`RADIOPHARMACEUTICALS
`Volume 4, Number 4, 1991
`Mary Ar.n Lieber!, Inc.,
`Publishers
`
`Development of Human Antimouse Antibodies
`
`(HAMA) After Single and Repeated
`
`
`Diagnostic Application of Intact Murine
`Monoclonal Antibodies
`
`PETER LIND,1 PETER LECHNER,2 BARBARA HAUSMANN,3
`MICHAEL G. SMOLA,4 PETER KOELTRINGER,1 PETER STEINDORFER,4
`HARALD CESNIK,2 RAINER PASSL,3 and OTTO EI3ER1
`
`1 Bannherzige Dnceder Graz-Eggenberg Hospital
`
`
`lntcmal Dcpa11111e111/N11clear Medicine
`
`22nd Department of Surgery, University of Graz
`JDepar1111e111 of Accident Surgery, Graz
`
`
`4lst Department of Surgery, University of Graz
`Austria
`
`ABSTRACT
`The HAMA response after diagnostic application of murine monoclonal antibodies was examined in
`67 patients. In 23 cases HAMA 's were determined before and three months after administration of I
`mg
`intact monoclonal anti-CEA antibody (MAb BW 431/26), in 29 patients before and three
`months after application of 0.5 mg intact monoclonal antigranulocyte antibody (MAb BW 250/183).
`In another group of 15 patients, RAMA 's were determined at three-week intervals before and after
`repeated application of 0.3 mg MAb BW 250/183. In all patients total HAMA response (IgM and
`IgG) as well as the proportion of antiidiotypic response (IgM and IgG) was determined by an
`enzyme immuno assay (Enzygnost HAMA micro, Behring, Marburg FRG). After single application
`of �.mg MAb BW 431/26, 7 out of 23 patients developed HAMA 's (6 patients with predominantly
`antnsotypic, I patient with predominantly antiidiotypic response). After administration of 0.5 mg
`Mt\b BW 250/183, 4 out of 29 patients developed HAMA's (3 patients with predominantly
`antusotypic, 1 patient with predominantly antiidiotypic response). At repeated application of0.3 mg
`MAb BW 250/183 none of the 15 patients developed human antimouse antibodies after the first
`application; after the second and third application one patient showed a clear increasing
`antiidiotypic response one patient a marginal antiidiotypic response, two patients an antiisotypic
`response and 4 patients a transient predominantly antiisotypic IgM response. Deterioration of i�age
`quality in repeated immunoscintigraphic investigations was observed in cases of high HAMA tilers
`and/or predominantly antiidiotypic HAMA response.
`
`/
`
`INTRODUCTION
`With the introduction of Tc-99m-labeled monoclonal mouse antibodies, irnmunoscintigraphy has
`become a routine method at a number of centres, especially in the diagnosis and follow-up of
`colorectal carcinomas and the identification of inflammatory processes. Its employment has been
`encouraged by the ready availability of Tc-99m as well as its high sensitivity and specificity
`(1,2,3,4,5). However, the utility of a method depends, inter alia, on whether it can be applied
`repeatedly also in the follow-up of diseases (e.g. colorectal recurrence, lymph node and distant
`metastases; Crohn's disease, ulcerative colitis, osteomyelitis). The formation of human antimouse
`antibodies (HAMA) could considerably reduce the potential of immunoscintigraphy for follow-up
`because of the deterioration of image quality. Literature provides greatly differing and sometimes
`confusing information on the actual frequency of HAMA response and on the consequences for
`repeated examinations. The results usually refer to therapeutic applications of monoclonal mouse
`antibodies, with dosages varying from 6 to 400 mg and 100 to 700 mg respectively in the groups
`investigated (6,7). As far as the results of diagnostic application of monoclonal mouse antibodies are
`concerned, data on the doses of antibodies administered is partly not available or HAMA response
`data of different monoclonal antibodies were subsumed (8,9).
`Thus it has been the aim of our study to investigate the frequency of HAMA response at defined
`diagnostic antibody administration after single and repeated applications.
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`PATIENTS AND METHODS
`In 67 patients the total HAMA response as well as the percentage of antiisotypic and antiidiotypic
`response after diagnostic immunoscintigraphy were determined.
`Group I: HAMA response after 1 mg MAb BW 431/26
`In 23 patients (15 females, 8 males; age 61 ± 11 years) with suspicious primary colorectal
`carcinomas or colorectal recurrences the HAMA values could be determined before and three
`months after application of 1 mg antibody (MAb BW 431/26, Behring Werke, Marburg, FRG).
