Jill Giles-Komar

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.

Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders.

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, “humanized” and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor β1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.

Development of the IL-12/23 antagonist ustekinumab in psoriasis: past, present, and future perspectives

The development of ustekinumab as a first‐in‐class anti‐interleukin (IL) 12/23p40 therapeutic agent for psoriasis represents an important example of modern and rational drug design and development. Psoriasis is a chronic, systemic, immune‐mediated skin disorder with considerable clinical, psychosocial, and economic burden. Ustekinumab is a human monoclonal antibody (mAb) that binds the p40 subunit common to IL‐12 and IL‐23, key cytokines in psoriasis pathogenesis. The therapeutic mAb was developed using human gamma‐1 immunoglobulin (IgG)‐expressing transgenic mice, which created a molecule with endogenous IgG1 biologic properties and low immunogenicity. Ustekinumab was well tolerated in clinical studies and yielded rapid, significant, and sustained efficacy plus improved quality of life/work performance and reduced depression/anxiety. Its pharmacologic properties afford the most convenient dosing regimen among approved biologics, representing a significant advancement in the treatment of moderate to severe psoriasis. Ustekinumab also holds promise for other immune‐mediated disorders with significant unmet need.

Chemokine (C-C motif) ligand 2 mediates direct and indirect fibrotic responses in human and murine cultured fibrocytes

BackgroundFibrocytes are a population of circulating bone-marrow-derived cells that express surface markers for leukocytes and mesenchymal cells, and are capable of differentiating into myofibroblasts. They have been observed at sites of active fibrosis and increased circulating numbers correlate with mortality in idiopathic pulmonary fibrosis (IPF). Inhibition of chemokine (C-C motif) receptor 2 (CCR2) during experimental models of lung fibrosis reduces lung collagen deposition, as well as reducing lung fibrocyte accumulation. The aim of the present study was to determine whether human and mouse fibrocytes express functional CCR2.ResultsFollowing optimized and identical human and murine fibrocyte isolation, both cell sources were shown to be positive for CCR2 by flow cytometry and this expression colocalized with collagen I and CD45. Human blood fibrocytes stimulated with the CCR2 ligand chemokine (C-C motif) ligand 2 (CCL2), demonstrated increased proliferation (P < 0.005) and differentiation into myofibroblasts (P < 0.001), as well as a chemotactic response (P < 0.05). Murine fibrocytes also responded to CCR2 stimulation, with CCL12 being more potent than CCL2.ConclusionsThis study directly compares the functional responses of human and murine fibrocytes to CCR2 ligands, and following comparable isolation techniques. We have shown comparable biological effects, strengthening the translatability of the murine models to human disease with respect to targeting the CCR2 axis to ameliorate disease in IPF patients.

Solubility evaluation of murine hybridoma antibodies

The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.

Development of the IL-12/23 antagonist ustekinumab in psoriasis: past, present, and future perspectives - an update: Ustekinumab development as psoriasis treatment

Since the original publication of the article “Development of the IL‐12/23 antagonist ustekinumab in psoriasis: Past, present and future perspectives” in March 2011 (see Appendix), 1 there have been several new publications and developments of note. A number of new reports from the ustekinumab psoriasis clinical development program have been published. The analysis of efficacy and safety in the PHOENIX 1 long‐term extension demonstrated that continuous stable maintenance dosing of ustekinumab was generally well tolerated and sustained durable efficacy through up to three years of treatment. 2 Pooled safety data from the phase 2 and phase 3 global trials showed that the safety profile of long‐term continuous ustekinumab treatment through up to three years 3,4 and four years 5 of follow‐up was favorable and comparable to what has been reported previously in the shorter‐term ustekinumab psoriasis studies. 6–8 This represents the greatest exposure and longest follow‐up of psoriasis patients treated with a biologic published to date. Additional phase 3 trials in Asian populations demonstrated similar high levels of efficacy and favorable safety profiles in Japanese, 9,10 Korean, 11,12 and Taiwanese 11,12 patients as those observed in trials conducted in mostly White populations in North America and Europe. 6–8 These data support the positive benefit:risk profile and consistency of response to ustekinumab over years of usage, and in multiple ethnic groups. Results from up to five years of treatment with ustekinumab in the long‐term extensions of the phase 3 trials, and the efficacy, safety, and effect on quality of life in Chinese patients will be available in 2012. In addition to clinical trials of ustekinumab for the treatment of psoriasis, 24‐week data from one phase 3 study of ustekinumab for the treatment of psoriatic arthritis has recently been presented 13 and another study is ongoing. A Phase 2b trial in Crohns disease has also been presented, 14 and three phase 3 studies in Crohns disease are currently in progress.

Pharmacodynamic enhancement of the anti-platelet antibody Fab abciximab by site-specific pegylation

Monoclonal antibodies have been firmly established as human therapeutics. Their high affinity and specificity for target antigens minimize adverse reactions and their molecular size results in extended circulation times relative to small molecule pharmaceuticals. The ability to customize the pharmacokinetics in a rational manner can enhance the potential for these and other classes of biologicals. We have systematically studied the effect of site-specific pegylation of the Fab‵ fragment of the anti-GPIIb/IIIa, αVβ3 antibody c7E3. Regardless of the molecular weight of the PEG molecules, the intrinsic affinity of the resulting constructs remained unchanged. However, in functional assays measuring inhibition of platelet aggregation, the calculated IC50 values of the conjugates decreased with increasing molecular weight of the conjugated PEG. It was determined that the molecular size of the conjugates affects antigen accessibility and whereas high levels of binding to antigen molecules on cells with high antigen density can be demonstrated with the Fab fragment, comparable levels are not achievable with large molecular weight conjugates. In spite of the inability of the larger PEG constructs to achieve saturation binding, functional inhibition of platelet aggregation consistent with saturation binding was demonstrated and the increased molecular size of the conjugates led to predictably prolonged inhibition of platelet aggregation.

Generation and Characterization of Rat Anti-mouse ST2L Monoclonal Antibodies

ST2L is a transmembrane receptor that belongs to the IL-1 receptor family. The receptor is expressed on various cell types including Th2 cells, mast cells, basophils, growth-activated fibroblasts, and vascular endothelial cells. ST2L activation by its ligand IL-33 has been implicated in Th2-mediated immunity, inflammation, and allergic responses in vivo. Inhibition of ST2L activity can attenuate Th2-dominated immune responses such as lung eosinophilia, airway hyper-responsiveness, and arthritis in animal models. Here we report the generation and in vitro characterization of a panel of rat anti-mouse ST2L monoclonal antibodies. We demonstrate that the antibodies specifically bind to recombinant receptor protein and that a subset of the binders inhibits mouse ST2L activity in multiple in vitro assays. Four of the identified anti-mouse ST2L antibodies were shown to prevent IL-33 from binding to ST2L, down-regulate IL-33-induced NF-κB signaling, and neutralize the ability of IL-33 to stimulate mouse Th2 cell proliferation. The characterized monoclonal antibodies are important tools that will be used to study mouse ST2L receptor functionality in vivo.

The Use of an Anti-CD40 Agonist Monoclonal Antibody During Immunizations Enhances Hybridoma Generation

ABSTRACT Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.