PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis.

INTRODUCTION: Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20- plasmablasts is noted. The strong expression of CD319 on CD20- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target. METHODS: PDL241, a novel humanized IgG1 monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys. RESULTS: PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319+ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG1 and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed. CONCLUSIONS: The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.

Dissociation of efficacy and cytokine release mediated by an Fc-modified anti-CD3 mAb in a chronic experimental autoimmune encephalomyelitis model.

Humanization and modification of the Fc region of anti-human CD3 mAbs have greatly expanded their potential use in chronic T cell mediated diseases. However, low levels of cytokine release and immunogenicity may still impact a chronic dosing strategy. We investigated the use of an Fc-modified murine chimeric anti-mouse CD3 (N297A) in the chronic MOG(35-55)-induced EAE mouse model of MS. Two daily doses of 10 microg at the onset of clinical symptoms led to both a reduction in T cell numbers in the blood and a significant, prolonged reduction in the symptoms. Histological examination of the spinal cords at the peak of efficacy confirmed a reduction of infiltrating T cells in the CNS. Analysis of the cerebral spinal fluid from EAE mice showed biologically active levels of N297A. Analysis of the cytokine/chemokine levels in cerebrospinal fluid showed a decrease in GM-CSF, IL-6 and IP-10. The combination of N297A dosing with cyclosporine A (CSA) pretreatment showed a significant decrease of TNFalpha, IL-6 and IP-10 without effect on clinical efficacy. However, pretreatment of CSA significantly reduced the immunogenic response observed following a second course of N297A treatment. Therefore, the side effects of an Fc-modified anti-CD3 mAb may be modulated without affecting efficacy.

Functional characterization of N297A, a murine surrogate for low-Fc binding anti-human CD3 antibodies.

Several low- or non-FcR binding anti-human CD3 monoclonal antibodies have been under investigation for the treatment of autoimmune diseases. To model the mechanism of action of these anti-human CD3 mAbs in the murine system, an Fc-modified anti-mouse CD3 antibody (N297A) was generated. N297A exhibited similar biological effects as Fc-modified anti-human CD3 antibodies including rapid, reversible reduction in peripheral leukocyte numbers, differential modulation of activated versus resting T cells, and reduced levels of induced cytokine release compared to the non-Fc-modified parent antibody. In an in vivo model of colitis induced by adoptive transfer of IL-10-deficient cells, administration of N297A significantly reduced body weight loss. As N297A shared many functional characteristics of non-FcR binding anti-human CD3 mAbs both in vitro and in vivo, it provides a means to model the mechanisms of action of Fc-modified anti-human CD3 antibodies in mouse.

Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu.

Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.

Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent.

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.

Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer.

Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor-like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.

E-selectin up-regulation allows for targeted drug delivery in prostate cancer.

We have used the Eos Hu03 GeneChip array, which represents over 92% of the transcribed human genome, to measure gene expression in a panel of normal and diseased human tissues. This analysis revealed that E-selectin mRNA is selectively overexpressed in prostate cancer epithelium, a finding that correlated strongly with E-selectin protein expression as assessed by immunohistochemistry. Antibodies against E-selectin that blocked function failed to impede cancer cell growth, suggesting that overexpression of E-selectin was not essential for cell growth. However, a novel auristatin E-based antibody drug conjugate (ADC), E-selectin antibody valine-citrulline monomethyl-auristatin E, was a potent and selective agent against E-selectin-expressing cancer cell lines in vitro, with the degree of cytotoxicity varying with surface antigen density. Interestingly, sensitivity to the ADC differed among cell lines from different tissues expressing similar amounts of E-selectin and was found to correlate with sensitivity to free auristatin E. Furthermore, E-selectin-expressing tumors grown as xenografts in severe combined immunodeficient mice were responsive to treatment with E-selectin antibody valine-citrulline monomethyl-auristatin E in vivo, with more than 85% inhibition of tumor growth observed in treated mice. These findings demonstrate that an E-selectin-targeting ADC has potential as a prostate cancer therapy and validates a genomics-based paradigm for the identification of cancer-specific antigens suitable for targeted therapy.

Beyond organisational design: moving from structure to service enhancement.

The Australian health care industry prior to the 1990s was notable for its relative stability and uniformity in relation to organisational design. Since then, new organisational designs have proliferated and a diversity of approaches is evident. The new fluidity in organisational design is particularly evident amongst the allied health professions. The aim of this paper is two-fold. Firstly, to summarise recent changes in organisational design as they relate to the allied health professions and secondly, to move beyond design issues to focus on service level enhancement in an organisational change context. This later aim is achieved by presenting data from an in-depth study of one institutions experience with wide-ranging organisational reforms. The recent formation of the National Allied Health Organisational Structures Network (NAHOSN) has given energy to the impetus of placing a research-based framework around the change experiences reported by Allied Health groups. An objective of the network is to foster research, rather than rely on commentary and anecdote, in the often highly contested arena of organisational design and reform.