Research and Development, Knoll Pharmaceuticals, Pennyfoot Street, Nottingham NG1 1GF, UK. E-mail: firstname.lastname@example.org
Objective: To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo.
Research Methods and Procedures: Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated.
Results: All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC50 values (maximum percentage of lysis in parentheses) were 0.680 μg/mL (63.2%), 0.546 μg/mL (72.4%), and 0.391 μg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5.
Discussion: Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.
Polyclonal antibodies raised against plasma membranes of adipocytes of several species have been shown to induce lysis of adipocytes both in vitro and in vivo (1, 2, 3, 4, 5, 6, 7, 8, 9). In some studies, the lysis of adipocytes by polyclonal antibodies was accompanied by a reduction of body weight and favorable changes in body composition in lean growing and obese rats (2, 4, 10). Monoclonal antibodies raised against pig adipocytes have been reported to induce lysis of cultured pig adipocytes and to significantly reduce inguinal fat pad weights in young growing rats (11). A monoclonal antibody against pig preadipocytes has also been shown to induce lysis and to reduce body fat mass (12). The potential, therefore, exists for the use of anti-adipocyte antibodies in the reduction of body weight and adiposity. However, any such use would be dependent on the preparation of anti-adipocyte monoclonal antibodies that react exclusively with adipose tissue.
In this study, we have used phage display technology to identify antibodies to human adipocytes that also recognize the rat adipocyte cell surface (13, 14). The ability of three such monoclonal anti-adipocyte antibodies, reformatted as rat chimeric IgG2bs, to lyse rat epididymal adipocytes in vitro was evaluated both singly and in combination. The lysis of adipocytes by both monoclonal and polyclonal anti-adipocyte antibodies has been shown to be through a complement-mediated mechanism (1). Therefore, we have also assessed the potential of these monoclonal antibodies to reduce serum complement levels in vivo in the rat.
Research Methods and Procedures
Identification of Anti-Adipocyte Single-Chain Variable Fragment of the Antibody Structure (scFv) Antibodies
A phage display library of over 1010 human antibodies (13) was used to isolate a panel of over 100 different monoclonal antibodies that recognize the cell surface of human adipocytes as previously detailed (14). The monoclonal anti-adipocyte antibodies designated Fat 13, Fat 37, and Fat 41 were identified in this manner. Both phage ELISA and immunohistochemistry using phage antibodies were performed as described previously (14). Binding of antibodies adipocytes was monitored by a horseradish peroxidase reaction using the substrate tetramethylbenzidine and the optical density of the color change read spectrophotometrically at 450 nm (OD 450).
Reformatting of Human scFv to Rat/Human Chimeric IgG2b
Human scFvs were reformatted to rat/human chimeric IgG2b by DNA cloning. The heavy and light domains (VH and VL, respectively) from the scFv were separately amplified by PCR and assembled with a mouse immunoglobulin secretory leader sequence. A rat G2b constant domain (courtesy of Dr. M. Bruggemann, Babraham Institute, UK) was cloned into the vector pEE6 (Lonza Biologics, Slough, UK). The leader-VH fragment was ligated to the G2b constant domain in the pEE6 vector to generate the heavy chain gene. The leader-VL fragment was ligated to a rat κ-constant domain (supplied by Dr. M. Neuberger; MRC-LMB, Cambridge, UK) in the pEE12 expression vector (Lonza Biologics) to generate the light chain vector. The plasmid pEE12 also contained the glutamine synthetase selectable marker gene (15). The control rat/human chimeric IgG2b (2G6) comprised a VH domain isolated from a human anti-NP (4-hydroxy-3-nitrophenolacetyl)-hapten immunoglobulin and a VL domain isolated from an anti-lysozyme human immunoglobulin that were fused to rat constant domains.
Cell Culture and Antibody Purification
Heavy and light chain vectors were co-transfected into NS0 mouse myeloma cells (Lonza Biologics) by electroporation. Transfected colonies were isolated by selection in glutamine-free culture medium containing 10% dialyzed fetal bovine serum (15). Colonies expressing rat IgG2b were detected by screening supernatants by ELISA. High-expressing colonies were identified and expanded into serum-free culture medium in roller bottles. Supernatant from saturated cultures was clarified by filtration and IgG was isolated by Protein-G sepharose chromatography. Purified IgG was diafiltered into phosphate buffered saline and concentrated to 10 mg/mL IgG by ultrafiltration and then 0.2-μm sterile filtered. IgG quality was verified by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography—high pressure liquid chromatography (SEC-HPLC), and limulus ameobcyte lysate (LAL) endotoxin assay. IgG potency was tested by binding to adipocyte membranes or human adipocyte sections.
