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Human myeloma IgG half-molecules. Catabolism and biological properties. H L Spiegelberg J Clin Invest. 1975;56(3):588-594. https://doi.org/10.1172/JCI108128. Research Article A human IgG1 myeloma protein that has a delection in the third constant domain of the heavy chain (Cgamma3) and forms two-chain half-molecules was studied for its in vivo turnover and its ability to fix C1q and hemolytic complement, to bind to human lymphocytes, neutrophils, and monocytes, and to induce a passive cutaneous reaction in guinea pigs. In both man and monkeys, the half-molecule was rapidly catabolized and in part excreted into the urine. The half-life in man was 4.3 days and the fractional turnover 165% per day; 7.6% of the intravascular pool was excreted into the urine per day. Although the 7S four-chain myeloma protein could not be obtained in a pure form, the elimination from the serum of a partially purified preparation suggested that it was also rapidly catabolized. The unaggregated half-molecule neither formed complexes with C1q, cound to human lymphocytes, neutrophils, and monocytes, nor elicited a reverse passive cutaneous reaction in guinea pigs. In contrast, the aggregated half-molecule fixed hemolytic complement and bound to the human white cells similarly to an intact IgG1 myeloma protein. In order to explain the biological activities of this half- moleculr, it is postulated that IgG1 may have several (at least two) submolecular sites for a given biological activity that are localized on both the Cgamma2 and Cgamma3 domains. Proteins having both sites […] Find the latest version: https://jci.me/108128/pdf Human Myeloma IgG Half-Molecules CATABOLISM AND BIOLOGICAL PROPERTIES HANs L. SPIEGELBERG From the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California 92037 A B S T R A C T A human IgG1 myeloma protein that first detected in the serum of colostrum-deprived pig- has a deletion in the third constant domain of the heavy lets (1, 2), and later IgA half-molecules were found in chain (CY3) and forms two-chain half-molecules was the urine of mice bearing certain mineral oil-induced studied for its in vivo turnover and its ability to fix IgA plasmacytomas (3). Hobbs and Jacobs reported Clq and hemolytic complement, to bind to human the first patient with a plasmacytoma who produced lymphocytes, neutrophils, and monocytes, and to in- half-molecules (4), and since then four more such pa- duce a passive cutaneous reaction in guinea pigs. In tients have been detected (5-7). The half-molecules pro- both man and monkeys, the half-molecule was rapidly duction does not appear to be associated with a distinct catabolized and in part excreted into the urine. The clinical syndrome since three patients had extramedul- half-life in man was 4.3 days and the fractional turn- lary plasmacytomas (5), one plasma cell leukemia (6), over 165% per day; 7.6% of the intravascular pool was and one classical multiple myeloma (M. Seligmann, excreted into the urine per day. Although the 7S four- personal communication). Nevertheless, all these pa- chain myeloma protein could not be obtained in a pure tients had in common that they excreted large amounts form, the elimination from the serum of a partially of half-molecules into their urine. Detailed structural purified preparation suggested that it was also rapidly analysis of the paraprotein of our patient (8) revealed catabolized. The unaggregated half-molecule neither that it lacked the noncovalent interactions characteristic formed complexes with Clq, bound to human lympho- for the Fc portion of the 'v-chain and that it had a de- cytes, neutrophils, and monocytes, nor elicited a reverse letion in the third constant domain (Cy3)' of the heavy passive cutaneous reaction in guinea pigs. In contrast, chain. The size of the deletion was 5,000-8,000 daltons. the aggregated half-molecule fixed hemolytic comple- It probably involved the intrachain disulfide loop of the ment and bound to the human white cells similarly to an C'v3 domain but not the carboxyterminal structure of the intact IgG1 myeloma protein. In order to explain the gamma chain. The half-molecule expressed the genetic biological activities of this half-molecule, it is postu- marker Gm (f) which is localized on the CO1 domain lated that IgG1 may have several (at least two) sub- but lacked the corresponding marker Gm(non-a) as well molecular sites for a given biological activity that are as another marker on the C'Y3 domain. The "hinge" localized on both the Cy2 and Cy3 domains. Proteins peptide containing the inter-heavy-heavy chain disulfide having both sites would be capable of binding to Clq bonds appeared intact and its amino acid composition and Fc cell receptors in unaggregated form whereas was identical to the corresponding peptide of IgG1 pro- proteins having a site on only one domain, such