`Group II: HAMA response after 0.5 mg MAb BW 250/183
`In 29 patients (16 females, 13 males; age 50 ± 9 years) the HAMA values were determined before
`and three months after application of 0.5 mg antibody (MAb BW 250/183; Behring Werke Marburg,
`FRG).
`Group III: HAMA response after repeated application of 0.3 mg MAb BW 250/183
`In 15 patients (11 males, 4 females; age 42 ± 12 years) with complicated bone fractures RAMA
`values were determined before and three weeks after first application, six weeks after first
`application and before second application respectively, three weeks after second application, six
`weeks after second application and before third application respectively, and three weeks after third
`application of 0.3 mg MAb BW 250/183. In addition, the liver uptake 18 hours post injection - liver
`activity/total injected activity - corrected for decay and background was determined during the first
`and third immunoscintigraphy in group III.
`Indirect enzyme immunoassay for RAMA determination
`Monoclonal antibodies (unspecific antibody; BW 431/26 = specific anti-CEA antibody; BW
`250/183 = specific anti-granulocyte antibody) were diluted to 1 ug/ml in the dilution medium and
`applied onto microtiter plates (Enzygnost HAMA micro; Behring Werke Marburg FRG) coated with
`goat polyclonal antibodies to mouse IgG. The plates were incubated at 370 C for one hour and then
`washed three times with 250 ul of diluted washing solution (phosphate buffer solution containing
`0.05%tween). 100 ul of the dilution medium, diluted negative control, positive control and patient
`s�rum (1: 10) were given into the wells, and the plates were incubated at 37oc with 100 ul of a 1 :50
`�Jluted IgG/POD (rabbit antihuman immunoglobulin/peroxidase) or lgM/POD (goat antihuman
`1mmunoglobulin/peroxidase) conjugate. The plates were then washed again three times, and 100 ul
`?f the TMB (3,3',5,5'tetramethylbenzidine) working solution were given into the wells and
`mcu�ated, protected from light, at +2QOC for 30 minutes. After addition of 100 ul of stopping
`solution (o.5 M sulfuric acid), the absorbance was determined at 436 nm. After reading the
`absorbance values, the HAMA factor was determined as follows: HAMA factor = serum samples -
`blank/ negative control - blank. For normal values the HAMA factor against unspecific monoclonal
`mouse antibodies and specific monoclonal antibody (BW 431/26, BW 250/183) were calculated in
`50 (B� 431/26) and 34 (BW 250/183) healthy subjects respectively (fable 1,2).
`.
`Baseline RAMA values (before immunoscintigraphy) were considered positive when their RAMA
`factor �as greater than the mean + 2s of the HAMA factor from the 50 and 34 healthy subjects
`respectively. HAMA response values after immunoscintigraphy were considered positive when
`Hi\�:.\ fac:tor was twofold higher than the baseline value. To differentiate between antiisotypic and
`ant11d1otyp1c HAMA response, the rnicrotiter plates were incubated with the unspecific (antiisotypic
`
`Table 1
`Normal values of total and antiisotypic HAMA factor against
`MAb BW 431/26 in 50 healthy subjects
`
`z
`
`IgM (total)
`
`2.42
`
`IgG (total)
`
`1.65
`
`IgM (iso)
`
`IgG (iso)
`
`2.40
`
`1.55
`
`s
`
`1.10
`
`0.52
`
`1.18
`
`0.44
`
`range
`
`0.22-4.62
`
`0.61-2.69
`
`0.04-4.76
`
`0.67-2.43
`
`HAMA: Human anti mouse antibody; total: antiiso- and antiidiotypic; iso: antiisotypic
`x = average; s = standard deviation
`
`.
`
`.
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`Table2
`Normal values of total and antiisotypic HAMA factor against
`MAb BW 250/183 in 3 4 healthy subjects
`
`lC
`
`II
`
`IgM (total)
`
`3.51
`
`IgG (total)
`
`2.05
`
`IgM (iso)
`
`2.39
`
`IgG (iso)
`
`1.32
`
`1. 68
`
`0.86
`
`1.02
`
`0.57
`
`range
`
`0.15-6.87
`
`0.33-3.97
`
`0.35-4.43
`
`0.18-2.50
`
`HAMA: Human anti mouse antibody; total: antiiso- and antiidiotypic; iso: antiisotypic
`x = average; s = standard deviation
`
`response) and the specific (antiisotypic and antiidiotypic response) monoclonal antibodies.
