CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
59, 89-103 (1991)
Human Anti-mouse Antibody Response Induced by Anti-CD4 Monoclonal Antibody Therapy in Patients with Rheumatoid Arthritis’ GERD HORNEFF, THOMAS WINKLER, JOACHIM R. KALDEN, FRANK EMMRICH,* AND GERD R. BURMESTER *Max Planck Clinical Research Institute of Clinical Immunology and Rheumatology, Department of Medicine Ill, Krankenhausstr. 12. University of Erlangen-NCrnberg, Federal Republic of Germany
Group,
The development of human anti-mouse monoclonal antibodies (HAMAs) was investigated in 10 patients with rheumatoid arthritis (RA) who had undergone an experimental therapeutic trial with an anti-CD4 monoclonal antibody. In this patient group, the antibody 16H5 of the IgGl isotype had been administered in a median total dosage of 140 mg per treatment cycle. Four patients took part in a second treatment regimen 6-8 weeks later. After the first treatment cycle, detectable HAMAs developed in 5 out of 10 patients. In 4 individuals undergoing a second course of therapy, increases of HAMAs were evident only in the 3 patients with previous HAMA responses. HAMAs were primarily of the IgG isotype, while the presence of rheumatoid factors usually interfered with the detectability of IgM HAMAs. However, using isolated Ftab), fragments of the monoclonal reagent used for therapy, HAMAs of the IgM isotype were also detectable. HAMAs of the IgG isotype did not exceed levels of 2.0 mg/hter after a single treatment cycle and 2.2 mg/hter after a repeated cycle. No IgE responses were detectable. Absorption experiments indicated that approximately 25% of the HAMA activity was directed against specific determinants of the 16HS monoclonal antibody, presumably including anti-idiotypic reactivities. These data demonstrate that HAMAs developed only in a proportion of RA patients treated with the anti-CD4 monoclonal antibody 16H5. However, the amounts were rather low compared to other monoclonal reagents used in cancer patients and were therefore allowed for repeated applications without an apparent loss
Of efficacy.
0 1991 Academic
PKS~, Inc.
INTRODUCTION Monoclonal antibody treatment and diagnostic procedures are increasingly used in cancer patients (1, 2), organ transplantation (3, 4), and certain diseases with autoimmune features, including rheumatoid arthritis (5-7). The development of human anti-mouse monoclonal antibodies (HAMAs) is of crucial importance with regard to repeated diagnostic and therapeutic procedures. Moreover, HAMAs interfere with in vitro assays using murine monoclonal antibodies in ELISA or IRMA systems (8, 9). Monoclonal antibody treatment has initially been used for cancer treatment administering reagents directed against tumor-associated antigens (1). Since these ’ Supported by the Federal Ministry of Research and Technology (grant “Lymphokine” 01 ZU 8601, No. 41) and the European Concerted Action on Immunopathogenesis and Immunotherapy of Chronic Arthritis. Dr. Homeff is a postdoctoral fellow of the Deutsche Forschungsgemeinschaft, Grant Ho143/1-2. 89 0090-1229191 $1.50 Copyright All tights
Q 1991 by Academic Press. Inc. of reproduction in any fan reserved.
