genes encoding these antibodies in mammalian cells. This chapter details a method for creation of a deimmunized antibody for production in mammalian cells.

New approaches to prediction of immune responses to therapeutic proteins during preclinical development.

Clinical studies show that immunogenicity observed against therapeutic proteins can limit efficacy and reduce the safety of the treatment. It is therefore beneficial to be able to predict the immunogenicity of therapeutic proteins before they enter the clinic. Studies using deimmunized proteins have highlighted the importance of T-cell epitopes in the generation of undesirable immunogenicity. In silico, in vitro, ex vivo and in vivo methods have therefore been developed that focus on identification of CD4+ T-cell epitopes in the sequence of therapeutic proteins. A case study of existing therapeutic proteins is presented to review these different approaches in order to assess their utility in predicting immunogenic potential.

Identification and removal of immunogenicity in therapeutic proteins.

The development of anti-therapeutic protein immune responses in patients can be a severe complication of treatment with this class of pharmaceuticals. Antibodies generated against therapeutic proteins limit the clinical efficacy of these agents by neutralizing their biological activity and/or enhancing their clearance. An assessment of the propensity of protein therapeutics to elicit immune responses in patients is likely to become an essential part of their preclinical development. It is clear that CD4+ T-cells are an important factor in the development of long-lived, class-switched, high-affinity antitherapeutic protein antibodies. The increased risk of immunogenicity that is attributed to the presence of T-cell epitopes in therapeutic protein sequences has led to the development of a variety of methods for locating T-cell epitopes and identifying binding peptide amino acids that are important for interacting with either major histocompatibility complex class II molecules or the T-cell receptors. Manipulation of these key residues to disrupt these interactions while maintaining biological activity can result in modified therapeutic proteins with reduced immunogenicity.

The development of a modified human IFN-alpha2b linked to the Fc portion of human IgG1 as a novel potential therapeutic for the treatment of hepatitis C virus infection.

Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-alpha2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-alpha alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-alphas currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-alpha2b.