We used 6- to 8-week-old, female C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA, Catalog 027), female F1(C57BL/6xDBA/2) (Jackson Laboratories, Bar Harbor, ME, USA, catalog 100006), FcγR2b-/- (Jackson, Catalog 002848), and TLR4-/- mice (Stefanie Vogel, University of Maryland) were used . All experiments, except those contracted to outside vendors, were approved by the Animal Care and Use Committee of the University of Maryland School of Medicine. Two contract research organizations paid by Gliknik, Inc., performed CIA experiments in separate mouse lines. Washington Biotechnology (Baltimore, MD, USA) used DBA1/J mice for their experiments, and Bolder-BioPATH (Boulder, CO, USA) used DBA/1OlaHsd mice (Harlan Laboratories, Frederick, MD, USA).
Generation of stradomer and IgG2a Fc cDNA constructs
The Fc regions of the immunoglobulin sequence were fused to MDs either by splicing by overlap extension PCR or by using existing compatible restriction sites . Polymerase chain reaction (PCR) primers were generated encoding the MD and an overlapping region of either the C or the N-terminus of the IgG Fc region and used for fusing the two sequences by PCR. The PCR products were then cloned into pcDNA3.3 by TA cloning (Invitrogen, Carlsbad, CA, USA) and confirmed by sequencing. Stradomers were transiently expressed in HEK293 cells after transfection.
Purification of stradomers
Purification was done by using automated chromatography (Aktaxpress, GE, Piscataway, NJ, USA) and Hitrap Mabselect, protein A-derived ligand columns (GE 28-4082-56), followed by desalting on a HiPrep 26/10 Column (GE 17-5087-01). Stradomers were separated into homodimeric and multimeric fractions by using the standard Aktaxpress purification protocol and a GE Hiload 16/60 Superdex 200-pg size exclusion column (GE 17-1069-01).
From 0.5 to 2 μg of nonreduced or reduced sample was loaded onto Nupage Novex 4-12% Bis-Tris Mini Gels (Invitrogen, Carlsbad, CA, USA, NPO322BOX) and were run according to the manufacturer's protocol (Invitrogen, IM-8042 Version H). Gels were washed with deionized water, stained for 30 minutes at room temperature with Bio-safe Coomassie Dye (Bio-Rad, Hercules, CA, USA, 161-0786), and destained with water.
Surface plasmon resonance
Surface Plasmon resonance (SPR) was performed on a GE Healthcare Biacore 3000 instrument or on a Biacore T100. Recombinant mouse FcγRI, FcγRIIb, FcγRIII, FcγRIV, and SIGN-R1 (RnD Systems; Minneapolis, MN, USA) were immobilized onto a CM4 (FcγRs) or CM5 (SIGN-R1) chip by amine coupling. Samples were serially diluted in HBS-EP (running buffer) from 1,000 nM to 0.5 nM and injected at 20 μl/min, followed by a dissociation phase. Flow cells were regenerated with 1 M MgCl at 100 μl/min and then washed with HBS-EP (FcγRI required 10 mM glycine pH 2.0 to regenerate). A blank flow cell was used as a reference. All samples were run in duplicate or triplicate. The KD was calculated by using a 1:1 Langmuir kinetic model. Unless otherwise specified, all reagents were from GE Healthcare (Piscataway, NJ, USA). Because many of the preparations used in our studies contained multimers of diverse molecular masses, we standardized our analysis by assigning a molecular mass of 50 kDa to homodimer isolates and 150 kDa to all other protein preparations.
Biolayer interferometry assay was performed on an Octet 96Red Instrument (ForteBio, Menlo Park, CA, USA) according to the manufacturer's instructions (Data Acquisition User Guide 6.4, ForteBio). For binding analysis, His-tagged mouse FcγRIIb (R&D Systems, Minneapolis, MN, USA, 1460-CD) and FcγRIII (R&D Systems 1960) were loaded on anti-penta-His Biosensors (ForteBio 18-5077) at 10 μg/ml in 1× kinetic analysis buffer (ForteBio,18-5032). After sensor-tip loading, protein association was measured by transfer of tips to preparations of either the monomeric (concentration 2,000 nM to 62.5 nM) or multimeric fractions (concentration, 100 nM to 3.13 nM) in 1× kinetics analysis buffer, and dissociation was measured by transfer of sensor tips to 1× kinetics buffer. Analysis was as described (Data Analysis User Guide 6.4ForteBio). Analysis was standardized, as described earlier by assigning a MW of 50 to homodimers and 150 to all other protein preparations.