Antibodies manufactured were the irrelevant, isotype-matched, control IgG2b rat/human chimeric antibody (2G6, batch 9807144A; 10.0 mg/mL) and the anti-adipocyte rat/human chimeric IgG2bs designated Fat 13 (batch 980611A; 7.908 mg/mL), Fat 37 (batch 980625A; 10.183 mg/mL), and Fat 41 (batch 980623B; 10.822 mg/mL). Monoclonal antibodies were prepared as 1-mL aliquots in sterile potassium-free phosphate buffered saline at pH 7.2 and stored at −70oC until required.
Male and female Wistar rats (180 to 325 g) were obtained from Charles River (Margate, Kent, UK). Animals were maintained on a 12-hour light–dark cycle and allowed free access to standard SDS CRM food and water. Animals were killed by carbon dioxide asphyxiation followed by cervical dislocation.
Isolation of Adipocytes and Lysis Assay
White adipocytes, isolated from epididymal fat pads were prepared as previously described using collagenase (Type II Sigma, C6885, lot 115H6855; Poole, Dorset, UK) digestion (16). Adipocyte numbers in the final adipocyte suspensions were approximately (8 × 106 cells/mL) and adipocyte diameter of 27 μm.
Adipocyte lysis was estimated by measuring lactate dehydrogenase (LDH) release as previously described using a commercial LDH kit (Sigma 500-C) (1). Adipocyte lysis in control tubes was determined in triplicate at the beginning and end of the assay. Samples of incubation buffer were also taken at time zero to allow a determination of basal LDH levels. One hundred percent lysis of adipocytes was determined by incubating adipocyte samples for 2 hours in the presence of 1% Triton X-100. Guinea pig serum was used as the source of complement and LDH activity differed markedly from batch to batch. To control this variability, batches were only used if the LDH activity was <50% of the total activity in the time zero assay tubes.
Data Manipulation and Statistical Analyses
The percentage of lysis was estimated by expressing the LDH activity in each assay as the percentage of the detergent disrupted cells (100% lysis) and the buffer control (defined as 0% lysis) after the 2-hour incubation. For each experiment, the EC50 (concentration causing 50% of the maximal response) and maximum percentage of lysis were calculated by nonlinear regression using Marquardt's compromise method. The estimates for each experiment were then combined by one-way ANOVA to obtain the average result of each treatment with 95% confidence intervals (CIs). The EC50 values and the maximum percentage of lysis values were compared with each other using the Newman–Keuls test.
Antibodies were administered by the intraperitoneal (IP) route to female animals and blood samples taken from the tail vein at times before and at 6, 24, 48, and 120 hours after dosing. Blood (100 μL) was collected into Microvette CB300 Z tubes (Sarstedt, Germany) allowed to clot for 20 minutes, centrifuged at 13,000 rpm for 1 minute, and serum stored at −20 °C until required for complement analysis.
Total complement (CH100) was assessed by measuring lysis of sensitized sheep red blood cells immobilized in agarose gels as previously described (17). Lysis assays were performed in radial immunodiffusion plates (IC003; Binding Site, Birmingham, UK) with 3-mm wells punched. Standard complement rat serum (S-3394; Sigma) was reconstituted using ice-cold deionized water and stored on ice until required. Serum samples (5 μL) were added to the wells of the assay plates. A rat serum complement standard curve was applied to each plate consisting of 1, 3, 5, and 7 μL of standard. The plates were incubated right-way-up at 4 °C for 16 hours and at 37 °C for 2 hours before measuring the ring diameters on an electronic plate reader (AD001; Binding Site). CH100 values were calculated from the standard curve using linear regression (5 μL standard is equivalent to 583 CH100U/mL serum, calculated from a known standard supplied by Binding Site). Complement levels were expressed as the percentage of the time 0 level for each individual animal.
Characterization of Monoclonal Anti-Adipocyte Antibodies
Fat 13, Fat 37, and Fat 41 were shown to selectively bind adipocyte plasma membranes (14). These three antibodies cross-react strongly with plasma membranes prepared from rat adipocytes (Figure 1). This cross-reactivity was also evident by immunohistochemistry, where all three antibodies were shown to bind both white and brown adipocytes in frozen sections of rat tissue. This is exemplified by Fat 13 (Figure 2). Thus, the reactivity of these three antibodies for rat adipocytes made them our preferred choice for the investigation of antibody-mediated complement lysis of adipocytes in the rat.
