Of Myeloma Proteins

[CANCER RESEARCH 28, 1351-1353,July 1968] The "Abnormality" of Myeloma Proteins

Henry G. Kunkel The Rockefeller University, New York, New York

SUMMARY is antibodies (9). These were not special protein products of malignant cells, but rather individual proteins which reflected The long-known phenomenon of individual antigenic speci the differences between individual antibodies. The principle has ficity of myeloma proteins, which suggested unique abnormali finally become accepted that the diversity of myeloma proteins ties, is now readily explainable on the basis of the homogeneity reflects the diversity of antibodies, and a very exciting area of of these proteins. Isolated antibodies of similar homogeneity biochemistry has developed involving determination of the have been obtained which show the same individual specificity. amino acid sequence of these useful tools which are providing All currently available criteria, including a large number of the answer to the chemistry of antibodies. Thus far, every piece genetic markers, indicate the extreme similarity of myeloma of information gathered from myeloma proteins has proven di proteins to individual normal y-globulins and individual anti rectly applicable to normal y-globulin and antibodies, although bodies. Evidence is accumulating that they may actually repre it is important to continue to be alert to possible differences. sent antibodies for which in most instances the antigen is unknown. SUBGROUPS AND GENETIC TYPES: MYELOMA The major protein defect thus far found for the malignant INCIDENCE VERSUS MOLECULAR INCIDENCE plasma cell involves asynchronous polypeptide chain synthesis. Decreased heavy chain synthesis is frequently observed, leading The delineation of the H and L chain structure of y-globulin, to an excess of uncombined light chains which is manifest as as well as the subgroups and genetic types, has resulted primar Bence Jones proteinuria. Light chain synthesis is only rarely ily from a study of myeloma proteins. The two types of L affected. chains, kappa and lambda, were discovered through the study of the natural L chains of myeloma proteins, the Bence Jones proteins. Chart 1 summarizes the major types of yG-globulin INTRODUCTION with respect to subgroups and genetic types for both the L and Initial studies of myeloma proteins from the antigenic stand H chains. Each myeloma protein consists of a single subgroup point lead most investigators to consider these proteins as ab and a single genetic type for each of the chains. For example, normal products of plasma cells or "paraproteins," as they were the commonest myeloma protein has kappa L chains of Inv called. Antisera were readily prepared in rabbits which gave (b) type and yGl H chains of Gm (fy) type. strong reactions with the myeloma protein used for immuniza The incidence of myeloma proteins of each subgroup and tion even after large absorptions with normal y-globulin or each genetic type obtained from studies of a large number of normal serum. Fig. 1 illustrates this individual specific reaction different patients closely parallels the molecular incidence of which was so impressive to the early workers and remains very such characteristics in the normal y-globulin of a single indi impressive to interested workers today (6). Myeloma protein vidual (4, 5, 11). In the study of 195 yG myeloma proteins, Ci which was used for immunization reacts much more strongly 67% contained kappa L chains and 33% contained lambda with this antiserum and spurs over five other myeloma proteins. chains. Careful quantitation of the percentage of kappa and In fact, when this antiserum was studied with 120 heterologous lambda molecules in normal y-globulin by the use of labeled proteins, none approached the specificity of Ci. proteins indicates percentages of 66 and 34 respectively. An Gradually over the past few years it has become apparent other example concerns the incidence of yG3 myeloma proteins that the basis for this remarkable individual specificity resides which has been studied recently. Eight percent of all yG pro in the properties of the normal products of plasma cells, that teins are of this subgroup. Quantitation of the yG3 level in

L Chain H Chain

Lambda JTGl Ã¯Gi /G3 JTG4 /\ /\ /\ Inv a Inv b Gm az Gm yf Gm n Gm n~ Gm b Gm g

Chart 1. Known differences between myeloma proteins with regard to subgroup of their H and L chains and genetic markers.