`Antiidiotypic HAMA response was calculated as percentage of the total HAMA response minus
`antiisotypic HAMA response. This calculated antiisotypic HAMA response was compared with the
`results after inhibition. In this experiment, inhibition of HAMA in the patients sera was performed
`with the specific antibody (total inhibition) and the unspecific antibody (isotypic inhibition) before
`the first incubation. The percentage of antiidiotypic HAMA response was calculated as total
`inhibition minus antiisotypic inhibition/total inhibition.
`
`RESULTS
`Group I: Three months after the application of 1 mg of MAb BW 431/26, 7 of the 23 patients
`developed human antimouse antibodies.
`In 6 cases the HAMA response (IgG) was primarily antiisotypic and in one case predominantly
`antiidiotypic (Table 3). In all 7 patients the IgM response was negative.
`
`Table3
`HAMA factors (IgG) and percentage of antiiso- and antiidiotypic response
`3 month after 1 mg MAb BW 431/26:
`
`A
`HAHA factors
`total
`iso
`
`B
`% response I
`iso
`id.lo
`
`c
`% response II
`iso
`idio
`
`5.93
`5.96
`8.64
`8.70
`13.76
`16.11
`25.58
`
`3.68
`5.73
`4.92
`7.25
`11.14
`4.65
`22.81
`
`62
`96
`56
`83
`81
`29
`98
`
`38
`4
`44
`17
`19
`71
`11
`
`83
`84
`58
`78
`83
`25
`81
`
`17
`16
`42
`22
`17
`75
`19
`
`pts
`
`1
`2
`3
`4
`5
`6
`7
`
`/
`
`Total and antiisotypic HAMA factors (A); calculated percentage of antiiso-and antiidiotypic
`response (B: % response I) and percentage of antiiso- and antiidiotypic response after total and
`isotypic inhibiton (C: % response II) in 7 patients with positive HAMA response.
`
`Fig. 1. shows the whole body distribution (5 1/2 hours post injection) of the antibody before and
`after development of predominantly antiidiotypic HAMA in a patient with inoperable recurrence
`after left hemicolektomia.
`Also in patients with high total HAMA factor and predominantly antiisotypic response the image
`quality is altered due to the high liver activity, but the demonstration of the lesions are still possible
`(Fig. 2, a, b ).
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`FIGURE 1: Whole body distribution of Tc-99m MAb BW 431/26 5 1/2 hours post injection in a
`patient with malignant recurrence after left hemicolektomia before (left side) and after (right side)
`dev<?lopme�t of predominantly antiidiotypic antibodies is shown. Whereas in the left image the
`malignant (inoperable) recurrence is clear demonstrated (arrow), the control scan after development
`of predominantly antiidiotypic HAMA 's failed to image the recurrence.
`
`For the pe�centage of antiidiotypic response there was a good correlation b�tween the values
`cal�.ulated. di;ec.tl� .from the total and antiisotypic response and the values deterrmned after total and
`ant11sotyp1c mh1b111on.
`Group II: Three months after application of 0.5 mg MAb BW 250/183, 4 of 29 patients ex�mined
`showed human antimouse antibodies. In 3 cases the RAMA response was predominantly
`antiiso�ic, in one case 50% antiisotypic and 50% antiidiotypic. Like in group I, there was a good
`correlation between the values determined directly and after inhibition (Table 4).
`
`Table4
`RAMA factors (IgG) and percentage of antiiso- and antiidiotypic response
`3 month after 0 . 5 mg MAb BW 250/183
`
`pts
`
`l
`2
`3
`4
`
`A
`KAMA factors
`total
`iso
`
`6.00
`9. 73
`17.30
`18.74
`
`4.30
`4. 18
`ll . 98
`18.71
`
`B
`Ill response :i:
`idio
`total
`
`71
`43
`70
`99
`
`29
`57
`30
`1
`
`c
`Ill response :i::i:
`iso
`total
`
`63
`49
`62
`96
`
`27
`51
`38
`4
`
`Total and antiisotypic HAMA factors (A); calculated percentage of antiiso-and antiidiotypic
`response (B: % res�onse I) and percentage of antiiso- and antiidiotypic response after total and
`isotypic inhibiton (C: % response II) in 4 patients with positive HAMA response.