antibodies are of xenogenic origin, the development of a strong immune respunsc against mouse reagents is not surprising. Ilnfortunately. this response inhibited further applications of mouse monoclonal antibodies in this patient group (2). This situation may be completely different in therapeutical trials where antibodies with highly immunosuppressive potential are used (IO, I I). The rational in these settings is that the immune system is severely suppressed by the antibody itself, thereby not allowing for a vigorous anti-mouse monoclonal antibody response. Especially, application of anti-CD4 and foreign antigens in parallel resulted in a persistant tolerance to the antigen in animal models (I I). However, studies using the OKT3 monoclonal reagent directed against all mature T cells for the treatment of organ rejection episodes have shown a considerable reactivity toward this reagent (12, 13). Therefore, the objective of the present study was to investigate the response against another immunomodulatory reagent directed against the CD4 antigen characteristic of the T helper cell subset. The data were derived from a therapeutic trial in patients with rheumatoid arthritis refractory to conventional therapy (5) treated with an anti-CD4 antibody in an attempt to induce a beneficial immunomodulation. MATERIALS
AND METHODS
Patients Patients undergoing the clinical trial with the anti-CD4 monoclonal antibody 16H5 suffered from severe and active rheumatoid arthritis according to the ACR criteria (14). The study protocol was designed according to the guide lines of the “Gesellschaft fur Immunologie” (Society for Immunology) and the Paul Martini Endowment. It was reviewed and approved by the ethical committee of the University of Erlangen-Ntirnberg. Written and informed consent was obtained from all patients after information about the possible risks of treatment. Effects seen in this patient group have been reported previously (5) and consisted of a persistent decrease of CD4 + cells, decreased CD4/CD8 ratios for 3 to more than 8 weeks, reduced lymphocyte proliferations induced either by mitogens (phythohemagglutinin, pokeweed mitogen, and concanavalin A) or antigens (tetanus toxoid, diphteria toxoid, and tuberculin), and reduced skin test responses along with significant clinical benefits. Side effects were mild elevations of body temperature in three cases, presumably due to cytokine release and an apparently allergic skin reaction in one individual. Concommitant therapy consisted of only nonsteroidal anti-inflammatory drugs (NSAIDs) in three patients, and NSAIDs and prednisone (less than 10 mg/day) in six patients. One patient was on a low dosage of cyclophosphamide (50 mg/day) in addition to 10 mg of prednisone due to severe vasculitis. Another individual had received a total nodal irradiation because of severe RA 5 years prior to this therapeutic trial. The antibody was administered intravenously in an approximate dosage of 0.3 mg/kg body weight during an infusion time of 30 min in the morning on 7 subsequent days. The total amount of antibodies applicated varied from 105 to 175 mg
HAMAs
AND
ANTI-CD4
TREATMENT
91
(median, 140 mg). Repeated treatment cycles were performed using the same dosage in three patients and a higher dose in one individual (total amount: 140 mg vs 10s mg). Sera from 10 patients (9 rheumatoid factor positive) treated with reagent 16H5, 18 additional RA patients (13 seropositive in the Waaler Rose test, range 50 to 800 IE) without antibody treatment, and 8 normal donors without a history of joint diseases or allergy were tested. All sera were separated immediately from peripheral blood and stored at - 20°C until used. Rheumatoid factors were determined using standard Waaler Rose and Latex agglutination assays. In addition, rheumatoid factors were measured by the ELISA technique. Absorption
of Sera
To determine the anti-idiotypic proportion of the anti-mouse response, sera were diluted 1:20 in phosphate-buffered saline (PBS), and absorption was performed using immobilized murine IgG (Pierce). Sera were incubated for 1 hr and subsequently separated by centrifugation. In parallel, absorbed sera and nonabsorbed sera were tested on plates coated with the monoclonal antibody 16H5 and the subclass control antibody as described below. Preparation
of Antibodies