Collagen-induced arthritis model
Collagen-induced arthritis experiments were performed by two contract research organizations (CROs; Washington Biotechnology, Inc., Columbia, MD, USA; Bolder BioPATH Inc., Boulder, CO, USA). All arthritis experiments, except for the dose-response data for 2A-2HC, were generated by Washington Biotechnology by using the following protocol: On day 0, DBA1/J mice were immunized with 50 μl of a 4-mg/ml solution of Type II bovine collagen (Chondrex, Inc., Redmond, WA, USA, Cat. 20021) emulsified with an equal volume of complete Freund's Adjuvant (Sigma, St. Louis, MO, USA, Cat. 5506). A second vaccination was given on day 21 by using the same dose of Type II bovine collagen emulsified with an equal volume of incomplete Freund's Adjuvant. Mice were scored daily for signs of arthritis. Each paw was scored, and the sum of all four scores was recorded as the Arthritic Index (AI). The maximum possible AI was 16, as follows: 0, no visible effects of arthritis; 1, edema and/or erythema of one digit; 2, edema and/or erythema of two joints; 3, edema and/or erythema of more than two joints; and 4, severe arthritis of the entire paw and digits, including limb deformation and ankylosis of the joint. Starting on day 28 (treatment day 0), 10 collagen-immunized mice (all manifesting disease) were sorted into each of the treatment groups based on average AI (3.3). AI was measured for 14 treatment days, after which mice were euthanized. For the positive control group, mice were dosed orally with 10 mg/kg prednisolone daily. For the 2A-2HC group, mice were dosed every fourth day (day 0, day 4, day 8, and day 12) with 400 μg 2A-2HC (17.4 mg/kg). Fractionated 2A-2HC was given at a lower dose (250 μg, 10.9 mg/kg).
A second contract research organization, Bolder BioPATH Inc. (Boulder, CO, USA), independently generated dose-response data to 2A-2HC in a CIA model by using a slightly different protocol. In brief, male DBA/1OlaHsd (Harlan Laboratories, Frederick, MD, USA) mice were anesthetized with isoflurane and injected with 150 μl of Bovine Type II collagen (Elastin Products, Owensville, MO, USA), in Freund's complete adjuvant on day 0 and day 21 (Freund's complete adjuvant [with supplemental Mycobacterium. tuberculosis, 4 mg/ml], Difco). On study days 24 through 27, onset of arthritis occurred, and mice were randomized to treatment groups. Animals were randomized to a treatment group only after swelling was obviously established in at least one of four paws (score of 1 for the paw, minimum mean score of 0.25 for the animal). Attempts were made to assure approximately equal mean scores across all groups at the time of enrollment. Treatment was initiated after enrollment (treatment day 0). Mice were treated with intravenous 2A-2HC given at varying doses (10 mg/kg, 20 mg/kg, and 40 mg/kg). The first dose was administered on day 0 and then every fourth day. IVIG was given at a dose of 2 g/kg by intraperitoneal injection on day 0 and every fourth day. Dexamethasone was given orally at a dose of 0.2 mg/kg daily beginning on day 0. During this time, clinical scores were given for each of the paws (right front, left front, right rear, left rear) as follows: 0, normal; 1, 1 hind- or forepaw joint affected or minimal diffuse erythema and swelling; 2, 2 hind- or forepaw joints affected or mild diffuse erythema and swelling; 3, 3 hind- or forepaw joints affected or moderate diffuse erythema and swelling; 4, marked diffuse erythema and swelling, or four digit joints affected; and 5, severe diffuse erythema and severe swelling of the entire paw, unable to flex digits, or ankylosis, if present. Under this protocol, the mean score of all four paw scores, rather than a sum, was used for the scoring index.
