ABSTRACT
Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.
Subject(s)
Antibodies, Bispecific , Neoplasms , Animals , Mice , Cell Line , Immunoglobulin G/geneticsABSTRACT
Based on results from a signal sequence trap, we investigated chemerin gene expression in brown adipose tissue. Male NMRI mice were exposed to 30, 22 or 4 °C for 3 weeks, or were fed control (chow) diet, cafeteria diet or high-fat diet at thermoneutrality for the same time. In brown adipose tissue, cold acclimation strongly diminished chemerin gene expression, whereas obesogenic diets augmented expression. Qualitatively, changes in expression were paralleled in brite/beige adipose tissues (e.g. inguinal), whereas white adipose tissue (epididymal) and muscle did not react to these cues. Changes in tissue expression were not directly paralleled by alterations in plasma levels. Both these intact animal studies and brown adipocyte cell culture studies indicated that the gene expression regulation was not congruent with a sympathetic/adrenergic control. The data are discussed in relation to suggested endocrine, paracrine and autocrine effects of chemerin.
Subject(s)
Acclimatization/genetics , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Cold Temperature , Gene Expression Regulation , Obesity/genetics , Acclimatization/drug effects , Adipocytes, Brown/drug effects , Adipocytes, Brown/metabolism , Adipose Tissue, Brown/drug effects , Adipose Tissue, White/drug effects , Animals , Cells, Cultured , Chemokines , Chemotactic Factors/blood , Chemotactic Factors/genetics , Diet, High-Fat , Gene Expression Profiling , Gene Expression Regulation/drug effects , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/genetics , Ion Channels/metabolism , Male , Mice , Mitochondrial Proteins/metabolism , Norepinephrine/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Uncoupling Protein 1 , Weight Gain/drug effects , Weight Gain/geneticsABSTRACT
PURPOSE: To determine if there is a different clinical outcome after cross-pin versus interference screw fixation in anterior cruciate ligament (ACL) reconstruction with hamstring tendons. TYPE OF STUDY: Prospective randomized clinical follow-up study. METHODS: Sixty-two patients were randomized into either TransFix cross-pin (Arthrex, Naples, FL) (TransFix group, n = 31) or metal interference screw femoral fixation (screw group, n = 31) in ACL reconstruction with hamstring tendons. The distal fixations were an AO screw with spiked washer post and metal interference screw. The evaluation methods were clinical examination, CA 4000 laxity (OSI, Hayward, CA), and Lido isokinetic muscle torque measurements (Lido MultiJoint II; Loredon, West Sacramento, CA) as well as International Knee Documentation Committee (IKDC), Tegner activity level, Lysholm knee, and Kujala patellofemoral scores. RESULTS: There were no differences between the study groups preoperatively. For the 2-year follow-up, 26 patients of the TransFix group and 30 patients of the screw group were available (90%). The evaluation methods disclosed no statistical differences between the groups at the 1- or 2-year follow-up examinations. At the 2-year follow-up, 22 (85%) of the TransFix and 22 (73%) of the screw group patients were in the IKDC A or B categories. There were significantly more additional procedures postoperatively in the TransFix group owing to the removal of the tibial fixation post hardware in 15 (48%) cases. CONCLUSIONS: There were no statistically or clinically relevant differences in the results 1 or 2 years postoperatively and both techniques seemed to improve patients' performance. LEVEL OF EVIDENCE: Level I.
Subject(s)
Anterior Cruciate Ligament Injuries , Anterior Cruciate Ligament/surgery , Bone Nails , Bone Screws , Tendons/transplantation , Adolescent , Adult , Female , Follow-Up Studies , Humans , Male , Middle Aged , Orthopedic Procedures , Prospective Studies , Time FactorsABSTRACT
BACKGROUND: Bone-patellar tendon-bone graft has been the most commonly used graft material in anterior cruciate reconstructions, but there has been increasing use of hamstring tendon grafts. However, no existing clinical studies show adequate support for the choice of one graft over the other. HYPOTHESIS: Hamstring tendons are equally as good as patellar tendon in anterior cruciate ligament reconstructions. STUDY DESIGN: Prospective randomized clinical trial. METHODS: Ninety-nine patients with laxity caused by a torn anterior cruciate ligament underwent arthroscopically assisted reconstruction with graft randomization according to their birth year. Grafts were either bone-patellar tendon-bone with metal interference screw fixation or double-looped hamstring tendons with metal plate fixation. There were no significant differences between the two groups preoperatively or at operation. Standard rehabilitation included immediate postoperative mobilization without a knee brace, protected weightbearing for 2 weeks, and return to full activity at 6 to 12 months. RESULTS: Forty-three patients in the patellar tendon group and 46 patients in the hamstring tendon group were available for clinical evaluation at a minimum of 21 months after surgery. No statistically significant differences were seen with respect to clinical and instrumented laxity testing, International Knee Documentation Committee Score ratings, isokinetic muscle torque measurements, and Kujala patellofemoral, Lysholm, and Tegner scores. CONCLUSION: Equal results were seen for patellar and hamstring tendon autograft anterior cruciate ligament reconstructions at 2 years after surgery. Both techniques seem to improve patients' performance.