The murine monoclonal antibody 16H5 (IgGl K) directed against the CD4 antigen present on the T-helper/inducer cell subset (15) had been purified for treatment according to the regimens of the European Community Committee for Proprietary Medicinal Products (June 1986,111/859/86EN). F(ab), fragments of this antibody were prepared by incubation of the antibody with immobilized Pepsin (Pierce) in 0.02 M sodiumacetate buffer (pH 4.2) for 4 hr. After neutralization with Tris-HCl and dialysis against PBS, F(ab), fragments were isolated from undigested IgG by affinity chromatography using a protein G column (Pharmacia, Uppsala, Sweden). Subsequently, dialysis against PBS with a membrane cutoff point of 30 kDa was performed to eliminate contaminating protein fragments. The presence of F(ab), fragments was verified by SDS gel electrophoresis. The purity of this preparation was tested by the ELISA technique which demonstrated a good reactivity with F(ab),-specific antisera, while there was no reactivity with an Fc-specific goat anti-mouse antiserum. The monoclonal anti-CD3 reagent 89bl (IgGl K) was used as an isotype control (16). After an ip injection of the mouse hybridoma 89b1, ascites was obtained from BALB/c mice. After precipitation with half-saturated ammonium sulfate solution and resuspension in PBS, the antibody was purified using a protein G fast flow column (Pharmacia). ELISA
The respective antigens were coated to standard ELISA plates (NUNC, Roskilde, Denmark) in a concentration of 10 kg/ml in 0.05 sodium carbonate (pH 9.6) for 18 hr at +4”C. Testing for coating efficacy using an alkaline phosphataseconjugated goat anti-mouse antiserum revealed equivalent amounts of murine IgG coated to the plates. The plates were washed three times with PBS containing 0.05% Tween (Sigma Chemical Co., Munich, FRG). Excess binding sites were
92
HORNEFF
El
Al
blocked by postcoating the wells with 10% (v/v) fetal calf serum in PBS-Tween for I hr at room temperature. Washing procedures were repeated. For comparison, on each plate standard curves were generated using a defined primate anti-mouse immunoglobulin solution (Medac, Hamburg, FRG) containing 220 rig/ml of antimouse antibodies. Experiments had been carried out to investigate the quality of this standard preparation. In these assays, dilutions of a human IgG preparation (Medac) were coated to an ELISA plate in concentrations from 500 down to 3.9 rig/ml). In parallel, the murine monoclonal l6H5 was coated in a concentration of 10 pg/ml to the same plate. In a second step, a serial dilution of the primate anti-mouse antibody preparation was added to the well coated with the murine monoclonal reagent. In a third step, both serial dilutions (the human IgG coated directly to the plastic, the primate IgG binding to the murine antibody) were incubated with a goat anti-human IgG alkaline phosphatase conjugate. Bound primate anti-mouse antibodies of the standard preparation used in the assays revealed equivalent optical densities in a range from 3 to 200 rig/ml compared to defined amounts of a human IgG preparation directly coated to the wells. Sera were diluted I :20 with PBS-Tween and incubated at room temperature for 90 min. After consecutive washing procedures, a 1: 1000 dilution of an alkaline phosphate-conjugated goat anti-human immunoglobulin specific for y or p, chains was added (Medac). After incubation for 90 min and consecutive washing procedures, staining was performed using p-nitrophenyl-phosphate (Sigma) in a concentration of 0.5 mg/ml in 0.05 M sodium bicarbonate solution containing 0.01 M magnesium chlorid. Substrate turnover was stopped after 10 min and subsequently determined at 405 nm versus 630 nm with an ELISA reader (Titertek multiskan, EFLAB, Finland). Optical densities were calculated to nanograms per milliliter using standard curves derived from each plate. For the detection of HAMAs of the IgE class, a three-step ELISA system including a biotin/streptavidine step (biotinylized goat anti-human IgE and a horseradish peroxidase-conjugated streptavidin, all Medac) with increased sensitivity was used. For the detection of rheumatoid factors by the ELISA technique, rabbit IgG was coated as described above. Sera were diluted 1: 10 for determining IgG rheumatoid factor and 1:50 for IgM rheumatoid factor. After incubation for 90 min and subsequent washing procedures, goat anti-human IgG and IgM were added, respectively. The quality of this assay was tested using sera of 10 normal donors, 14 seronegative, and 25 seropositive (according to the Waaler Rose and Latex assay) RA patients. There was a correlation coefficient to the Latex assay of 0.53 (P < 0.005) for the IgG and 0.68 (P < 0.0001) for the IgM rheumatoid factor determined by the ELISA technique. In the IgG assay, normal donors revealed extinctions of 535 t 113 versus 397 & 188 in seronegative RA patients and 971 ‘-t- 142 of seropositive RA patients. In the IgM assay, these values were 260 ‘-’ 40, 275 * 108, and 609 + 82, respectively. Differences of seropositive RA patients vs seronegative and normal donors were highly significant (P < 0.0001). Statistical Methods Correlations between quantitative