Induction and assessment of ITP
Platelet counts were measured by serial tail-vein nicking on days 1 through 5. To measure platelet counts, 10 μl of blood was diluted in 15 μl citrate buffer (0.105 M,///3.2%). The samples were analyzed for absolute platelet count on a Drew Scientific HemaVet 950FS hemocytometer that was standardized daily. Control-group mice received no pretreatment and no platelet depletion. ITP mice received only platelet depletion with MWReg30 α-platelet antibody (BD Biosciences, San Jose, CA, USA). Mice in the experimental groups received pretreatment with stradomers, murine IgG2a Fc, or IVIG on day 1, administered 16 to 24 hours before platelet depletion with MWReg30 each evening on days 2 through 4 (2 μg diluted in 200 μl PBS, given intraperitoneally). Mice pretreated with murine IgG2a Fc or stradomers received only a single dose (400 μg intravenous) on day 1. Mice pretreated with IVIG received daily doses (2 g/kg by intraperitoneal injection every morning on days 1 through 5).
In some experiments, administration of 2A-2HC on day 1 caused an initial decrease in platelet count before administration of MWReg30. Therefore, percentage platelet decrease is shown relative to the baseline measurement on day 2 by using the formula: % = measured platelet count/baseline platelet count on day 2. Experiments were done in replicate, as indicated, and all mice were included in pooled statistical analysis except for the following: for experiments testing the efficacy of the homodimeric versus multimeric fractions of 2A-2HC in preventing ITP, three replicate experiments were done with six mice per group. One mouse in the ITP group was excluded because of spurious platelet values. Two of 18 mice in the 2A-2HC group were excluded from day 5 analysis because of equipment failure that prevented measurement of platelet count.
The GVHD model was previously described . On day 0, BDF1 mice were given 4 × 107 C57BL/6 splenocytes and lymphocytes by tail-vein injection. Mice received no treatment, IgG2a Fc, or 2A-2HC (both given at 400 μg IV) on day 0 and day 4. In the preventive study, 2A-2HC was administered before adoptive transfer of B6 splenocytes and lymphocytes. On day 9, donor anti-host CTL activity was tested by 51Cr-release assay against H2d-expressing P815 cells (positive target) and EL4s that do not express H2d (negative control).
Injection of opsonized red blood cells (oRBCs)
One day before injecting oRBCs, mice were given either 40 mg of IVIG by intraperitoneal injection, 400 μg of murine IgG2a Fc by intravenous injection, or 400 μg of 2A-2HC by intravenous injection. Fluorescence-labeled oRBCs were generated as described by Song et al. (2003) . Erythrocytes were then washed 3 times with PBS and resuspended in 5 ml of PBS. Mice were given tail-vein injections of 200 μl of oRBCs. Mice were then bled by tail-vein nicking at 3, 30, 60, 90, and 165 minutes after injection. Blood (10 μl) was collected into a tube containing an equal volume (10 μl) of 1% EDTA in PBS. After mixing, 1 μl of the sample was diluted in 300 μl of FACS buffer and used for flow cytometry. The percentage of PKH26-labeled oRBCs in circulation was measured with flow cytometry. The percentage of fluorescent erythrocytes at the 3-minute time point was considered the baseline (100%).
Statisticians had access to all primary data. For the platelet data, Wilcoxon rank-sum tests were used to compare percentages between groups. For the Arthritis Index data, linear mixed-effects models with either one or two slopes were used to compare the Arthritis Index over time between groups. For the platelet and the RBC data, percentages of baseline values were calculated for each post-baseline time point and presented in the plots as means ± SEM. Wilcoxon rank-sum tests were used to compare percentages between groups at some later time points at which we expected differences to have developed between groups (days 4 and 5 for platelet data, and 60 minutes, 90 minutes, and 165 minutes after injection for RBC data). Unless otherwise indicated, all comparisons reported were made against the diseased with no-treatment group (ITP, CIA, or injected oRBCs with no pretreatment). Bonferroni's correction was used for significance where multiple comparisons were made, with the intended number of comparisons reported.