`
`Group �II: In a group of 15 patients examined after triple application (0.3 mg MAb BW 250/183),
`one p�uent developed increasingly antiidiotypic IgG human antimouse antibodies after the second
`and third application. The liver uptake rose from 3% to 24% in this patient (Fig. 3a, b; Tab. 5).
`In � second patient the RAMA value was just slightly elevated after second application, in two
`pattents the IgG response was predominantly antiisotypic; in 4 patients there was only a transient
`predominantly antiisotypic IgM response. The remaining seven patients of group ill developed
`neither transient nor permanent RAMA's up to three weeks after third application (15 weeks after
`first application). In these patients the liver uptake (basal values between 2% and 4%) did not show
`any significant increase after repeated immunoscintigraphy.
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`FIGURE 2a: Whole body scan ofTc-99m MAb BW 431/26 5 1/2 h post injection after development
`of predominantly antiisotypic HAMA 's. The bone metastases in the right humerus, the left femur
`and the pelvis are still shown on the image.
`
`FIGURE2b:
`Coronal SPECT slices 6 h post injection: same patient as figure 2a.
`
`Tables
`Total and antiisotypic HAMA factors (lgG, IgM) after repeated application
`of 0.3 mg MAb BW 250/183 in a patient with positive response
`
`weeks
`
`0 (IS)
`3
`6 (IS)
`9
`12 (IS)
`15
`
`tota1
`IgG
`
`IgM
`
`4.65
`7.24
`6.64
`6 .06
`6.34
`6.07
`
`1.52
`2.12
`3.44
`8.12
`9.38
`13.83
`
`iso
`
`IgG
`
`1. 42
`2.28
`2.06
`3.08
`3.30
`3.70
`
`IgM
`
`4.65
`6. 75
`5.04
`4.99
`3.59
`4.70
`
`Total: Antiiso- and antiidiotypic; iso: antiisotypic; IS: immunoscintigraphy
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`FIGURE3a:
`Liver uptake of a patient before the development of anti-mouse antibodies (3%)
`
`FIGURE3b:
`Clearly increase of the liver uptake (24%) after development of human anti-mouse antibodies
`
`Although none of the patients developed allergic reactions, the interpretation of scintiscans is clearly
`limited in patients with total RAMA factors above 10 and/or predominantly antiidiotypic response.
`
`DISCUSSION
`Most of the literature on the development of antimouse antibodies refers to the application of
`therapeutic doses of murine monoclonal antibodies. Bilchler et al. detected a positive RAMA
`response three weeks after completion of therapy in all of the 8 patients (100 %) who had been
`treated with MAb BW 494 (180 to 340 mg) for advanced pancreas carcinoma ( 10). DeFreitas et al.
`(7) reported a RAMA response of 84% (16 of 19 patients) after administration of antibodies in doses
`between 100 and 700 mg (MAb 17 lA). Abdel-Nabi et al described three different patterns of
`RAMA response: no response, transient response, and permanent response. The authors did,
`however, not indicate how many of the 17 patients showed a positive HAMA response after 40 mg
`
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`---- ··-· ----·· -- --· ·-····-·--··
`
`. . -- . . --
`
`.... --·--- ---··· -·· ----
`
`MAb ZCE 025 ( l l). Most publications provide informa1ion only on the total HAMA response; a
`differentiation between antiisotypic and antiidiotypic response is usually not made (12,13,14,15).
`Herlyn et al reported a HAMA response rate between 36% and 73% at single dose application, and
`between 92% and 100% after repeated application. In this paper the antiidiotypic share of the total
`response was between 21 % and 80% (16). Our results obtained from 67 patients show that the
`HAMA response after first application ranges up to 30%. A reduction of the amount of the
`adminstered antibody (0.5 to 0.3 mg) seems to diminish the HAMA response. As shown by Joseph
`et al (17) an antibody amount of 0.2 mg (Tc-99m labeled MAb BW 250/183) is sufficient for
`diagnostic imaging. After repeated application a permanent response was observed in 4 and a
`transient response in 4 out of 15 patients investigated. It is only in case of a higher HAMA factor
`and predominantly antiidiotypic response that the evaluation of the immunoscin1igraphic images is
`affected. With the determination of the total and antiisotypic HAMA response the antiidiotypic
`response can be assessed with sufficient accuracy. Inhibition tests with unspecific and specific in
`vivo adminis1ered antibodies are in good agreement with the antiidiotypic response assessed from
`the total iUld antiisotypic response. In our experience (more than 200 immunoscintigraphies since
`1988), no allergic reactions were observed, not even when scintiscans were performed repeatedly.