variables were tested by means of simple
HAMAs
AND ANTI-CD4
93
TREATMENT
linear regression analysis. The differences seen in normal donors and RA patients were compared using the Student t test after testing for normal distribution using the Kolmogoroff Smirnov test. RESULTS Preexisting HAMA Responses in RA Patients and Normal Donors
The detection of a newly formed human antibody response against xenogenic antibodies is influenced by preexisting anti-immunoglobulin antibodies, especially of the rheumatoid factor type which is a human anti-IgG antibody primarily detectable in the IgM isotype fraction. Figure 1 demonstrates the reactivity of sera from normal donors and untreated patients with rheumatoid arthritis. Anti-murine immunoglobulin antibodies were detected in the ELISA system using plates coated with either the whole antibody 16H5 or the F(ab), fragment. Using the whole antibody as antigen coupled to the plates, there was a significant correlation between the IgM responses toward 16H5 and the titers of rheumatoid factor in the Waaler Rose test (r = 0.76, P < 0.001). In contrast, there were no significant correlations between titers of rheumatoid factors and the IgM responses against the F(ab), fragment (r = 0.06, P = 0.81) or the IgG responses against both the whole antibody (Y = 0.19, P = 0.71) or the F(ab), fragment (r = -0.16, P = 0.53). Therefore, in all subsequent experiments, IgG responses were tested against both the whole antibody and the F(ab), fragment and IgM responses were investigated against the F(ab), fragment only. The rheumatoid factors determined by the Latex agglutination test or the
IgG
‘gM
”
16H5 Normal
F(ab)Z Donors
16H5
F(ab)2 RA
Patients
16H5 Normal
F(ab)Z Donors
16H5
F(ab)P
RA
Patients
FIG. 1. Human anti-mouse response measured in the sera of 9 normal donors and 18 untreated RA patients (IgG in mg/liter, IgM in OD). A considerable HAMA activity of the IgM class may be due to the presence of rheumatoid factors as indicated by the marked reduction of HAMA activity against the F(ab), fragment as compared to the whole antibody. There was a significant difference of HAMA responses between the normal donors and RA patients, both against the whole antibody and the F(ab), fragment (P < 0.01, asterisks). Preexisting HAMA activity of the IgG class did not correlate to rheumatoid factors and was found in normal donors as well as in RA patients without a significant difference (P > 0.10).
94
HORNkFl,
1.1
‘\I
Waaler Rose test were decreased in 7 out of 9 rheumatoid factor-positive patients immediately at the end of the treatment period, while in three individuals titers of rheumatoid factors were slightly increased (one dilution step) during the whole observation period. Thus. there was no evidence of increasing levels of rheumatoid factor resulting in positive HAMA levels. Especially. no correlation was found between the levels of rheumatoid factor determined by Waaler Rose or Latex agglutination and the IgG or IgM HAMAs against F(ab), fragments in the sera used in this investigation. In addition, rheumatoid factors were evaluated by the ELISA technique as described above. In Fig. 2, levels of HAMAs of the IgG (A) and 1gM (B) isotype were compared to rheumatoid factors determined by the ELISA technique in the sera of a patient who had undergone a repeated treatment regimen. There was no increase of either the IgG or the IgM response to rabbit IgG in contrast to a demonstrable anti-mouse response. HAMA
Responses
in RA Patients
after Treutnrent
a*ith an Anti-CD4
Antibody
Figure 3 demonstrates the IgG responses of two individuals after a single treatment (A) and a second treatment cycle (B) against the therapeutical antibody l6H5, the isotype control, and the F(ab)? fragment of the 16H5 reagent. There was a low preexisting reactivity against both complete antibodies. The patient in Fig. 3A was rheumatoid factor negative as determined by the Waaler Rose and the Latex agglutination assays, while the patient in Fig. 3B had a high titer (800 IU). Concommitant therapy consisted of NSAIDs and 9 mg (patient A) or 8 mg (patient B) prednisone. In both patients, HAMAs developed 2-3 weeks after the treatment had been started. HAMAs were detectable for IO-12 weeks after a single treatment. There was a parallel development of HAMAs against the whole antibody and the F(ab)? fragment with a higher response against the antibody used for therapy compared to the isotype control antibody. A second treatment cycle (Fig. 3B) resulted in enhanced levels of HAMAs which included reactivities against the epitopes present on the F(ab), fragment. Figure 4 shows the HAMA responses against the F(ab), fragment of 16H5 of all patients treated. There was a demonstrable elevation in 5 out of IO individuals after a single treatment (A and B), but in three cases only did HAMAs reach levels higher than the untreated healthy control population (mean t SD was 0.23 ? 