Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 x 10(8) paraprotein-antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely

Braun

Colwell

Dellagi

Dighiero

Friedman

Jack

Kilgore

Konrad

Kosmas

Kritzman

Mills

Prabhala

Redmon

Sahin

Sahota

Seligmann

Suarez

Trimarchi

Preuss, Klaus-Dieter

Held, Gerhard

Kubuschok, Boris

Hung, Chun-Zhu

Malatsidze, Natalia

Wagner, Mathias

Pfreundschuh, Michael

Scientific documents from the Saarland University,

SHORT REPORT
Identiﬁcation of antigenic targets of paraproteins by expression cloning does not
support a causal role of chronic antigenic stimulation in the pathogenesis of
multiple myeloma and MGUS
Klaus-Dieter Preuss1, Gerhard Held1, Boris Kubuschok1, Chun-Zhu Hung1, Natalia Malatsidze1, Mathias Wagner2
and Michael Pfreundschuh1*
1Klinik f€ur Innere Medizin I, Saarland University Medical School, Homburg/Saar, Germany
2Institute of Pathology, Saarland University Medical School, Homburg/Saar, Germany
Antigenic targets of monoclonal gammopathy of undetermined
signiﬁcance (MGUS) and multiple myeloma (MM) paraproteins
have been suggested to play an important role as growth stimula-
tors in the pathogenesis of these neoplasms. To identify such
targets, we screened cDNA libraries from human testis, lung and
breast cancer, bovine and porcine muscle and wheat germ for
reactivity with paraproteins in the sera from 115 patients with
MGUS and MM. Of >6 3 108 paraprotein–antigen interactions
screened, an IgA paraprotein from a female patient bound to
sperm-speciﬁc cylicin-2, and 3 IgG paraproteins bound to tripep-
tidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-pro-
tein-2 (IGFBP-2) and porcine kinesin. Speciﬁcity was conﬁrmed
by reverse Western blots using recombinant antigens. The broad
spectrum of auto-, allo- and heteroantigens as targets of human
paraproteins in patients without signs of chronic antigenic stimu-
lation renders a causal role of the antigenic stimulus in the patho-
genesis of MGUS and MM unlikely.
' 2007 Wiley-Liss, Inc.
Key words: multiple myeloma; monoclonal gammopathy of un-
determined signiﬁcance; antigenic stimulation; antigen identiﬁcation
The identiﬁcation of the antigenic stimuli of B-cell neoplasms
might have considerable impact, because a causal relationship
between these neoplasms and antigenic stimulation has been sug-
gested.1,2 To date, antigenic targets of paraproteins were discovered
accidentally due to clinical symptoms caused by the paraprotein
(e.g., chronic cold agglutinin disease or cryoglobulinemia3 or bleed-
ing disorder4), because of interference of the paraprotein with labo-
ratory tests ordered for the clinical work-up of the patient (e.g.,
HIV-1 p24 antigen in an HIV-infected patient with myeloma5) or by
screening paraproteins against predeﬁned antigens (e.g., anti-strepto-
lysin, anti-DNA, anti-IgG3). Systematic searches covering a broad
spectrum of potential antigens have not been reported to date.
SEREX (serological identiﬁcation of antigens by expression
cloning) allows for the systematic screening of putative antibody–
antigen interactions, even if neither the antigen nor the antibody
are known.6 We therefore used SEREX for the identiﬁcation of
antigenic targets of paraproteins in expressed cDNA libraries
derived from human, animal and plant tissues.
Patients, material and methods
Patients
The study was approved by the local ethical review board
(‘‘Ethikkommission der €Arztekammer des Saarlandes’’) and con-
ducted according to the Declaration of Helsinki. Recombinant DNA
work was performed with permission and according to the regulations
of local authorities (Government of Saarland). Human materials
were obtained during routine diagnostic or therapeutic procedures
after obtaining written informed consent and stored at280C.
SEREX
cDNA expression libraries were established as described.6 A
wheat germ cDNA library was obtained from Sylvia de Pater
(Utrecht, Netherlands). The phage assay6 was used for the screen-
ing of sera from 115 patients with (multiple myeloma) MM or
(monoclonal gammopathy of undetermined signiﬁcance) MGUS
at a dilution of 1:1,000,000. Positive clones were sub-cloned to
