Aaron P. Yamniuk

Engineering of a Novel Anti-CD40L Domain Antibody for Treatment of Autoimmune Diseases

CD40–CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a “domain Ab” (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb–Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb–Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin–induced Ab responses, alloantigen-induced T cell proliferation, “heart-to-ear” transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

Drug Excipient Interactions

Unintended physicochemical interaction of an excipient with a drug substance in a dosage form can result in the complexation or binding of the drug, resulting in slow and/or incomplete drug release in a dissolution medium. It is important to assess the risk whether such interactions would reduce oral bioavailability of a drug from its dosage form. This chapter describes the development of a methodology to assess the biorelevance of the drug release impact of drug-excipient binding interactions using a model compound, brivanib alaninate. This methodology was developed using a combination of modeling and simulation tools as well as experimental data generated in vitro and in vivo. In addition, general application of this principle and methodology to other drug substances and binding affinities of drugs with excipients as a function of dose is described.

An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(+) iTreg in the absence and presence of CTLA-4 Ig.

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc‐mediated deletion of CD154‐expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti‐CD154‐induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti‐CD154 therapy, two alternative means of targeting the CD40–CD154 pathway were used: a nonagonistic anti‐CD40 antibody and an Fc‐silent anti‐CD154 domain antibody. We compared these therapies to an Fc‐intact anti‐CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti‐CD40 mAbs as well as Fc‐silent anti‐CD154 domain antibodies were equivalent to Fc‐intact anti‐CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen‐specific T cells and promote the conversion of Foxp3+ iTreg. Importantly, iTreg conversion observed with Fc‐silent anti‐CD154 domain antibodies was preserved in the presence of CTLA4‐Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti‐CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti‐human CD154 domain antibody (dAb, BMS‐986004). The anti‐CD154 dAb effectively blocked CD40‐CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti‐CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti‐CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti‐CD154 dAb treatment increased the frequency of CD4+CD25+Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti‐CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti‐CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Detection of drug specific circulating immune complexes from in vivo cynomolgus monkey serum samples

BACKGROUND Administration of a biotherapeutic can result in the formation of anti-drug antibodies (ADAs). The resulting ADA can potentially form immune complexes (ICs) with the drug leading to altered pharmacokinetic (PK) profiles and/or adverse events. Furthermore the presence of such complexes may interfere with accurate PK assessment, and/or detection of ADA in immunogenicity assays. Here, we present two assays to detect the presence of drug-ADA immune complexes in cynomolgus monkeys. RESULTS Serum samples were analyzed for IC formation in vivo. 8/8 tested animals were positive for drug specific IC. Depending on the time point tested 4/8 or 7/8 animals tested positive for ADA during drug dosing. All 8 animals were confirmed positive for ADA during the washout phase, indicating drug interference in the bridging assay. Relative amount of IC over time was determined and its correlation with PK and ADA was then assessed. Multivariate data analysis demonstrates good correlation between signals obtained from the anti-drug and FcγRIIIa based capture assays, although due to its biological characteristic FcγRIIIa based assay captured only a subset of drug specific IC. In one animal IC remained in circulation even when the drug levels decreased below detection limit. CONCLUSION Results from this study indicate the presence of IC during administration of an immunogenic biotherapeutic. Potential application of these assays includes detection of ADA in an IC during high drug levels. The results on the kinetics of IC formation during ADA response can complement the understanding of PK and ADA profiles. Moreover, the presence of IC indicates possible ADA interference in standard PK assays and potential underestimation of total drug exposure in toxicology studies. In addition this study also highlights the need to understand downstream in vivo consequences of drug-ADA IC as no animals under investigation developed adverse events.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Role of Self-Association and Supersaturation in Oral Absorption of a Poorly Soluble Weakly Basic Drug

