1 of 8 celltrion, inc., exhibit 1066

ANTIBODY, IMMUNOCONJUGATES, ANO 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.

811

/

1 of 8 Celltrion, Inc., Exhibit 1066

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 s range

IgM (total) 2.42 1.10 0.22-4.62

IgG (total) 1.65 0.52 0.61-2.69

IgM (iso) 2.40 1.18 0.04-4.76

IgG (iso) 1.55 0.44 0.67-2.43

HAMA: Human anti mouse antibody; total: antiiso- and antiidiotypic; iso: antiisotypic x = average; s = standard deviation

. . 1112


Table2 Normal values of total and antiisotypic HAMA factor against

MAb BW 250/183 in 34 healthy subjects

lC II range

IgM (total) 3.51 1. 68 0.15-6.87

IgG (total) 2.05 0.86 0.33-3.97

IgM (iso) 2.39 1.02 0.35-4.43

IgG (iso) 1.32 0.57 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.

pts

1 2 3 4 5 6 7

Table3 HAMA factors (IgG) and percentage of antiiso- and antiidiotypic response

3 month after 1 mg MAb BW 431/26:

A B c HAHA factors % response I % response II total i s o i s o id.lo i so idio

5.93 3.68 62 38 83 17 5.96 5.73 96 4 84 16 8.64 4.92 56 44 58 42 8.70 7.25 83 17 78 22

13.76 11.14 81 19 83 17 16.11 4.65 29 71 25 75 25.58 22.81 98 11 81 19

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 ).

'" .. _____ ,Jlll ...... ,

/


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).

pts

l 2 3 4

Table4 RAMA factors (IgG) and percentage of antiiso- and antiidiotypic response

3 month after 0.5 mg MAb BW 250/183

A B c KAMA factors Ill response :i: Ill response :i::i: total iso total idio total iso

6.00 4.30 71 29 63 27 9. 73 4.18 43 57 49 51

17. 30 l l.98 70 30 62 38 18 . 7 4 18 .71 99 1 96 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.

~ .. ____ J}J,4 -- ---·--


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

tota1 iso weeks IgM IgG IgM IgG

0 (IS) 4.65 1.52 4.65 1. 42 3 7.24 2.12 6. 75 2.28 6 (IS) 6.64 3.44 5.04 2.06 9 6 .06 8.12 4.99 3.08 12 (IS) 6.34 9.38 3.59 3.30 15 6.07 13.83 4.70 3.70

Total: Antiiso- and antiidiotypic; iso: antiisotypic; IS: immunoscintigraphy


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

. U16


- --- ··-· ----·· -- --· ·-····-·--·· . . -- .. -- .... --·--- ---··· - ·· ----

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!.

REFERENCES 1. Lind, P., Langsteger W, KOltringer P, Lechner, P., Beham, A., Arian-Schad, K. & Eber 0. T~-99m labeled monoclonal anti carcinoembryonic antigen antibody (BW 431/26). Clinical Results m the detection of colorectal carcinomas and recurrences. Scand J Gastroentero/ 24: 1205-12 l l ( 1989).

2. Lind P, Langsteger W, Koltringer P., Dimai, H.P., Passi, R & Eber 0. Immunoscintigraphy of inflammatory processes with a Tc-99m labeled monoclonal antigranulocyte antibody (MAb BW 250/183). J Nucl Med 31:417-423 (1990).

3. Kroiss A, Schuller J, Tuchmann A, Weiss, W., Wirth, M, Auinger, Ch., Dinstl, K. & Neumayr, A. lmmunoscintigraphy in patients with colorectal cancer recurrences. Pp. 570-572 in Schmidt HAE, Chambron J.(eds) Nuc/ Med (1990).

4. Kroiss A, Kolb! Ch, Tuchmann A, Auinger, Ch., Weidlich, G., Weiss, W. & Neumayr, A .. Jmmunszintigraphie bei entziindlichen Erkrankungen mit Tc-99m MAk BW 250/183. In: Eber 0, Lind P, Langsteger W, eds. Workshop Jmmunoscintigraphy Sonderheft: Acta Med Austriaca 11 (1989).

5. Baum RP, Hertel A, Lorenz N, Hottenrott, C., Schwarz, A., Maul, F.D. & Hor, G. Tc-99m labeled intact monoclonal anti-CEA antibody for sucessfull localization of tumor recurrences. In: Schmidt HAE, Buraggi GL. Nuc/ Med 515-518 (1989).

6. Schroff RW, Foon KA, Beatty SM, Oldham, R.K. & Morgen, A.C. Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cane Res 45:879-885 (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 a gastrointestinal carcinoma antigen. Current Topics Microbiol lmmuno/ 119:75-89 (1985).

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).

T'r.. l .. --· --illil.7. __ ---!-~


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-111ZCE025 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 156 Immunszintigraphien mit 6 verschiedenen monoklonalen Antikorpern. Nucl Med 28:67 (abstr.) (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 infusions of murine monoclonal antibodies: pharmacokinetic analysis following retreatment. J Nucl 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).

16. Herlyn_ D, Lubeck M, Sears H,& Koprowski H. Specific detection of anti-idiotypic immune 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).

Submitted: November 20, 1990 Accepted: February 13, 1991

Authors address: Peter Lind Barmherzige Brader Eggenberg Hospital Nuclear Medicine Bergstrasse 27 A-8021 Graz Austria Telefax: 00431316155110-155


1 of 8 celltrion, inc., exhibit 1066

Documents