monoclonality and the nucleotide sequence of cDNA inserts was
determined as described before.6 Antibodies against the identiﬁed
antigens were probed in 1:100 diluted control sera.
Reverse western blot
Patient’s serum was subjected to electrophoresis and transferred
to nitrocellulose membranes. Membranes were incubated with
recombinant GST-TPP-2 fusion protein that had been produced by
cloning TPP2 into a pGEX4T1 vector according to the manufac-
turer’s (Pharmacia, Uppsala, Sweden) recommendations. Recombi-
nant GST-SCP-1 fusion protein served as negative and a mouse
antibody against the respective light chain (Dianova, Hamburg,
Germany) as positive control.
Immmunohistochemistry
Deparafﬁnized bone marrow sections were incubated with
recombinant GST-TPP-2, followed by mouse anti-GST antibody
and visualization by the APAAP technique according to the manu-
facturer’s (Dako-Cytomation, Glostrup, Denmark) recommendations.
Results and discussion
Identiﬁcation of paraprotein-binding clones
At least 1 3 106 clones from each cDNA expression library
derived from human testis, nonsmall cell lung cancer, breast can-
cer, bovine and porcine muscle as well as wheat germ were
screened with 1:1,000,000 diluted sera from 115 patients with
MGUS or MM (55 IgGj, 33 IgGk and 11 IgAj, 9 IgAk, 5 IgGj1k,
1 IgAk 1 IgGk, 1 IgAj 1 IgGj; male: 69, female: 46, median age:
65 years, range: 33–90 years). A testis library was chosen because
a large proportion of the human genome is expressed in testis due
to its genome-wide hypomethylation. Non-small-cell lung and
breast cancer libraries served as sources for putative tumor-associ-
ated, and bovine and porcine muscle (beef and pork), as well as
wheat germ as sources for food-associated antigens.
The screening of >6 3 108 potential paraprotein/antigen inter-
actions revealed 39 positive clones, 1 reacting with an IgA and all
others reacting with IgG paraproteins. The positive clones coded
for cylicin-2 (target of an IgA paraprotein), SOX6, metalloprotei-
nase 17, tripeptidyl-peptidase-II (TPP2), insulin-like growth-factor
Grant sponsor: Deutsche Krebshilfe; Grant number: 10-1811-Pf 5.
*Correspondence to: Innere Medizin I, Universit€at des Saarlandes,
D-66421 Homburg, Germany. Fax:149-6841-162-3101.
E-mail: inmpfr@uniklinikum-saarland.de
Received 23 September 2006; Accepted after revision 7 February 2007
DOI 10.1002/ijc.22686
Published online 20 March 2007 inWiley InterScience (www.interscience.
wiley.com).
Int. J. Cancer: 121, 459–461 (2007)
' 2007 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
binding-protein-2 (IGFBP-2), porcine kinesin and 33 unknown
sequences or proteins with unknown function.
Demonstration of paraprotein-mediated reactivity
Reactivities at high dilutions of the respective paraprotein-con-
taining sera were detected against TPP-2 (1:1010), IGFBP-2
(1:109), porcine kinesin (1:109) and cylicin-2 (1:109, as previously
described7). In contrast, only low-titered reactivities (<1:102)
were observed against the latter 3 antigens, and no reactivity at all
against cylicin-2 in normal male and female sera. The reactivities
being mediated by paraproteins was conﬁrmed by Western blot-
ting with the recombinant GST fusion antigens (Fig. 1). The anti-
gen-binding bands were identical with the bands detected by the
anti-sera used for the demonstration of monoclonality of the para-
proteins by immunoﬁxation. Moreover, recombinant GST-TPP-2
stained plasma cells in the bone marrow of the MGUS patient
with the anti-TPP-2 reactive paraprotein (Fig. 2). All other anti-
gens reacted with MM and MGUS sera at dilutions <1:105, and
antibodies against these antigens were detected in normal sera up
to dilutions of 1:104, making a paraprotein-mediated reactivity
unlikely.
TPP-2, detected in a testis-derived cDNA and IGFBP-2, which
was detected in a lung-cancer derived cDNA, are widely ex-
pressed human autoantigens. Cyclicin-2, which was also detected
in a testis-derived cDNA, is a speciﬁc component of the sperm
head cytoskeleton and as such an alloantigen for the female patient
with the reactive paraprotein.7 Porcine kinesin meets the deﬁnition
