Eva Emmell

CNTO 95, a fully human monoclonal antibody that inhibits αv integrins, has antitumor and antiangiogenic activity in vivo

Integrins of the αv family, such as αvβ3 and αvβ5, are implicated in tumor‐induced angiogenesis; but their role in tumor growth has not been fully explored. CNTO 95 is a fully human antibody that recognizes the αv family of integrins and is likely to be less immunogenic in humans compared to chimeric or humanized antibodies. CNTO 95 bound to purified αvβ3 and αvβ5 with a Kd of approximately 200 pM and to αv integrin–expressing human cells with a Kd of 1–24 nM. In vitro, CNTO 95 inhibited human melanoma cell adhesion, migration and invasion at doses ranging 7–20 nM. In a rat aortic ring sprouting assay, CNTO 95 (approx. 70 nM) completely inhibited sprouting. Using a human melanoma xenograft model in nude mice wherein CNTO 95 recognized αvβ3 and αvβ5 on human tumor cells but not mouse angiogenic integrins, CNTO 95 (10 mg/kg, 3 times/week) inhibited growth of human melanoma tumors in nude mice by approximately 80% (p = 0.0005), suggesting that CNTO 95 inhibited human tumor growth independently of its antiangiogenic activity. In a nude rat human xenograft model where CNTO 95 binds and blocks both tumor and host integrins, this antibody (10 mg/kg once/week) reduced final tumor weight by >99% (p < 0.0001). Based on these preclinical data, a dose‐escalating phase I clinical trial in cancer patients has been initiated. To our knowledge, CNTO 95 is the first fully human MAb to αv integrins that has potent antitumor and antiangiogenic properties in in vivo preclinical models.

Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Anti-TNF-α antibody allows healing of joint damage in polyarthritic transgenic mice

Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-α (hTNF-α) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-α antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-α and local expression of various proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming a critical role of hTNF-α in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-α treatment was observed in young mice but not in aged mice.

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.

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor‐α transgenic mice

TNF‐α is a pleitropic cytokine that expresses both pro‐ and anti‐inflammatory activity and transgenic mice expressing human tumor necrosis factor‐α (TNF‐α) exhibit a progressive polyarthritis that models rheumatoid arthritis (RA). One of the common comorbidities of RA is anemia of chronic disease (ACD). The purpose of these experiments was to study the changes in the bone marrow and peripheral blood that accompany polyarthritis in TNF‐α transgenic mice in an effort to better understand the pathogenesis of myelodysplasia and ACD. Polychromatic cytometry, hematology and serum cytokine analysis were used to study the pathogenesis of ACD in human TNF‐α transgenic mice. Our hematological evaluation revealed a mild, compensated, microcytic hypochromic anemia, and monocytosis. In the bone marrow, we observed alterations in cell kinetics, decreased relative expression of transferrin receptor and increased apoptosis and cell death in several late precursor cell populations. Although significant levels of human TNF‐α were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL‐β, IL‐5, IL‐6, IL‐10, IL‐12(p70), IL‐17, TNF‐α, IFNγ, GM‐CSF, MIP‐1αJE, MCP‐5 was observed. Tg197 mice develop a compensated, microcytic, hypochromic anemia, and a functional iron deficiency by 9 weeks of age. Changes in peripheral blood are reflected in alterations in cell kinetics, transferrin receptor expression and markedly increased apoptosis and cell death in the bone marrow indicating that TNF‐α may contribute to myelodysplasia in ACD. Moreover, since human TNF‐α can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD

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.

Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo 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.