Characterization of a Novel Bispecific Antibody That Activates T Cells In Vitro and Slows Tumor Growth In Vivo.

Although CD3 T cell redirecting antibodies have been successfully utilized for the treatment of hematological malignancies (blinatumomab), the T cell signaling pathways induced by these molecules are incompletely understood. To gain insight into the mechanism of action for T cell redirection antibodies, we created a novel murine CD3xEpCAM bispecific antibody that incorporates a silent Fc to dissect function and signaling of murine CD8 OT1 T cells upon stimulation. T cell-mediated cytotoxicity, cytokine secretion, expression of activation markers, and proliferation were directly induced in T cells treated with the novel CD3xEpCAM bispecific molecule in vitro in the presence of epithelial cell adhesion molecule (EpCAM) expressing tumor cells. Nanostring analysis showed that CD3xEpCAM induced a gene expression profile that resembled antigen-mediated activation, although the magnitude was lower than that of the antigen-induced response. In addition, this CD3xEpCAM bispecific antibody exhibited in vivo efficacy. This is the first study that investigates both in vitro and in vivo murine CD8 T cell function and signaling induced by a CD3xEpCAM antibody having a silent Fc to delineate differences between antigen-independent and antigen-specific T cell activation. These findings expand the understanding of T cell function and signaling induced by CD3 redirection bispecific antibodies and may help to develop more efficacious CD3 redirection therapeutics for cancer treatment, particularly for solid tumors.

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.

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

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.

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.

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.

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.

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.

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor-alpha transgenic mice.

TNF-alpha is a pleitropic cytokine that expresses both pro- and anti-inflammatory activity and transgenic mice expressing human tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha 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-alpha were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL-beta, IL-5, IL-6, IL-10, IL-12(p70), IL-17, TNF-alpha, IFNgamma, GM-CSF, MIP-1alphaJE, 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-alpha may contribute to myelodysplasia in ACD. Moreover, since human TNF-alpha can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD.

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses.

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.

Modulation of antigen-specific T cell response by a non-mitogenic anti-CD3 antibody.

Suppression of T cell response is the key to enhance graft survival and control autoimmune diseases. A mitogenic anti-CD3 monoclonal antibody (mAb), OKT3, has been used for decades to control acute rejection in organ transplantation. Although effective, the clinical use was limited by its side effects, such as cytokine release mediated by T cell activation. A low mitogenic humanized OKT3 with reduced FcR-binding (hgammaOKT3 Ala-Ala) was generated and tested in several clinical studies. Although hgammaOKT3 Ala-Ala demonstrated maintained efficacy and better safety it still activated T cells. To investigate if a non-mitogenic anti-CD3 mAb can be equally effective in immune suppression, a chimeric non-FcR-binding anti-mouse CD3 mAb (anti-CD3 IgG2a Ala-Ala) was generated. Unlike the hgammaOKT3 Ala-Ala, the mouse IgG2a Ala-Ala anti-CD3 mAb did not induce T cell activation as measured by proliferation, cytokine production and apoptosis. Nevertheless, the IgG2a Ala-Ala anti-CD3 mAb was equally effective in the inhibition of antigen-specific CD4+ T cell activation in vitro to that of the mitogenic anti-CD3 mAb (Anti-CD3 IgG2a). In vivo, the IgG2a Ala-Ala anti-CD3 mAb only induced transient reduction of peripheral and spleen T cells and did not trigger detectable cytokine release. Nonetheless, this non-mitogenic anti-CD3 mAb significantly prolonged islet graft survival as effectively as the mitogenic anti-CD3 mAb in an allogenic islet transplantation model. These results demonstrated that a non-mitogenic anti-CD3 mAb could be used as an effective immune modulator. It may also indicate that a true non-mitogenic version of OKT3 could further improve its safety profile for clinical use.

Therapeutic dosing with anti-interleukin-13 monoclonal antibody inhibits asthma progression in mice.