of a heteroantigen. Remarkably, despite the high homology
between human, bovine and porcine kinesin, the patient’s parapro-
tein did not cross-react with the recombinant human or bovine
homologue (data not shown). Thus, 2 autoantigens, 1 allo-antigen
and 1 hetero- or food-associated antigen were identiﬁed in this
study as targets of more than 100 paraproteins tested. Notably,
none of the 4 patients had clinical signs of chronic antigenic stim-
ulation or associated morbidities. The female patient with the anti-
bodies against the sperm-head associated cylicin-2 had given birth
to 2 children, and the patient with the anti-porcine kinesin did not
have any symptoms of hypersensitivity reactions against pork or
other porcine products.
Several previous reports have identiﬁed myeloma paraproteins
directed against various infectious agents, including bacteria3 and
the p24 antigen of the human immunodeﬁciency virus-1.5 In
another case, a patient developed a serum M-component with
speciﬁcity for horse a2-macroglobulin 30 years after receiving
passive serotherapy with horse antiserum to tetanus.8 Even though
few of these studies convincingly proved that the observed phe-
nomena were indeed caused by binding of the respective parapro-
tein to its speciﬁc antigenic target (e.g., by ‘‘reverse Western’’
blotting or sequencing of the target antigen as done in this study),
these selective observations are often cited to support a causal
relationship between the development of an MGUS or MM clone
and chronic antigenic stimulation.1–5,9–16 In contrast to these
previous reports, our unbiased systematic study did not only con-
vincingly prove the paraprotein-mediated reactivity against the
identiﬁed antigens, it also revealed a broad spectrum of antigenic
targets of paraproteins. The fact that of >6 3 108 possible para-
protein/target-antigen interactions screened only 4 were identiﬁed
as paraprotein-mediated indicates that the antigenic sources tested
(which included a large proportion of the expressed human ge-
nome as possible sources for autoantigens as well as the major
sources for food allergens) represent only a small part of the entire
antigenic target spectrum of human paraproteins.
We have identiﬁed autoantigens in patients without signs of
chronic antigenic stimulation, and heteroantigens in patients without
FIGURE 1 – Demonstration of the paraprotein-mediated reactivity against identiﬁed antigens. Western blot using recombinant GST-TPP-2.
Left panel: serum from an MGUS patient with an IgGj paraprotein without anti-TPP-2 reactivity in the phage assay; right panel: serum from the
patient with TPP-2 reactive IgGj paraprotein; 1: anti-human-IgG; 2: anti-mouse-IgG; 3: TPP-2-GST fusion protein (500 lg/ml); 4: GST-SCP-1
fusion protein (500 lg/ml).
FIGURE 2 – Immunohistologic demonstration of intracytoplasmic
anti-TPP-2 antibodies using GST-TPP-2. A plasma cell in the bone
marrow of the MGUS patient with the anti-TPP-2 reactive paraprotein
stains positive with recombinant GST-TPP-2. GST-TPP2 stained cells
were conﬁrmed as plasma cells by staining an adjacent section with
the respective anti-light chain antibodies. APAAP stain, Fast Red; ca.
3400 magniﬁcation.
460 PREUSS ET AL.
animal allergies. Thus, this unbiased systematic study lends support
to the view that the role of chronic antigenic stimulation in the
pathogenesis of MGUS and MM has probably been overestimated
in the past. The view that many—if not any—auto-, allo- and hetero-
antigen-reactive B-cell clones can become the random target of ma-
lignant transformation is also supported by the fact that there is no
convincing evidence for the preferential use of V genes in MGUS
and MM,17,18 which contrasts with the selective immunoglobulin V
gene use in other B-cell malignancies, e.g., marginal zone lympho-
mas associated with chronic infection or autoimmunity.19 The
knowledge of the antigenic target structures of paraproteins allows
to address in more detail tumor-host interactions in the presence and
absence of speciﬁc antigens in the respective patients, and to study
more speciﬁcally the role of immunoregulatory deﬁciencies, such as