0. I I mg/liter). A maximum level of 0.8 mg/liter developed above baseline, and elevations of HAMAs were detectable for l&12 weeks. In 3 out or 4 patients, a rise in the HAMA titer was developed after the second treatment cycle (Fig. 4B, arrows). HAMAs reached levels of less than I.7 mg/Iiter after the booster. The I individual without a demonstrable HAMA response (upper curve) had a remarkable preexisting activity without a booster reaction. In contrast, in this patient the level of HAMA activity decreased after each treatment cycle, possibly due to a consumption of preexisting anti-mouse antibodies upon therapy. The HAMA response of the IgM isotype directed against the F(ab), determinants obtained in four patients treated twice is shown in Fig. 5. The development of this type of IgM HAMAs was comparable to the IgG isotype in this group and was the most pronounced after the second treatment cycle (B,D). In six patients
HAMAs
AND
ANTI-CD4
95
TREATMENT
0.6
0
a
0 0
20
40
60
60
100
120
80
100
120
timefdays) 6)
1200: 4 1000
8
,,I
600
piiq
C
400
-
0
20
40
60
time(dsys)
of IgG (A) and IgM (B) HAMAs and rheumatoid factor activity in a patient with rheumatoid arthritis who had undergone a repeated anti-CD4 treatment. HAMA responses to the whole antibody (a)+ HAMA response to the F(ab), fragment (m), and response to rabbit IgG (A) (0) 0% (0). FIG. 2. Kinetics
who received a single treatment cycle, IgM HAMAs directed against the F(ab), fragment were not demonstrable (data not shown). IgE responses were not detectable in the patients treated twice nor in the one patient who had developed skin urticaria (the limit of sensitivity of the assay was 1.5 x&ml). Also, this patient did not show a preexisting IgG or IgM response to the monoclonal antibody and did even not develop demonstrable HAMAs.
96
HORNEFF
A)
ET AL.
IgG HAMA
000
20
40
60
80
100
120
80
100
120
time(days)
B)
0
20
40
60 time(days)
FIG. 3. IgG HAMA responses after a single (A) or repeated (B) treatment regimen against the whole antibody 16H5, the F(ab), fragment, and an isotype control antibody. The figure illustrates the two individuals with the highest amounts of IgG HAMAs that developed after treatment. The first 7-day treatment was started at t = 0 days, the repeated treatment regimen was started at t = 32 days (B) in this patient. There was a preexisting elevated IgG HAMA activity against both whole antibodies as compared to the F(ab), fragment. The patient in A was rheumatoid factor negative while the patient in B was highly positive (800 W/ml).
Anti-idiotypic
Response
Compared to the response against the isotype control antibody investigated in parallel, the HAMA reactivity developing against the 16H5 antibody was higher in each case. Absorption of the sera by immobilized murine IgG resulted in an unresponsiveness to the subclass control while the response to 16H5 was reduced to 24% at the time point of the maximal HAMA response as shown in Fig. 6. In Table 1, the maximal values of HAMAs of the IgG isotype against both complete antibodies are shown. The preexisting reactivities of all 6 patients’ sera outlined
HAMAs
4
AND
Single
ANTI-CD4
treatment
97
TREATMENT
cycle
0.8
0.6
-,
0.4
F
-
mean +2SD
-
+lSD
0.2
-
0 20
40
60
80
100
time(days)
Two
8)
treatment
cycles
1.8
1.5
0.6
0
20
40
60
80
100
120
140
tlme(days)
FIG. 4. IgG HAMA development after a single or repeated treatment regimens against the F(ab), fragment. Seven-day treatment cycles are indicated by arrows. There was a remarkable HAMA development above the means -+ I SD of 18 untreated RA patients in 2 out of 6 patients after a single treatment (A) and in 3 or 4 patients who had undergone a repeated treatment cycle (B).
in Table I were comparable (0.66 +- 0.12 mg/liter for the 16H5 antibody and 0.68 5 0.15 mgiliter for the isotype control antibody). After treatment, HAMAs of the IgG isotype were detectable in 5 out of 10 individuals as judged by values higher than the means 5 SD of untreated RA patients and the healthy control population.