JULY 1968 1351

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1968 American Association for Cancer Research. Henry G. Kunkel normal sera by radial diffusion studies shows a mean value of lysin (20) and anti-dinitrophenyl (DNP) (3), have been 7%, very close to the myeloma incidence figures (19). found in clearly invasive situations. The anti-DNP activity is The incidence of a specific genetic type of myeloma protein of particular interest because this was studied by specialized also closely approximates that expected from studies of gene methods which gave all the criteria of known DNP antibodies. frequencies in normal populations and subgroup levels (10, 12, 13). Thus, for a given population, the frequency with which a ANTIBODIES RESEMBLING MYELOMA given genetic type of myeloma might be expected is defined as PROTEINS IN HOMOGENEITY follows: Myeloma Frequency = Gene Frequency X Molecular Frequency of Subgroup. These observations strongly suggest Although the vast majority of antibodies to the usual antigens that a random process is involved in the selection of the malig are extremely heterogeneous, a few, particularly to carbohy nant cell line. No deviation in genetic markers from that ex drate antigens, show surprising homogeneity. An anti-levan pected from the phenotype of the individual has been observed antibody produced in response to the injection of levan has in a study of several hundred myeloma proteins. It is apparent been studied in considerable detail (IS) and has all the proper that no unique coding is involved in the synthesis of these pro ties of a myeloma protein except that it is present in relatively tein products. low concentration. It consists entirely of kappa light chains Studies of the genetic markers in myeloma proteins also indi and only of yG2 heavy chains; it shows sharp banding of light cated that in heterozygous individuals the myeloma cells pro chains in gel electrophoresis after reduction and alkylation; duced only the protein product of one of the two allelic genes individually specific antibodies are readily produced in rabbits; instead of both products as in most other cells (7, 10, 12). Indi a defined mobility in the y-globulin spectrum is readily demon viduals who were Gm (az) X Gm (yf) with respect to their strated. Other human antibodies to carbohydrates show similar normal â€¢y-globulinproduced only myeloma proteins of one homogeneity. In rabbit antibodies to cell wall, carbohydrates genetic type. This allelic exclusion phenomenon was at first also give rise to homogeneous antibody populations (15). Rab thought to be specific for myeloma. However, evidence was bit anti-haptene antibodies are usually extremely heterogeneous, obtained from human antibody studies indicating that in many but recently a monoclonal type anti-benzoate antibody has instances these also exhibited such properties. This is more been produced (14). All these clearly defined antibodies are clearly evident from fluorescent antibody studies of single cells difficult to distinguish from myeloma proteins by chemical, in rabbits (16). Only one product is found per cell in heter physical, and antigenic methods of analysis. ozygous individuals. Thus another unusual feature of myeloma proteins was found to be characteristic of antibodies. THE PROTEIN DEFECT OF MYELOMA "ANTIBODY" ACTIVITY OF MYELOMA PROTEINS With all these similarities between myeloma proteins and antibodies, is there any defect apparent in these malignant cells AND WALDENSTRÃ–M MACROGLOBULINS in terms of their protein product? Table 2 shows a classification An increasing body of evidence is accumulating indicating of different myeloma cases compared to the normal situation that myeloma proteins may actually be antibodies. Antibody- in terms of the synthesis of light and heavy chains and in terms like activity has long been known for WaldenstrÃ¶m macro- of the protein products produced. Recent work has demon globulins (8), but similar findings for the myeloma proteins, strated some excess of light chain synthesis in the normal the product of more malignant cells, has been much more re process (2, 17), with excretion of free light chains in the urine. cent (20). Table 1 lists some of the activities associated with The current concept of the ribosomal synthesis of y-globulin monoclonal bands from patients with macroglobulinemia and is that the light chains are formed first in slight excess, and from patients with multiple myeloma. Not all of these activities these aid in removal of heavy chains from the heavy chain have been clearly associated with invasive types of malignant ribosomes. An analogous situation appears to exist for many change in the cells giving rise to the sharp bands. This dis myeloma cells as indicated for myeloma 1. However, in the tinction is frequently difficult, and a benign form of monoclonal case of myeloma types 2 and 3, a marked excess of light chains hyperglobulinemia is widely prevalent which appears to be asso are produced which is manifest by the appearance of Bence ciated relatively frequently with biologic activity of the pro Jones proteins, the equivalent of free light chains, in the urine. tein peak. However, some of the activities, namely anti-strepto- In fact, in myeloma 3 there is no evidence of any heavy chain synthesis. Occasionally multiple myeloma is seen without syn Table 1 thesis of either chain as exemplified by myeloma 4. The figures given in Table 2 are approximate, as obtained 1. Cold agglutinins 2. Anti-7-globulins from the literature and from our personal experience. More 3. "Old cell" agglutinin than one-half of the patients with multiple myeloma show a 4. Anti-cytoplasm defect in chain synthesis, and this primarily involves the heavy 5. Anti-lipoproteins chains, although both chains may rarely be involved. In a 6. Anti-streptolysin given individual, the defect in chain synthesis remains con 7. Anti-staphylolysin stant with time, and the serum level of myeloma protein in 8. Anti-dinitrophenyl relation to Bence Jones protein excretion shows little variation "Antibody" activities in myeloma proteins and WaldenstrÃ¶m that cannot be explained on such secondary factors as the macroglobulins. status of renal function.

1352 CANCER RESEARCH VOL. 28

2ConditionNormal Table synthesisL product7-Globulin chains > H chains + trace L chains Myeloma 1 (48%) L chains > H chains Myeloma protein -f- trace L chains (30%)MyelomaMyeloma 2 L chains > H chains Myeloma protein -f Bence Jones protein 3 (20%) L chainsOo Bence Jones protein Myeloma 4 (2%)Chain oProtein 0 Four types of myeloma classified according to differences in the synthesis of H and L chains of 7-globulin. The normal situation is shown for comparison.