In vivo models have demonstrated that interleukin-13 (IL-13) plays an important role in asthma; however, few studies have evaluated the effect of inhibition of IL-13 on established and persistent disease. In the present study, we have investigated the effect of a therapeutic dosing regimen with an anti-IL-13 monoclonal antibody (mAb) in a chronic mouse model of persistent asthma. BALB/c mice were sensitized to allergen [ovalbumin (OVA); on days 1 and 8] and challenged with OVA weekly from day 22. Anti-IL-13 mAb or vehicle dosing was initiated following two OVA challenges when disease was established. At this time, mice exhibited airway hyperresponsiveness (AHR), increased mucus production, inflammation, and initiation of subepithelial fibrosis compared with saline-challenged mice. Mice received four additional OVA challenges. Treatment with anti-IL-13 mAb inhibited AHR and prevented the further development of subepithelial fibrosis and progression of inflammation. Furthermore, mAb treatment reversed the mucus hyperplasia to basal levels. These effects were associated with an inhibition of cytokines, chemokines, and matrix metalloproteinase-9. These data demonstrate that neutralization of IL-13 can inhibit the progression of established disease in the presence of repeated allergen exposures.

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling.

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.

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

Integrins of the alphav family, such as alphavbeta3 and alphavbeta5, 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 alphav family of integrins and is likely to be less immunogenic in humans compared to chimeric or humanized antibodies. CNTO 95 bound to purified alphavbeta3 and alphavbeta5 with a Kd of approximately 200 pM and to alphav 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 alphavbeta3 and alphavbeta5 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 alphav integrins that has potent antitumor and antiangiogenic properties in in vivo preclinical models.

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

Anti-tumor-necrosis-factor-alpha (TNF-alpha) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-alpha (hTNF-alpha) 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-alpha antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-alpha treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-alpha prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-alpha and local expression of various proinflammatory mediators were all diminished by anti-TNF-alpha treatment, confirming a critical role of hTNF-alpha 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-alpha treatment was observed in young mice but not in aged mice.

Multiple roles for platelet GPIIb/IIIa and alphavbeta3 integrins in tumor growth, angiogenesis, and metastasis.

In vivo tumor cells interact with a variety of host cells such as endothelial cells and platelets, and these interactions are mediated by integrins GPIIb/IIIa and alphavbeta3. We used chimeric (c) 7E3 Fab (ReoPro) and murine (m) 7E3 F(ab')(2) to elucidate the role of these integrins in angiogenesis, tumor growth, and metastasis. These antibodies are potent inhibitors of GPIIb/IIIa and alphavbeta3. c7E3 Fab inhibited alphavbeta3-mediated human umbilical vein endothelial (HUVEC) and melanoma cell adhesion, migration, invasion, and basic fibroblast growth factor stimulated proliferation of HUVECs (IC(50) values range from 0.15 to 5 microg/ml for different assays). In an in vitro angiogenesis assay, c7E3 Fab inhibited basic fibroblast growth factor and platelet-stimulated capillary formation of HUVECs (IC(50) = 10 microg/ml and 15 microg/ml, respectively), demonstrating that endothelial alphavbeta3 is important for sprouting, and platelet-stimulated sprouting is mediated by GPIIb/IIIa. In an experimental metastasis assay, a single pretreatment of human melanoma cells with c7E3 Fab (2.5 microg/ml) inhibited lung colonization of the tumor cells in severe combined immunodeficient mice. In vivo, m7E3 F(ab')(2) partially inhibited growth of human melanoma tumors in nude mice compared with control-treated animals. These data suggest that tumor cell-expressed integrins are important but not the only component involved in tumor growth. Because c7E3 Fab and m7E3 F(ab')(2) do not cross-react with murine integrins, this inhibition of metastasis and tumor growth is attributable to direct blockade of human tumor alphavbeta3 integrins. m7E3 F(ab')(2) completely blocked tumor formation and growth of human melanoma tumors growing in nude rats. In this xenograft model, m7E3 F(ab')(2) simultaneously binds to both human tumor and host platelet GPIIb/IIIa and endothelial alphavbeta3 integrins, thus participating as an antiangiogenic and an antitumor agent. Collectively, these results indicate that combined blockade of GPIIb/IIIa and alphavbeta3 affords significant antiangiogenic and antitumor benefit.