98
HORNEFf
El Al
‘loo B
+ 30” ff I i
FIG. 5. IgM HAMA development against the F(ab)2 fragment in four patients who had undergone a repeated treatment regimen. Seven-day treatment cycles are indicated by arrows. There was a detectable HAMA response of the IgM class in three out of the four patients who had undergone a repeated treatment cycle.
In one additional individual (No. I), low levels of HAMAs were detectable, not exceeding the mean baseline of untreated RA patients. In all of these 6 individuals, the difference in response to either the monoclonal reagent used or to the subclass control was 0.36 k 0.06 mg/liter (26 k 5% of the total response). In 3 out of 4 individuals who underwent a repeated trial, a rise in HAMA levels was detectable, and the difference in response was 0.39 +- 0.08 mg/liter (20 + 6%) of the total reactivity. DISCUSSION
The investigation of the development of human anti-mouse antibodies in patients with rheumatoid arthritis treated with an anti-CD4 monoclonal antibody led to the following principle observations: 1. In RA patients, preexisting antibodies of the IgM class with an anti-mouse antibody activity showed strong correlations to the presence of IgM rheumatoid factors. 2. In contrast, IgG antibodies with HAMA activity did not show correlations to rheumatoid factors. 3. Specific HAMA activity was primarily of the IgG isotype, indicating a secondary immune response. Interestingly, increased levels of specific HAMA activity of the IgM-isotype against the F(ab)* part were the most pronounced after the second treatment cycle. 4. Treatment with an anti-CD4 monoclonal antibody resulted in only low
HAMAs
0
AND ANTI-CD4
1 0
20
I
40
.
1
60
.
1
80
99
TREATMENT
.
I
100
I
120
.
140
I
I
160
180
time(days)
0
] 0
,
I 20
40
'
I. 60
I 80
*
I. 100
I 120
-
I 140
'
I. 160
I 180
time(days)
FIG. 6. Response to the whole antibody 16H5 (A) and the subclass control (B) before (0) and after absorption (0) on immobilized murine IgG in a patient who had undergone a repeated treatment cycle (arrows).
amounts of HAMAs, even after a second treatment course, and these were demonstrable only in 5 out of 10 subjects. 5. Approximately 25% of HAMA activities are directed against specific determinants of the 16H5 antibody, including anti-idiotypic reactivities. The study of human anti-mouse immunoglobulin antibodies appears to be a particular problem in individuals with rheumatoid arthritis, a patient group that is characterized by the presence of high titers of antibodies toward the Fc portion of immunoglobulins, i.e., the rheumatoid factors. These antibodies are not only directed toward antigenic determinants of the human Fc part, but also react with antigens present on rodent antibodies, as has been thoroughly studied with rabbit sera (for review see Ref. (17)). Indeed, the Waaler Rose test, commonly used to identify rheumatoid factors, uses rabbit IgG bound to sheep erythrocytes in the
100
HORNEFF El AL. ‘TABLE
DIFFERENCES
IN HAMA
I
ACTIVITY AGAINST THE ANTIBODY ISOTYPE CONTROL ANTIBODY
USED
FOR TREATMENT
AND
AN
I6h5 (mg/liter)
Control tmgiliter)
Difference tmg/liter)
‘$,
A. IgG-HAMA activity (mg/liter) after a single treatment cycle I” 2 3 4 5 6 Mean SEM
o.88h 1.25 1.61 1.66 1.56 I .98 1.49 0.14
0.46 0.87 1.48 1.17 1.18 1.61 1.11 0.15
0.42 0.38 0.13 0.49 0.38 0.37 0.36 0.06
48 30 I 30 24 19 26 5
B. IgG-HAMA activity (mgkter) after the second treatment 4” 5 6
1.69 2.16 2.22
1.12 1.80 1.99
0.57 0.36 0.23
34 17 10
2.02 0.14
1.64 0.22
0.39 0.08
20 6
Mean
SEM
(1Only patients with detectable HAMA development are outlined above. ’ Maximal values which developed against the whole antibodies are shown. Patients 1, 2, and 3 were treated once, and patients 4, 5. and 6 were retreated.