Western blots were performed for all three antibodies. However, only Fat 37 bound to adipocyte plasma membrane preparations and in these experiments Fat 37 recognized a protein of 35 kDa. The antigen bound by Fat 37 has not been characterized.
The collagenase digestion technique used resulted in predominantly spherical cells containing a single large fat depot with an associated halo. Isolated adipocytes can be fragile and may be susceptible to spontaneous lysis during the 2-hour incubation. On average, ∼20% spontaneous lysis was observed in the control tubes during the 2-hour incubation. Spontaneous lysis did, however, show considerable batch-to-batch variation (5% to 30%).
Effect of Monoclonal Anti-Adipocyte Antibodies on Adipocyte Lysis In Vitro
All three monoclonal anti-adipocyte antibodies showed concentration-dependent lysis of adipocytes in the presence of guinea pig complement (Figure 3). The calculated mean EC50 values from individual experiments (n = 3 to 4 with 95% CI in parentheses) were 0.680 μg/mL (0.376 to 1.23), 0.546 μg/mL (0.275 to 1.08) and 0.391 μg/mL (0.197 to 0.78) for Fat 13, Fat 37, and Fat 41, respectively. The corresponding extrapolated mean maximum percentages of lysis obtained were 63.2% (43.6 to 82.8), 72.4% (49.7 to 95.0), and 73.7% (51.0 to 96.3) for Fat 13, Fat 37, and Fat 41, respectively. The maximum percentage of lysis values or EC50 values did not significantly differ. The control isotype-matched antibody, 2G6, had no significant effects to promote adipocyte lysis when used at concentrations of up to 100 μg/mL (Figure 3).
Combinations of the monoclonal anti-adipocyte antibodies also produced concentration-dependent lysis of adipocytes. The calculated EC50 values (with 95% CI) were 1.231 μg/mL (0.680 to 2.23), 0.657 μg/mL (0.331 to 1.30), and 0.974 μg/mL (0.490 to 1.93) for the combinations Fat 13 + Fat 41, Fat 37 + Fat 41, and Fat 13 + Fat 37 + Fat 41, respectively. The maximum percentage of lysis obtained with the antibodies were 64.4% (44.8 to 84.0), 76.1% (53.5 to 98.7), and 67.0% (44.4 to 89.7) for Fat 13 + Fat 41, Fat 37 + Fat 41, and Fat 13 + Fat 37 + Fat 41, respectively. None of the combination maximum percentage of lysis or EC50 values was significantly different from each other or to the individual monoclonal antibodies.
Effect of Monoclonal Anti-Adipocyte Antibodies on Serum Complement Levels In Vivo
IP administration of individual monoclonal antibodies produced quantitatively similar dose- and time-dependent reductions in serum complement (Table 1). For example, Fat 41 at 2 mg/kg IP caused a 20% reduction in complement at 6 hours that had recovered to normal levels by 24 hours after dosing. At higher doses (5 and 10 mg/kg IP), Fat 41 caused 100% reduction of serum complement 6 hours after dosing. Serum complement levels recovered more quickly with the lower dose but were still reduced by 50% and by 96% 48 hours after dosing with 5 and 10 mg/kg IP, respectively. Serum complement levels had recovered to normal values by 120 hours after dosing. The control isotype matched antibody 2G6 (2, 5, and 10 mg/kg IP; data not shown) had no significant effect on serum complement levels.
Table 1. Effect of anti-adipocyte antibodies on serum complement levels in Wistar rats after injection (individual and total combination dose)*
Values (mean ± SD, n = 3 to 4)are percentage of the 0-hour values.
119.4 ± 8.9
112.6 ± 12.0
124.6 ± 15.3
96.45 ± 1.4
Fat 13, 10 mg/kg intraperitoneal (IP)
2.0 ± 3.0
0.0 ± 0.0
11.0 ± 17.0
107.0 ± 13
Fat 13, 5 mg/kg IP
3.0 ± 5
25 ± 15
61 ± 41
104 ± 20
Fat 13, 2 mg/kg IP
77 ± 36
97 ± 15
100 ± 18
108 ± 26
Fat 37, 10 mg/kg IP
10.7 ± 9.6
25.6 ± 31.0
45.1 ± 41.6
97.4 ± 23.2
Fat 41, 10 mg/kg IP
1.7 ± 2.9
0.0 ± 0.0
11.4 ± 16.7
106.6 ± 23.4
Fat 13+ 37, 10 mg/kg IP
0.0 ± 0.0
0.0 ± 0.0
19.9 ± 19.0
109.9 ± 17.5
Fat 13+ 41, 10 mg/kg IP
27.5 ± 47.4
1.1 ± 1.9
6.4 ± 11.1
101.4 ± 11.3
Fat 37+ 41, 10 mg/kg IP
26.9 ± 6.4
27.8 ± 5.7
40.1 ± 3.1
105.2 ± 8.0
Fat 13+ 37+ 41, 10 mg/kg IP
26.9 ± 6.4
27.8 ± 5.7
40.1 ± 3.1
105.2 ± 8.0
Combinations of monoclonal antibodies demonstrated very similar dose- and time-dependent reductions in serum complement as that observed for the individual monoclonal antibodies. No synergistic effects were noted.