the recently reported dysfunction of regulatory T cells20 in patients
with MGUS and multiple myeloma.
Acknowledgements
The authors thank Ms. Natalie Fadle, Ms. Maria Kemele, Ms.
Evi Regitz, and Ms. Claudia Schormann for excellent technical as-
sistance.
References
1. Jack HM, Beck-Engeser G, Lee G, Wofsy D, Wabl M. Tumorigenesis
mediated by an antigen receptor. Proc Natl Acad Sci USA 1992;
89:8482–6.
2. Friedman DF, Cho EA, Goldman J, Carmack CE, Besa EC, Hardy RR,
Silberstein LE. The role of clonal selection in the pathogenesis of an
autoreactive human B cell lymphoma. J Exp Med 1991;174:525–37.
3. Seligmann M, Brouet JC. Antibody activity of human myeloma glob-
ulins. Semin Hematol 1973;10:163–77.
4. Colwell NS, Tollefsen DM, Blinder MA. Identiﬁcation of a monoclo-
nal thrombin inhibitor associated with multiple myeloma and a severe
bleeding disorder. Br J Haematol 1997;97:219–26.
5. Konrad RJ, Kricka LJ, Goodman DB, Goldman J, Silberstein LE.
Brief report: myeloma-associated paraprotein directed against the
HIV-1 p24 antigen in an HIV-1-seropositive patient. N Engl J Med
1993;328:1817–19.
6. Sahin U, Tureci O, Schmitt H, Cochlovius B, Johannes T, Schmits R,
Stenner F, Luo G, Schobert I, Pfreundschuh M. Human neoplasms
elicit multiple speciﬁc immune responses in the autologous host. Proc
Natl Acad Sci USA 1995;92:11810–13.
7. Xie X, Schmits R, Renner C, Preuss D, Kubuschok B, Pfreundschuh
M. Systematic search and molecular characterization of the antigenic
targets of myeloma immunoglobulins: a monoclonal IgA from a
female patient targeting sperm-speciﬁc cylicin II. Cancer Immun
2001;1:11.
8. Seligmann M, Sassy C, Chevalier A. A human IgG myeloma protein with
anti-2 macroglobulin antibody activity. J Immunol 1973;110:85–90.
9. Dighiero G, Guilbert B, Fermand JP, Lymberi P, Danon F, Avrameas S.
Thirty-six human monoclonal immunoglobulins with antibody activity
against cytoskeleton proteins, thyroglobulin, and native DNA: immuno-
logic studies and clinical correlations. Blood 1983;62:264–70.
10. Dellagi K, Brouet JC, Perreau J, Paulin D. Human monoclonal IgM
with autoantibody activity against intermediate ﬁlaments. Proc Natl
Acad Sci USA 1982;79:446–50.
11. Braun PE, Frail DE, Latov N. Myelin-associated glycoprotein is the
antigen for a monoclonal IgM in polyneuropathy. J Neurochem
1982;39:1261–5.
12. Kritzman J, Kunkel HG, McCarthy J, Mellors RC. Studies of a
Waldenstrom-type macroglobulin with rheumatoid factor properties.
J Lab Clin Med 1961;57:905–17.
13. Kilgore LL, Patterson BW, Parenti DM, Fisher WR. Immune complex
hyperlipidemia induced by an apolipoprotein-reactive immunoglobu-
lin A paraprotein from a patient with multiple myeloma. Characteriza-
tion of this immunoglobulin. J Clin Invest 1985;76:225–32.
14. Redmon B, Pyzdrowski KL, Elson MK, Kay NE, Dalmasso AP, Nut-
tall FQ. Hypoglycemia due to an insulin-binding monoclonal antibody
in multiple myeloma. N Engl J Med 1992;326:994–8.
15. Mills LE, Brettman LR, Jentoft JE, Viner ED, Bernier GM. Crystal-
locryoglobulinemia resulting from human monoclonal antibodies to
albumin. Ann Intern Med 1983;99:601–4.
16. Trimarchi F, Benvenga S, Fenzi G, Mariotti S, Consolo F. Immuno-
globulin binding of thyroid hormones in a case of Waldenstrom’s
macroglobulinemia. J Clin Endocrinol Metab 1982;54:1045–50.
17. Sahota SS, Leo R, Hamblin TJ, Stevenson FK. Myeloma VL and VH
gene sequences reveal a complementary imprint of antigen selection
in tumor cells. Blood 1997;89:219–26.
18. Kosmas C, Stamatopoulos K, Stavroyianni N, Zoi K, Belessi C, Vin-
iou N, Kollia P, Yataganas X. Origin and diversiﬁcation of the clono-
genic cell in multiple myeloma: lessons from the immunoglobulin
repertoire. Leukemia 2000;14:1718–26.
19. Suarez F, Lortholary O, Hermine O, Lecuit M. Infection-associated
lymphomas derived from marginal zone B cells: a model of antigen-
driven lymphoproliferation. Blood 2006;107:3034–44.
20. Prabhala RH, Neri P, Bae JE, Tassone P, Shammas MA, Allam CK,
Daley JF, Chauhan D, Blanchard E, Thatte HS, Anderson KC, Munshi
NC. Dysfunctional T regulatory cells in multiple myeloma. Blood
2006;107:301–4.
461PARAPROTEIN TARGETS


Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUS

Universaar

Scientific publications of the Saarland University

SHORT REPORTIdentiﬁcation of antigenic targets of paraproteins by expression cloning does notsupport a causal role of chronic antigenic stimulation in the pathogenesis ofmultiple myeloma and MGUSKlaus-Dieter Preuss1, Gerhard Held1, Boris Kubuschok1, Chun-Zhu Hung1, Natalia Malatsidze1, Mathias Wagner2and Michael Pfreundschuh1*1Klinik f€ur Innere Medizin I, Saarland University Medical School, Homburg/Saar, Germany2Institute of Pathology, Saarland University Medical School, Homburg/Saar, GermanyAntigenic targets of monoclonal gammopathy of undeterminedsigniﬁcance (MGUS) and multiple myeloma (MM) paraproteinshave been suggested to play an important role as growth stimula-tors in the pathogenesis of these neoplasms. To identify suchtargets, we screened cDNA libraries from human testis, lung andbreast cancer, bovine and porcine muscle and wheat germ forreactivity with paraproteins in the sera from 115 patients withMGUS and MM. Of >6 3 108 paraprotein–antigen interactionsscreened, an IgA paraprotein from a female patient bound tosperm-speciﬁc cylicin-2, and 3 IgG paraproteins bound to tripep-tidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-pro-tein-2 (IGFBP-2) and porcine kinesin. Speciﬁcity was conﬁrmedby reverse Western blots using recombinant antigens. The broadspectrum of auto-, allo- and heteroantigens as targets of humanparaproteins in patients without signs of chronic antigenic stimu-lation renders a causal role of the antigenic stimulus in the patho-genesis of MGUS and MM unlikely.' 2007 Wiley-Liss, Inc.Key words: multiple myeloma; monoclonal gammopathy of un-determined signiﬁcance; antigenic stimulation; antigen identiﬁcationThe identiﬁcation of the antigenic stimuli of B-cell neoplasmsmight have considerable impact, because a causal relationshipbetween these neoplasms and antigenic stimulation has been sug-gested.1,2 To date, antigenic targets of paraproteins were discoveredaccidentally due to clinical symptoms caused by the paraprotein(e.g., chronic cold agglutinin disease or cryoglobulinemia3 or bleed-ing disorder4), because of interference of the paraprotein with labo-ratory tests ordered for the clinical work-up of the patient (e.g.,HIV-1 p24 antigen in an HIV-infected patient with myeloma5) or byscreening paraproteins against predeﬁned antigens (e.g., anti-strepto-lysin, anti-DNA, anti-IgG3). Systematic searches covering a broadspectrum of potential antigens have not been reported to date.SEREX (serological identiﬁcation of antigens by expressioncloning) allows for the systematic screening of putative antibody–antigen interactions, even if neither the antigen nor the antibodyare known.6 We therefore used SEREX for the identiﬁcation ofantigenic targets of paraproteins in expressed cDNA librariesderived from human, animal and plant tissues.Patients, material and methodsPatientsThe study was approved by the local ethical review board(‘‘Ethikkommission der €Arztekammer des Saarlandes’’) and con-ducted according to the Declaration of Helsinki. Recombinant DNAwork was performed with permission and according to the regulationsof local authorities (Government of Saarland). Human materialswere obtained during routine diagnostic or therapeutic proceduresafter obtaining written informed consent and stored at280C.SEREXcDNA expression libraries were established as described.6 Awheat germ cDNA library was obtained from Sylvia de Pater(Utrecht, Netherlands). The phage assay6 was used for the screen-ing of sera from 115 