REFERENCES 12. Martensson, L. Genes and Immunoglobulines. Vox Sanguinis, //: 521-545, 1966. 1. Allen, J. C., Kunkel, H. G., and Rabat, E. A. Studies on 13. Natvig, J. B., Kunkel, H. G., and Gcdde-Dahl, Jr. T. Genetic Human Antibodies. II. Distribution of Genetic Factors. J. Studies of the Heavy Chain Subgroups of -yG Globulin. Re Exptl. Med., 119: 453-465, 1964. combination between the Closely Linked Cistrons. In: Johan 2. Askonas, B. A., and Williamson, A. R. Balanced Heavy and Killander (ed.), Nobel Symposium 3, pp. 313-328. Stockholm: Light Chain Synthesis in Immune Tissue and Bisulphide Bond Formation in IgG Assembly. In: Johan Killander (ed.), Nobel Almqvist and Wiksell, 1967. Symposium 3, pp. 369-383. Stockholm: Almqvist and Wiksell, 14. Nisonoff, A., Zappacosta, S., and Jureziz, R. Properties of a Crystallized Rabbit Anti-p-az-benzoate Antibody. Cold Spring 1967. 3. Eisen, H. N., Little, J. R., Osterland, C. K., and Simms, E. S. Harbor Symp. Quant. Biol., SS: 89, 1967. A Myeloma Protein with Antibody Activity. Cold Spring 15. Osterland, C. K., Miller, E. J., Karakawa, W. W., and Krause, Harbor Symp. Quant. Biol., 32: 75-81, 1967. R. M. Characteristics of Streptococcal Group-specific Anti 4. Fahey, J. L. Two Types of 6.6S y-Globulins, /32A-Globulins and body Isolated from Hyperimmune Rabbits. J. Exptl. Med., 18S 7-Macroglobulins in Normal Serum and y-Microglobulins 123: 599-614, 1966. in Normal Urine. J. Immunol., 91: 438-447, 1963. 16. Pernis, B., Chiappino, G., Kelus, A. S., and Gell, P. G. H. 5. Grey, H. M., and Kunkel, H. G. H Chain Subgroups of Mye Cellular Localization of Immunoglobulins with Different Allo- loma Proteins and Normal 7S y-Globulin. J. Exptl. Med., ISO: typic Specificities in Rabbit Lymphoid Tissues. J. Exptl. Med., 253-266, 1964. 128: 853-876, 1965. 6. Grey, H. M., Mannik, M., and Kunkel, H. G. Individual 17. Scharff, M. D. The Assembly of Gamma Globulin in Relation Antigenic Specificity of Myeloma Proteins. Characteristics and to its Synthesis and Secretion. In: Johan Killander (ed.), Localization to Subunits. J. Exptl. Med., 1Â®1:561-575, 1965. Nobel Symposium 3, pp. 385-398. Stockholm: Almqvist and 7. Harboe, M., Osterland, C. K., Mannik, M., and Kunkel, H. G. Wiksell, 1967. Genetic Characters of Human y-Globulins in Myeloma Pro 18. Yount, W. J., Dorner, M. M., Kunkel, H. G., and Kabat, E. A. teins. J. Exptl. Med., 118: 719-738, 1962. Studies on Human Antibodies. VI. Selective Variations in 8. Kritzman, J., Kunkel, H. G., McCarthy, J., and Mellors, R. C. Subgroup Composition and Genetic Markers. J. Exptl. Med., Studies of a Waldenstrom-type Macroglobulin with Rheuma W: 633-646, 1968. toid Factor Properties. J. Lab. Clin. Med., 57: 905-917, 1961. 19. Yount, W. J., Kunkel, H. G., and Litwin, S. D. Studies of the 9. Kunkel, H. G. Myeloma Proteins and Antibodies. Harvey Lec tures, Series 59, pp. 219-242. New York: Academic Press, 1965. Vi (72c) Subgroup of 7-Globulin. A Relationship between Con 10. Kunkel, H. G, Allen, J. C., and Grey, H. M. Genetic Charac centration and Genetic Type among Normal Individuals. J. ters and the Polypeptide Chains of Various Types of Gamma- Exptl. Med., 126: 177-190, 1967. globulin. Cold Spring Harbor Symp. Quant. Biol., SO: 443- 20. Zettorvall, O. Some Properties of Human G Myeloma Globu 447, 1964. lins with Antibody Activity. In: Johan Killander (ed.), Nobel 11. Mannik, M., and Kunkel, H. G. Two Major Types of Normal Symposium 3, pp. 349-357. Stockholm: Almqvist and Wiksell, 7S 7-globulin. J. Exptl. Med., 117: 213-230, 1963. 1967.

Fig. 1. The individual antigenic specificity of myeloma protein Ci. Strong spurs are seen for protein Ci over heterologous mye loma proteins in the presence of anti-Ci antiserum. The subsi diary spurs represent subgroup differences (5).

=Anti-Ci

JULY 196S 1353

Henry G. Kunkel

Cancer Res 1968;28:1351-1353.

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