Kerry Brosnan

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)2 fragments, but not for full-length IgG or for closely-related F(ab’)2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)2 fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)2 fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative. The purpose of these investigations was to use transplanted tumor models in immunocompetent, syngeneic mice to gain insight into the failure of the 2-year bioassay to show an increased incidence of neoplasia with CSA. C3H HeN mice were used in a battery of assays with a transplanted squamous cell carcinoma (SCC VII cells) or a B-cell, lymphoma (38C13 cells) cells to study effects of CSA on local growth and metastases, experimental metastases, and progression of established metastases. Mice received CSA twice weekly by subcutaneous (SC) injection at doses of 0.5, 5, or 50 mg/kg; controls received the CSA vehicle. CSA had a modest inhibitory effect on SC tumors initiated by 38C13 cells and on intramuscular tumors initiated by SCC VII cells. CSA also decreased the number of lung colonies and decreased the size, growth fraction and vascularity of established lung metastases initiated by SCC VII cells. In contrast, CSA increased progressive growth of metastases to the sentinel lymph node from an intramuscular SCC VII tumor, but had no effect cellular traffic to the node. In conclusion, CSA at doses up to 50 mg/kg did not facilitate tumor progression and it partially inhibited tumor growth, suggesting that suppression of tumor progression may partially explain the failure of CSA to act as a carcinogen in 2 year bioassays.

Development of a murine model of lymph node metastases suitable for immunotoxicity studies.

INTRODUCTION Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice. METHODS SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated. RESULTS SCC VII cells express RAE-1, an NKG2D ligand, and were sensitive to natural killer (NK) cells in vitro. QDs were stable in SCC VII cells and showed no evidence of toxicity to the cells. In vivo, confocal microscopy showed that QD-labeled SCC VII cells could migrate to the draining node and microfluorimetry showed progressive traffic of QDs to the node. There was no evidence of systemic toxicity of QDs. Primary immunosuppression in SCID and SCID-beige mice and treatment of normal mice with immunosuppressive agents (anti-asialoGM1 and cyclophosphamide) can enhance traffic of QDs and/or metastases to the draining lymph node. In contrast, cyclosporine had no effect on traffic or metastases. CONCLUSION This model of local invasion and metastases may be useful in immunotoxicology for identifying and characterizing the hazard posed by selective immunosuppressive drugs.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding

ABSTRACT The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.

Development of a squamous cell carcinoma mouse model for immunotoxicity testing

Abstract An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds. Thus, there is a need for alternative testing strategies. A novel 3-stage tumor model in syngeneic C3H/HeN mice was evaluated here to study the effects of immunosuppressive drugs on tumor promotion and progression in vivo. The model employed a skin squamous cell carcinoma cell line (SCC VII) due to the increased prevalence of squamous cell carcinoma (SCC) in humans associated with immunosuppression after transplants. Local invasion, colonization and tumor progression were evaluated. The validation set of immunosuppressive drugs included: Cyclosporin (CSA), cyclophosphamide (CTX), azathioprine, etanercept, abatacept and prednisone. Local invasion was evaluated by histological assessment as well as fluorescence trafficking from Qdot®-labeled tumor cells from the site of inoculation to the draining lymph node. Colonization was evaluated by lung colony counts following intravenous inoculation. Tumor progression was assessed by morphometric analysis of lesion area, angiogenesis and growth fraction of established metastatic neoplasia. Immunosuppressive drugs in the validation set yielded mixed results, including decreased progression. The methods and results described herein using an in vivo syngeneic mouse tumor model can provide insight about the assessment of immunosuppressive drugs in carcinogenicity risk assessment.

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact

Abstract Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.

Abstract B58: Early safety assessment of single and combination therapy using TDAR