patients with (multiple myeloma) MM or(monoclonal gammopathy of undetermined signiﬁcance) MGUSat a dilution of 1:1,000,000. Positive clones were sub-cloned tomonoclonality and the nucleotide sequence of cDNA inserts wasdetermined as described before.6 Antibodies against the identiﬁedantigens were probed in 1:100 diluted control sera.Reverse western blotPatient’s serum was subjected to electrophoresis and transferredto nitrocellulose membranes. Membranes were incubated withrecombinant GST-TPP-2 fusion protein that had been produced bycloning TPP2 into a pGEX4T1 vector according to the manufac-turer’s (Pharmacia, Uppsala, Sweden) recommendations. Recombi-nant GST-SCP-1 fusion protein served as negative and a mouseantibody against the respective light chain (Dianova, Hamburg,Germany) as positive control.ImmmunohistochemistryDeparafﬁnized bone marrow sections were incubated withrecombinant GST-TPP-2, followed by mouse anti-GST antibodyand visualization by the APAAP technique according to the manu-facturer’s (Dako-Cytomation, Glostrup, Denmark) recommendations.Results and discussionIdentiﬁcation of paraprotein-binding clonesAt least 1 3 106 clones from each cDNA expression libraryderived from human testis, nonsmall cell lung cancer, breast can-cer, bovine and porcine muscle as well as wheat germ werescreened with 1:1,000,000 diluted sera from 115 patients withMGUS or MM (55 IgGj, 33 IgGk and 11 IgAj, 9 IgAk, 5 IgGj1k,1 IgAk 1 IgGk, 1 IgAj 1 IgGj; male: 69, female: 46, median age:65 years, range: 33–90 years). A testis library was chosen becausea large proportion of the human genome is expressed in testis dueto its genome-wide hypomethylation. Non-small-cell lung andbreast cancer libraries served as sources for putative tumor-associ-ated, and bovine and porcine muscle (beef and pork), as well aswheat germ as sources for food-associated antigens.The screening of >6 3 108 potential paraprotein/antigen inter-actions revealed 39 positive clones, 1 reacting with an IgA and allothers reacting with IgG paraproteins. The positive clones codedfor cylicin-2 (target of an IgA paraprotein), SOX6, metalloprotei-nase 17, tripeptidyl-peptidase-II (TPP2), insulin-like growth-factorGrant sponsor: Deutsche Krebshilfe; Grant number: 10-1811-Pf 5.*Correspondence to: Innere Medizin I, Universit€at des Saarlandes,D-66421 Homburg, Germany. Fax:149-6841-162-3101.E-mail: inmpfr@uniklinikum-saarland.deReceived 23 September 2006; Accepted after revision 7 February 2007DOI 10.1002/ijc.22686Published online 20 March 2007 inWiley InterScience (www.interscience.wiley.com).Int. J. Cancer: 121, 459–461 (2007)' 2007 Wiley-Liss, Inc.Publication of the International Union Against Cancerbinding-protein-2 (IGFBP-2), porcine kinesin and 33 unknownsequences or proteins with unknown function.Demonstration of paraprotein-mediated reactivityReactivities at high dilutions of the respective paraprotein-con-taining sera were detected against TPP-2 (1:1010), IGFBP-2(1:109), porcine kinesin (1:109) and cylicin-2 (1:109, as previouslydescribed7). In contrast, only low-titered reactivities (<1:102)were observed against the latter 3 antigens, and no reactivity at allagainst cylicin-2 in normal male and female sera. The reactivitiesbeing mediated by paraproteins was conﬁrmed by Western blot-ting with the recombinant GST fusion antigens (Fig. 1). The anti-gen-binding bands were identical with the bands detected by theanti-sera used for the demonstration of monoclonality of the para-proteins by immunoﬁxation. Moreover, recombinant GST-TPP-2stained plasma cells in the bone marrow of the MGUS patientwith