as the teins. This explained why the patient formed also a half-molecule, must be aggregated in order to obtain small amount of four-chain 7S type myeloma protein. this binding. In order to investigate the biological properties of this unusual myeloma protein, we measured its rates of elimi- nation from the circulation and excretion into the urine, INTRODUCTION its capacity to fix complement, and its ability to bind Immunoglobulin half-molecules consisting of one heavy to the Fc receptors of human white cells and guinea and one light chain are rare. IgG half-molecules were pig mast cells. This is Scripps Clinic and Research Foundation Publi- 1 Abbreviations used in this paper: C,2, C,3, the second cation No. 953. and third constant domains, respectively, of the heavy Received for publication 6 March 1975 and in revised chain; RPCA, reverse passive cutaneous anaphylaxis; TCA, form 28 April 1975. trichloroacetic acid. 588 The Journal of Clinical Investigation Volume 56 September 1975 588-594 METHODS The plasma half-lives and the percent intra- and extra- vascular distribution was calculated from the semilogarith- Isolation of proteins. The myeloma protein of patient K. mic plot of the plasma elimination curve. The total body N. was isolated from his plasma and urine by a combination half-life in man was calculated from the slope of the semi- of DEAE-cellulose chromatography and Sephadex G-200 logarithmic plot of the radioactivity retained in the body gel filtration (8). The half-molecule was obtained from (total injected radioactivity minus accumulated radioactivity either serum or urine in a relatively pure form whereas the excreted into the urine). The whole body elimination could 7S IgG serum fraction contained only about 35% myeloma not be determined in this manner in monkeys because com- protein, the remaining portion being normal IgG (8). IgG1 plete daily urine collections could not be made. The frac- (Ma) and IgG2 (Do) myeloma proteins and normal human tional turnover rate was calculated from the radioactivity IgG were isolated by DEAE-cellulose chromatography excreted into the urine per day expressed as percent of the using a 0.01 M phosphate buffer, pH 8.0. An IgM macro- average intravascular pool in the same 24-h period (15). globulin (Vi) was isolated by a combination of euglobulin In order to determine the molecular size of the protein precipitation, Pevikon block electrophoresis, and Sephadex precipitable with TCA in the urine, the urine of a monkey G-200 gel filtration. The Fab fragment was isolated from injected with 'I-labeled half-molecule was collected during either normal IgG or the half-molecule after digestion the first 6 h after injection. 2 ml monkey serum from the with papain for 18 h (8). same animal was added as protein carrier; the mixture was Aggregation of proteins. Three methods were used to then dialyzed overnight against 0.001 M phosphate buffer, aggregate the proteins for the different test methods. For pH 8, containing toluene as a preservative and lyophilized. complment fixation, the proteins were aggregated either by The lyophilized material was dissolved in saline and applied addition of 200 pLg/5 mg of bis-diazotized benzidine (9) or to a Sephadex G-200 column. by heating at 630C for 5-30 min until the solution became Complement fixation. The ability of the proteins to bind slightly turbid. For the experiments designed to measure the complement component Clq was determined by analyti- the binding to different cell types, the proteins were aggre- cal ultracentrifugation.2 The immunoglobulin preparation gated with a F(ab')2 preparation of a rabbit anti-human was first cleared of aggregates by centrifugation at 100,000 Fab fragment antiserum (10). The F(ab')2 anti-Fab anti- g for 60 min in a swinging bucket rotor of a Beckman serum precipitated immunoglobulins of all classes and light model L preparative ultracentrifuge (Beckman Instru- chain types when tested by double gel diffusion analysis. ments, Inc., Spinco Div., Palo Alto, Calif.) and then mixed Radioiodination. All proteins were radioiodinated with with purified Clq kindly provided by Dr. H. J. Muller- either 'I or 'I by using modifications of the chloramine Eberhard. The final protein concentrations were 3 mg T procedure. For turnover studies, protein in 5-mg aliquots myeloma protein preparation and 1 mg Clq. The mixture was dissolved in 1 ml 0.05 M phosphate buffer, pH 7.0, and was then centrifuged at 52,000 rpm in a Beckman model E was labeled with 10 pg of chloramine T/mg by incubating analytical ultracentrifuge equipped with schlieren optics. for 5 min (11). The uptake of radioactivity varied between The sedimentation rates were calculated by standard pro- 35 and 60% and the specific radioactivity was about 0.1 cedures, corrected for temperature and solvent but not to pCi/pg.

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