The discovery of targeted therapies against tumors offers promise of therapeutic benefit to patients however, it poses challenges in assessing immunologic and toxicologic risk during drug development. We determined that early safety assessment of combination therapy, prior to later stage toxicology studies, can be used to support Go/No-Go NME decision and thereby reduce time, efficiency, and resources used to choose and develop lead candidates. The rodent T-cell-dependent antibody response (TDAR) assay is used to assess the effect of candidate therapeutic agents on the immune system by measuring primary and secondary IgM and IgG antibody responses to exogenous antigen challenge. TDAR responses require intact function of multiple immune cells including antigen presenting cells and T and B lymphocytes, as well as a cytokine-dependent isotype class switch from IgM to IgG, resulting in production of an antigen-specific antibody response. Alterations in the amount of antibody produced therefore can reflect effects on any or all cell populations involved in TDAR. TDAR is commonly used in preclinical drug development especially where increased cause for concern exists (ICH guideline S8). Development of combination therapy that engages multiple targets impacting the immune system poses unique opportunity for increased efficacy but also unique risk for increased immunotoxicity including immune stimulation. For this work, a mouse TDAR evaluating primary and secondary KLH antibody responses in a KLH-Specific IgM and IgG sandwich enzyme-linked immunosorbent assay (ELISA) was first validated and then used to assess immunotoxicologic potential of multiple single immunosuppressive agents (cyclophosphamide (CTX), abatacept, azathioprine (AZT), etanercept, cyclosporine (CsA), and prednisone) and biological therapies (A, B, C, D, E, and F) and combination biologic therapies ( A+B, C+D, E+F). Doses of 100 mg/kg CsA, 250 mg/kg abatacept, 125 mg/kg etanercept, 100 mg/kg AZT, 20 mg/kg prednisone administered subcutaneously (s.c.) on Days 1 and 3 had mild immunosuppressive effects. 200 mg/kg of CTX administered s.c. had an expected robust immunosuppressive effect that was statistically significant than control. Combination of biologic therapies did not result in enhanced TDAR immunotoxicity compared to single biologic therapy alone for the molecules evaluated. Tier 1 immunotoxicology assessments similar to those in standard toxicity studies were added to the TDAR assessment. Together, these data support the use of the TDAR assay for early safety assessment of potential combination therapies against tumors. Citation Format: Amy L. Volk, Mindi Walker, Kerry Brosnan, Dorie Capaldi, Patricia Rafferty, Daniel Weinstock, Eva Emmell. Early safety assessment of single and combination therapy using TDAR. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B58.

Abstract LB-312: Bispecific Centyrin simultaneously targeting EGFR and c-Met demonstrates improved activity compared to the mixture of single agents.

Many tumors compensate with a resistance mechanism once a signaling pathway is inhibited, as evidenced in patients treated with small molecule inhibitors of EGFR which frequently show increased expression of c-Met or HGF. Since both EGFR and c-Met signal through the same survival and anti-apoptotic pathways, inhibition of the pair of receptors could improve overall efficacy. This was demonstrated clinically, where the combination of onartuzumab and erlotinib showed promising Phase II trials in lung cancer patients. Centyrins are small proteins (~10 kDa) of an emerging class of proteins termed alternative scaffolds. They are engineered to bind to target molecules with high specificity and affinity, but are structurally simpler molecules than antibodies in that that they are single chain, unglycosylated proteins that lack disulfide bonds. In addition, monomeric Centyrins can be linked together such that one molecule can bind to and inhibit multiple targets. Anti-EGFR and anti-c-Met Centyrins were selected for their ability to inhibit ligand-induced phosphorylation. Molecules with a wide range of affinities were identified and bispecific molecules were generated. Phosphorylation of EGFR, c-Met, and ERK were monitored in cells treated with individual monomeric Centyrins, a mixture of monomeric Centyrins, or linked bispecific Centyrins. One bispecific EGFR/c-Met Centyrin provided a 134-fold increase in potency in inhibition of phosphorylation of c-Met compared to the mixture of the two Centyrins. The increase in potency observed in cell signaling translated to enhanced anti-proliferative activity with a potency >100-fold compared to that of the mixture of monospecific Centyrins. The bispecific EGFR/c-Met Centyrins were produced as fusion proteins linked to an albumin binding domain in order to reduce kidney filtration and evaluated in SCID-beige mice implanted with tumor cells engineered to express human HGF. Interestingly, all of the Centyrins tested significantly reduced the size of the tumors compared to the control and the degree of tumor growth inhibition correlated with the affinity to both EGFR and c-Met. In a second tumor model, complete tumor regressions were observed in all mice treated with the bispecific EGFR/c-Met Centyrin. The Centyrin platform could offer benefits over currently available treatment options. Our data demonstrate that in addition to inhibiting two targets simultaneously, a single bispecific molecule allows for significant avidity at the cellular level. This could translate into a lower dosing regimen that allows for improved efficacy through avidity and decreased toxicity. We anticipate that avidity will allow for improved specificity for tumor compared to normal tissue. In addition, the small size of Centyrins may provide an advantage for tumor targeting and accumulation. Citation Format: Donna Klein, Steve Jacobs, Moores Sheri, Mark Anderson, Ricardo Attar, Alexander Barnakov, Kerry Brosnan, Barbara Bushey, Kristen Chevalier, Diana Chin, Carla Cornejo, Mike Diem, Linus Hyun, Elise Kuhar, Francis McCabe, Kristen Picha, Tracy Spinka-Doms, Edward Swift, Karyn O9Neil. Bispecific Centyrin simultaneously targeting EGFR and c-Met demonstrates improved activity compared to the mixture of single agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-312. doi:10.1158/1538-7445.AM2013-LB-312