the anti-TPP-2 reactive paraprotein (Fig. 2). All other anti-gens reacted with MM and MGUS sera at dilutions <1:105, andantibodies against these antigens were detected in normal sera upto dilutions of 1:104, making a paraprotein-mediated reactivityunlikely.TPP-2, detected in a testis-derived cDNA and IGFBP-2, whichwas detected in a lung-cancer derived cDNA, are widely ex-pressed human autoantigens. Cyclicin-2, which was also detectedin a testis-derived cDNA, is a speciﬁc component of the spermhead cytoskeleton and as such an alloantigen for the female patientwith the reactive paraprotein.7 Porcine kinesin meets the deﬁnitionof a heteroantigen. Remarkably, despite the high homologybetween human, bovine and porcine kinesin, the patient’s parapro-tein did not cross-react with the recombinant human or bovinehomologue (data not shown). Thus, 2 autoantigens, 1 allo-antigenand 1 hetero- or food-associated antigen were identiﬁed in thisstudy as targets of more than 100 paraproteins tested. Notably,none of the 4 patients had clinical signs of chronic antigenic stim-ulation or associated morbidities. The female patient with the anti-bodies against the sperm-head associated cylicin-2 had given birthto 2 children, and the patient with the anti-porcine kinesin did nothave any symptoms of hypersensitivity reactions against pork orother porcine products.Several previous reports have identiﬁed myeloma paraproteinsdirected against various infectious agents, including bacteria3 andthe p24 antigen of the human immunodeﬁciency virus-1.5 Inanother case, a patient developed a serum M-component withspeciﬁcity for horse a2-macroglobulin 30 years after receivingpassive serotherapy with horse antiserum to tetanus.8 Even thoughfew of these studies convincingly proved that the observed phe-nomena were indeed caused by binding of the respective parapro-tein to its speciﬁc antigenic target (e.g., by ‘‘reverse Western’’blotting or sequencing of the target antigen as done in this study),these selective observations are often cited to support a causalrelationship between the development of an MGUS or MM cloneand chronic antigenic stimulation.1–5,9–16 In contrast to theseprevious reports, our unbiased systematic study did not only con-vincingly prove the paraprotein-mediated reactivity against theidentiﬁed antigens, it also revealed a broad spectrum of antigenictargets of paraproteins. The fact that of >6 3 108 possible para-protein/target-antigen interactions screened only 4 were identiﬁedas paraprotein-mediated indicates that the antigenic sources tested(which included a large proportion of the expressed human ge-nome as possible sources for autoantigens as well as the majorsources for food allergens) represent only a small part of the entireantigenic target spectrum of human paraproteins.We have identiﬁed autoantigens in patients without signs ofchronic antigenic stimulation, and heteroantigens in patients withoutFIGURE 1 – Demonstration of the paraprotein-mediated reactivity against identiﬁed antigens. Western blot using recombinant GST-TPP-2.Left panel: serum from an MGUS patient with an IgGj paraprotein without anti-TPP-2 reactivity in the phage assay; right panel: serum from thepatient with TPP-2 reactive IgGj paraprotein; 1: anti-human-IgG; 2: anti-mouse-IgG; 3: TPP-2-GST fusion protein (500 lg/ml); 4: GST-SCP-1fusion protein (500 lg/ml).FIGURE 2 – Immunohistologic demonstration of intracytoplasmicanti-TPP-2 antibodies using GST-TPP-2. A plasma cell in the bonemarrow of the MGUS patient with the anti-TPP-2 reactive paraproteinstains positive with recombinant