`Further optimization of immunoscintigraphy is, however, to be aimed at inasmuch as the applied
`doses of antibodies should be as low as possible (between 0.3 and 0.5 mg) or one uses the fragment.
`Prior to each second application, the result of the HAMA response and the percentage of the
`antiidiotypic fraction should be available, since at higher HAMA factors and predominantly and/or
`antiidiotypic response the information gained from the second examination is limited and thus its
`diagnostic value is questionable.
`
`ACKNOWLEDGMENTS
`We thank Mrs. Burgi Holler for preparing the manuscript.
`This publication would not have been possible without 1he technical work of Ms.Dagmar Peroutka,
`Mrs.Elfriede Brunner, Ms.Gabriele Kirchbaumer, Mrs. Maria Schwar and Ms. Birgit Prass!.
`
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`
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`
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`
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`
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`(1985).
`
`7. DeFreitas E, Suzuki H, Herlyn D, Lubeck, M., Sears, H., Herlyn, M. & Koprovski, H. Human
`antibody induction to the idiotypic and anti-idiotypic determinants of a monoclonal antibody against
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`
`8. Hertel A, Baum RP, Auerbach B, Hemnann, A. & Ht>r, G. Klinische Relevanz humaner anti
`Maus Antikt>rper (HAMA) in der Immunszintigraphie. Nucl Med29:46-41 (abstr.) (1990).
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`T'r..l .. --·--illil.7. __ ---!-�
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`9. Counenay-Luck NS, Epenetos AA, Moore R, Larche, M. & P�tasi.de, D. �evelope!11ent <?f
`primary and secondary immune responses to mouse monoclonal anubodies used in the diagnosis
`and therapy of malignant neoplasms. Cane Res 46:6489-6493 (1986).
`
`10. Biichler M, Kiibel R, Malfertheiner P, Friess, H., Schulz, G., Bosslet, K. & Berger, H.G.
`Immunotherapy of advanced pancreatic carcinoma with the monoclonal antibody BW 494. Dtsch
`Med Wschr 113:374-380 (1988).
`
`11. Abdel-Nabi H, Smith L, Unger B, Roth, S.C. & Merchant, B. Patterns of HAMA developement
`following repeated infusions of In-111ZCE 025 MoAb in recurrent colorectal carcinoma patients. J
`Nucl Med 30:906 (abstr) (1989).
`
`12. Hertel, A., Baum, R.P., Hermann, A. & Hor, G. Frequenz humaner anti-mouse Antikorper nach
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`(1989).
`
`13. Zimmer AM, Kazikiewicz JM, Kaplan EH, Lurain, J, Miller, D.S., Webber, D.I., Patel, P.A.,
`Goldman-Leiken, R.E., Manzel, L., Gilyon, K., Radosevich, J.A., Spies, W.G., Spies, S.M. &
`Rosen, S.T. Human antimurine antibody responses following intraperitoneal radioimmunotherapy
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`Med 30:827 (abstr.) (1989).
`
`14. Larsen SM, Brown JB, Wright PW., Carasquillo, J.A., Hellstroem, I. & Hellstroem, K.E.
`Imaging of melanoma with 1-131-labeled monoclonal antibodies. J Nucl Med 24: 123-129 (1983).
`
`15. Reynolds JC, Carasquillo JA, Lora ME, Sugarbaker, D., Abrams, P., Foon, K., Roth, J,
`Mulshine, J, Colcher, D., Schlom, J. & Larson, S.M . Measurement of human anti murine antibodies
`in patients for received radio labeled monoclonal antibodies. Nucl Med Suppl. 23:425-427 (1987).
`
`D, Lubeck M, Sears H,& Koprowski H. Specific detection of anti-idiotypic immune
`16. Herlyn
`_
`responses m cancer patients treated with murine monoclonal antibody. J lmmunol Meth 85:27-38
`(1985).
`
`17. Joseph K, Hoffken H, Damann V. In vivo Markierung von Granulozyten mit Tc-99m markierten
`monoklonalen Antikorem: Erste klinische Ergebnisse. Nuc Compact 18:223-229 (1987).
`
`Authors address:
`Peter Lind
`Barmherzige Brader Eggenberg Hospital
`Nuclear Medicine
`Bergstrasse 27
`A-8021 Graz
`Austria
`Telefax: 00431316155110-155
`
`Submitted: November 20, 1990
`Accepted: February 13, 1991
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