A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

Relationship of occupational therapy inpatient rehabilitation interventions and patient characteristics to outcomes following spinal cord injury: the SCIRehab project.

BACKGROUND/OBJECTIVE: Describe associations of occupational therapy (OT) interventions delivered during inpatient spinal cord injury (SCI) rehabilitation and patient characteristics with outcomes at the time of discharge and 1-year post-injury. METHODS: Occupational therapists at six inpatient rehabilitation centers documented detailed information about treatment provided. Least squares regression modeling was used to predict outcomes at discharge and 1-year injury anniversary for a 75% subset; models were validated with the remaining 25%. Functional outcomes for injury subgroups (motor complete low tetraplegia and motor complete paraplegia) also were examined. RESULTS: OT treatment variables explain a small amount of variation in Functional Independence Measure (FIM) outcomes for the full sample and significantly more in two functionally homogeneous subgroups. For patients with motor complete paraplegia, more time spent in clothing management and hygiene related to toileting was a strong predictor of higher scores on the lower body items of the self-care component of the discharge motor FIM. Among patients with motor complete low tetraplegia, higher scores for the FIM lower body self-care items were associated with more time spent on lower body dressing, manual wheelchair mobility training, and bathing training. Active patient participation during OT treatment sessions also was predictive of FIM and other outcomes. CONCLUSION: OT treatments add to explained variance (in addition to patient characteristics) for multiple outcomes. The impact of OT treatment on functional outcomes is more evident when examining more homogeneous patient groupings and outcomes specific to the groupings. Note: This is the third of nine articles in the SCIRehab series.

The SCIRehab project: treatment time spent in SCI rehabilitation. Occupational therapy treatment time during inpatient spinal cord injury rehabilitation.

BACKGROUND: Occupational therapy (OT) is a critical component of the rehabilitation process after spinal cord injury (SCI), the constitution of which has not been studied or documented in full detail previously. OBJECTIVE: To describe the type and distribution of SCI rehabilitation OT activities, including the amount of time spent on evaluation and treatment, and to discuss predictors (patient and injury characteristics) of the amount of time dedicated to OT treatment activities. METHODS: Six inpatient rehabilitation centers enrolled 600 patients with traumatic SCI in the first year of the SCIRehab. Occupational therapists documented 32 512 therapy sessions including time spent and specifics of each therapeutic activity. Analysis of variance and contingency tables/chi-square tests were used to test differences across neurologic injury groups for continuous and categorical variables. RESULTS: SCIRehab patients received a mean total of 52 hours of OT over the course of their rehabilitation stay. Statistically significant differences among four neurologic injury groups were seen in time spent on each OT activity. The activities that consumed the most OT time (individual and group sessions combined) were strengthening/endurance exercises, activities of daily living (ADLs), range of motion (ROM)/stretching, education, and a grouping of 'therapeutic activities' that included tenodesis training, fine motor activities, manual therapy, vestibular training, edema management, breathing exercise, cognitive retraining, visual/perceptual training desensitization, and don/doff adaptive equipment. Seventy-seven percent of OT work occurred in individual treatment sessions, with the most frequent OT activity involving ADLs. The variation in time (mean minutes per week) spent on OT ROM/stretching, ADLs, transfer training, assessment, and therapeutic activities can be explained in part by patient and injury characteristics, such as admission Functional Independence Measure (FIM) score, neurologic injury group, and the medical severity of illness score. CONCLUSION: OT treatment patterns for patients with traumatic SCI show much variation in activity selection and time spent on activities, within and among neurologic level of injury groups. Some of the variation can be explained by patient and injury characteristics. Almost all patients with SCI participated in strengthening/ endurance and ROM/stretching exercises during OT treatment and these two activities are where the most time was spent when therapy provided in individual and group settings was combined. ADL work consumed the most time in individual therapy sessions.

SCIRehab Project series: the occupational therapy taxonomy.

BACKGROUND/OBJECTIVE: Lack of a classification system for occupational therapy (OT) rehabilitation interventions for traumatic spinal cord injury (SCI) rehabilitation in the United States makes conducting outcomes research difficult. This article describes an OT SCI rehabilitation taxonomy (system to categorize and classify treatments). METHODS: OT clinicians and researchers from 6 SCI rehabilitation centers developed a taxonomy to describe details of each OT session. This effort is part of the SCIRehab study, which uses the practice-based evidence, observational research methodology to examine current treatment processes without changing existing practice. RESULTS: The OT taxonomy consists of 26 OT activities (eg, training on activities of daily living, communication, home management skills, wheelchair mobility, bed mobility, transfers, balance, strengthening, stretching, equipment evaluation, and community reintegration). Time spent on each activity is documented along with therapeutic interventions used to facilitate the activity. Treatment descriptions are enhanced further with identification of assistance needs, patient direction of care, and family involvement, which help to describe and guide OT activity selection. The OT taxonomy documentation process includes all OT rehabilitation interventions for patients with SCI while maintaining efficiency in data collection. CONCLUSION: The electronic documentation system is being used at 6 centers for all OT sessions with 1,500 patients with acute traumatic SCI. It is the largest known attempt to document details of the comprehensive OT rehabilitation process for patients with SCI in the United States.

Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

A novel method for increasing the expression level of recombinant proteins.

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

Designing HER2 vaccines.

HER2/neu is a compelling cancer vaccine candidate because it is overexpressed on some cancer cells relative to normal tissues, it is known to be immunogenic in both animal models and in humans, and it is already known to be targetable by the antibody component of the immune system in the form of monoclonal antibody therapy with trastuzumab. Vaccines offer the theoretical advantage of being able to elicit T-cell responses in addition to antibody responses. HER2 vaccines have been shown to provide benefit in animal models and to be immunogenic in humans. However, the optimal vaccine formulation is not yet known and the therapeutic efficacy of the vaccines in humans has not yet been evaluated. HER2 vaccine approaches currently being tested include peptide-based, DNA plasmid-based, and protein-based vaccines. Our group has developed and started testing a protein-based vaccine composed of both the extracellular domain of HER2 and the carboxyl terminal autophosphorylation portion of the intracellular domain. The extracellular domain was retained to provide for antibody targeting. The kinase domain of the intracellular domain was excluded because of its high degree of homology to other human kinases. The carboxyl terminal autophosphorylation domain was retained because it is the most unique and possibly most immunogenic portion of the HER2 molecule with the least homology to other members of the HER family. The vaccine, termed dHER2, is immunogenic in mice and primates. In animal models it can elicit CD8 and CD4 T-cell responses as well as antibody responses that suppress the growth of HER2-positive cancer cells in vitro and in vivo. Vaccine trials are contemplated in patients with breast cancer that will determine whether the vaccine construct is similarly immunogenic in humans.