GST-TPP-2. GST-TPP2 stained cellswere conﬁrmed as plasma cells by staining an adjacent section withthe respective anti-light chain antibodies. APAAP stain, Fast Red; ca.3400 magniﬁcation.460 PREUSS ET AL.animal allergies. Thus, this unbiased systematic study lends supportto the view that the role of chronic antigenic stimulation in thepathogenesis of MGUS and MM has probably been overestimatedin the past. The view that many—if not any—auto-, allo- and hetero-antigen-reactive B-cell clones can become the random target of ma-lignant transformation is also supported by the fact that there is noconvincing evidence for the preferential use of V genes in MGUSand MM,17,18 which contrasts with the selective immunoglobulin Vgene use in other B-cell malignancies, e.g., marginal zone lympho-mas associated with chronic infection or autoimmunity.19 Theknowledge of the antigenic target structures of paraproteins allowsto address in more detail tumor-host interactions in the presence andabsence of speciﬁc antigens in the respective patients, and to studymore speciﬁcally the role of immunoregulatory deﬁciencies, such asthe recently reported dysfunction of regulatory T cells20 in patientswith MGUS and multiple myeloma.AcknowledgementsThe authors thank Ms. Natalie Fadle, Ms. Maria Kemele, Ms.Evi Regitz, and Ms. Claudia Schormann for excellent technical as-sistance.References1. 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Myelin-associated glycoprotein is theantigen for a monoclonal IgM in polyneuropathy. J Neurochem1982;39:1261–5.12. Kritzman J, Kunkel HG, McCarthy J, Mellors RC. Studies of aWaldenstrom-type macroglobulin with rheumatoid factor properties.J Lab Clin Med 1961;57:905–17.13. Kilgore LL, Patterson BW, Parenti DM, Fisher WR. Immune complexhyperlipidemia induced by an apolipoprotein-reactive immunoglobu-lin A paraprotein from a patient with multiple myeloma. Characteriza-tion of this immunoglobulin. J Clin Invest 1985;76:225–32.14. Redmon B, Pyzdrowski KL, Elson MK, Kay NE, Dalmasso AP, Nut-tall FQ. Hypoglycemia due to an insulin-binding monoclonal antibodyin multiple myeloma. N Engl J Med 1992;326:994–8.15. Mills LE, Brettman LR, Jentoft JE, Viner ED, Bernier GM. Crystal-locryoglobulinemia resulting from human monoclonal antibodies toalbumin. Ann Intern Med 1983;99:601–4.16. Trimarchi F, Benvenga S, Fenzi G, Mariotti S, Consolo F. Immuno-globulin binding of thyroid hormones in a case of Waldenstrom’smacroglobulinemia. J Clin Endocrinol Metab 1982;54:1045–50.17. Sahota SS, Leo R, Hamblin TJ, Stevenson FK. Myeloma VL and VHgene sequences reveal a complementary imprint of antigen selectionin tumor cells. Blood 1997;89:219–26.18. Kosmas C, Stamatopoulos K, Stavroyianni N, Zoi K, Belessi C, Vin-iou N, Kollia P, Yataganas X. Origin and diversiﬁcation of the clono-genic cell in multiple myeloma: lessons from the immunoglobulinrepertoire. Leukemia 2000;14:1718–26.19. Suarez F, Lortholary O, Hermine O, Lecuit M. Infection-associatedlymphomas derived from marginal zone B cells: a model of antigen-driven lymphoproliferation. Blood 2006;107:3034–44.20. Prabhala RH, Neri P, Bae JE, Tassone P, Shammas MA, Allam CK,Daley JF, Chauhan D, Blanchard E, Thatte HS, Anderson KC, MunshiNC. Dysfunctional T regulatory cells in multiple myeloma. Blood2006;107:301–4.461PARAPROTEIN TARGETS

Klaus-Dieter Preuss

Gerhard Held

Boris Kubuschok

Chun-Zhu Hung

Natalia Malatsidze

Mathias Wagner

Michael Pfreundschuh

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Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUS

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