Phage display technology was used to produce human antibodies that bound selectively to adipocyte plasma membranes (14). To investigate potential effector functions of these antibodies, we have studied their ability to induce complement-mediated lysis of rat epididymal adipocytes in vitro and also to reduce complement levels in vivo in the rat. For this study three anti-adipocyte scFvs, all of which bound to human and rat adipocytes, were cloned into a rat IgG2b backbone and were expressed as rat/human chimeric antibodies. These antibodies were then used directly in studies using rat adipocytes.
The relatively low spontaneous lysis of adipocytes (∼20%) during the 2-hour incubation suggests that the isolated adipocyte preparation was sufficiently stable over this time-span for lysis experiments.
There were clear concentration- and time-dependent effects of the monoclonal anti-adipocyte antibodies (both singly and in combination) to induce cell lysis in vitro, whereas an isotype-matched control antibody (2G6) was without significant effect, ruling out nonspecific lysis as a mechanism.
Complement-dependent lysis of porcine adipocytes using monoclonal antibodies has been observed previously (11, 12). Although the earlier study (11) had indicated low and variable lysis of adipocytes with individual monoclonal antibodies and that multiple antibodies (2 to 4) were more effective, a later study demonstrated lysis of porcine adipocytes with a single monoclonal antibody, although this was not concentration-dependent (12). Although there is some debate on the effectiveness of single monoclonal antibodies to activate complement, we have clearly shown in this study that several individual monoclonal antibodies demonstrate impressive concentration-dependent complement-mediated cell lysis.
Although the specific adipocyte antigen or antigens have not been identified, the efficiency of adipocyte lysis by single monoclonal antibodies would indicate that the antigen is either sufficiently abundant or colocated to allow antibody cross linking and activation of the membrane attack complex. The maximum percentage of lysis observed with the monoclonal antibodies was between 70% and 75% compared with detergent-mediated (Triton-X) adipocyte lysis. The less than complete cell lysis obtained with the anti-adipocyte antibodies may indicate that there is a small population of adipocytes (10% to 20%) resistant to complement induced lysis or may not possess the same antigens.
Intraperitoneal dosing of monoclonal adipocyte antibodies either individually or in combination was well-tolerated and resulted in significant dose- and time-dependent reductions in serum complement. The antibodies were extremely effective in activating the complement; in almost all cases complement levels were reduced to near zero levels over a 24-hour period. Serum complement levels recovered to normal levels within 5 days. No synergistic effect was noticed when two or more monoclonal antibodies were injected together. Although the antibodies caused adipocyte lysis in vitro, it cannot be assumed that the reduction of plasma complement in vivo is solely due to exclusive action on adipocytes. However, given the selectivity of the antibodies toward adipocyte cell membranes, it is considered that the reduction in plasma complement is likely to be primarily due to the binding to adipocytes.
In summary, we have described for the first time the production and functional effects of human anti-adipocyte monoclonal antibodies raised against human white adipocyte cell membranes. In vitro studies demonstrated that singly or in combination, the monoclonal antibodies caused complement-dependent lysis of rat adipocytes in a concentration- and time-dependent manner. There were no significant differences in the potency or maximum percentage of lysis of the antibodies when tested alone or in combination. An isotype-matched antibody produced no significant lysis. Furthermore, when dosed intraperitoneally to rats, these monoclonal antibodies (both singly or in combination) effected a dramatic time- and dose-dependent reduction of serum complement.
The fact that these antibodies lyse adipocytes in vitro and activate complement in vivo clearly demonstrates their potential utility in controlling body fat mass. Further studies are aimed at assessing this possibility in diet-induced obese rat models.
This work was jointly funded by Knoll Limited, UK, and Cambridge Antibody Technology, UK.