PurposePrecipitation of weakly basic drugs in intestinal fluids can affect oral drug absorption. In this study, the implications of self-association of brivanib alaninate in acidic aqueous solution, leading to supersaturation at basic pH condition, on its solubility and oral absorption were investigated.MethodsSelf-association of brivanib alaninate was investigated by proton NMR spectroscopy, surface tension measurement, dynamic light scattering, isothermal titration calorimetry, and molecular modeling. Drug solubility was determined in various pH media, and its tendency to supersaturate upon pH shift was investigated in buffered and biorelevant aqueous solutions. Pharmacokinetic modeling of human oral drug absorption was utilized for parameter sensitivity analyses of input variables.ResultsBrivanib alaninate exhibited continuous, and pH- and concentration-dependent self-association. This phenomenon resulted in positive deviation of drug solubility at acidic pH and the formation of a stable supersaturated drug solution in pH-shift assays. Consistent with the supersaturation phenomenon observed in vitro, oral absorption simulations necessitated invoking long precipitation time in the intestine to successfully predict in vivo data.ConclusionsSelf-association of a weakly basic drug in acidic aqueous solution can increase its oral absorption by supersaturation and precipitation resistance at the intestinal pH. This consideration is important to the selection of parameters for oral absorption simulation.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Abstract 2586: Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR) are transmembrane receptor tyrosine kinases that mediate proliferative and invasive cell signaling in cancer. Inhibition of either receptor reduces tumor growth in both mouse models and in human clinical studies. Blocking the EGFR pathway can induce compensatory activation of the IGFR pathway to drive tumor growth and IGFR inhibition can result in activation of EGFR signaling in preclinical models. Therefore, blocking both receptors simultaneously may achieve superior efficacy to blocking either pathway alone. We developed individual optimized Adnectins™ specific for blocking either EGFR or IGFR signaling and engineered them into a single protein that linked both Adnectins together to construct a bi-specific Adnectin targeting the EGFR and IGFR (EI-tandem). The bifunctional molecule blocked activation of EGFR and IGFR, inhibited both EGF and IGF-induced down-stream cell signaling (MAPK and AKT pathways) and was antiproliferative in human cancer cell lines. Potency of the EI-tandem was comparable to anti-EGFR and anti-IGFR antibodies. The EI-tandem demonstrated a synergistic inhibition of IGFR phosphorylation and down-stream cell signaling compared to Adnectins specific for only EGFR or IGFR alone. Although Adnectins bound to the EGFR at a site distinct from the clinically approved anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, they still blocked binding of EGF to the EGFR. PEGylated EI-tandem inhibited the growth of human tumor xenografts driven by both EGFR and IGFR signaling, degraded EGFR and IGFR, and reduced phosphorylation of EGFR in tumors. Treatment of mice with EI-tandem caused increases in levels of the circulating ligands TGFα and IGF1 resulting from blockade of their respective receptors and provided convenient soluble biomarkers of target suppression. These results show that a bifunctional Adnectin can confer improved biological activity compared to monospecific biologics in tumors where growth is driven by multiple growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2586.

Abstract 1476: A therapeutic antibody that inhibits CD73 activity by dual mechanisms

CD73 has a central role in dictating the adenosine concentration within the tumor as it is the final step in converting extracellular ATP to adenosine. Thus, substantial reduction of CD73 enzymatic activity has the potential to reduce immunosuppression of effector immune cells within the tumor. We present data describing an anti-human CD73 antibody that suppresses CD73 by two mechanisms: 1. direct inhibition of enzymatic activity upon binding to CD73 and 2. rapid, near-complete internalization of the enzyme. Durable reduction of cell-surface CD73 was observed in multiple tumor cell lines both in vitro and in vivo. The unique properties of this antibody are a result of the use of a human IgG2-IgG1 hybrid antibody with effector function eliminated by specific mutations of the Fc. The IgG2 sequence of this antibody drives superior internalization of CD73 and enhanced CD73 inhibition. Syngeneic tumor models demonstrate that CD73 contributes to resistance to anti-tumor therapy. Combination therapy with PD-1 blockade and a surrogate anti-mouse-CD73 antibody resulted in a better anti-tumor efficacy than either treatment alone. Finally, we demonstrate a novel technique for assessing CD73 enzymatic activity in situ that has potential for clinical application. These data support antibody-based anti-CD73 therapy in cancer and highlight a novel mechanism for inhibition of CD73 enzymatic activity. Citation Format: Bryan C. Barnhart, Emanuela Sega, Aaron Yamniuk, Sandra Hatcher, Ming Lei, Haben Ghermazien, Anne Lewin, Xi-Tao Wang, Haichun Huang, Pingping Zhang, Alan Korman. A therapeutic antibody that inhibits CD73 activity by dual mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1476.