detection system. Therefore, it was not surprising in the present investigation that there was a strong correlation between the titers of IgM rheumatoid factors and a preexisting HAMA activity of the IgM class in RA patients. However, it was an unexpected observation that no such correlations existed between rheumatoid factors and the IgG HAMA activity since rheumatoid factors of the IgG isotype are a typical finding in RA patients. Previous reports on HAMA activities in normal donors and cancer patients have proposed that the IgG HAMA activity seen in these groups may be attributable either to “multireactive” antibodies (18) or to low levels of IgG rheumatoid factors (2). The present investigation lends little support to the latter hypothesis unless one assumes that rheumatoid factors of the IgG isotype have a different distribution or are directed against completely different epitopes on mouse immunoglobulins as compared to their IgM counterparts. Alternative explanations for the presence of preexisting HAMA-like activities may include the contact to rodents as pets-particularly known to elicit IgEmediated allergic reactions. Indeed the only patient with an allergic skin reaction in our treatment series had kept a squirrel as a pet. The failure to detect HAMAs of the IgE isotype in this individual may be due to the low sensitivity of the assay system used.
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AND
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101
One way to circumvent the problem of preexisting HAMA activities in monitoring patients undergoing therapy with monoclonal murine antibodies appears to be the utilization of F(ab), fragments in the ELBA system as shown in the present investigation. Even though the sensitivity of detection will decrease-since the Fc part represents an important immunogenic region-the specificity appears to be greatly enhanced, especially in patient groups with high levels of rheumatoid factor activity. This is suggested by the observation in the present study that the development of IgM HAMA activity could be shown in the patients treated with the monoclonal reagent (Fig. 5). Therefore, these data suggest that using F(ab), fragments of the therapeutic antibody will be of great advantage in monitoring specific responses to murine antibodies. The present investigation demonstrated that therapy with the anti-CD4 antibody 16H5 led to the development of small amounts of HAMAs in five out of eight patients without a concomitant additive immunosuppressive treatment (four of them received 4 to 9 mg of prednisonelday, one had none). Only one of the three patients without the development of HAMAs was treated with prednisone (5 mg/day). One additional patient treated simultaneously with 50 mg of cyclophosphamid per day and a second subject who had received a total nodal radiation 5 years prior to antibody therapy did not develop HAMAs either of the IgG or IgM isotype. In all patients the IgG HAMA response did not exceed levels higher than 1.012.0 mg/liter after the first and 1.7/2.2 mg/liter after the second course of therapy as measured against the F(ab), fragment and the complete monoclonal antibody 16H5, respectively. This HAMA response developed during a phase of decreased counts of CD4 + T cells and inversed CD4KDS ratios in the peripheral blood which generally persisted 3-4 weeks after the anti-CD4 treatment (5). With regard to the high HAMA response of more than 100 mg/liter in immunocompetent patients treated with monoclonal murine antibodies against cancer antigens (1, 2, 19), the amounts of HAMAs found in our study were very low. Parallel investigations of the cellular immune system in the RA patients treated demonstrated a drastic and persistent depletion of CD4 + cells, a decreased antigen and mitogen-induced lymphocyte proliferation, and a decreased delayed-type hyperreactivity during therapy. Therefore, the low amounts of HAMAs detected here may be explained by a significant immunosuppression due to the anti-CD4 treatment. In addition, cell-mediated immune responses to murine immunoglobulines were determined by lymphocyte proliferation assays. There was a moderate rise in the proliferation index compared to the pretreatment situation. However, responses to common antigens like tetanus toxoid and diphteria toxoid demonstrated significant changes following anti-CD4 treatment with paradoxically elevated levels of T cell response in certain patients (5). Therefore, it was not possible to assess T cell reactivity to mouse immunoglobulins in a simple way. This will require further analysis on a clonal level. The HAMA response detected was primarily of the IgG isotype. Newly developed HAMA reactivity of the IgM isotype primarily occurred after a repeated treatment cycle. In general, the kinetics of the anti-F(ab), IgM response were comparable to the IgG HAMA activity, with lower amounts. The differences in the reactivities developed against the complete antibody
IO?
HORNEFF
L-f
Al
16H5 and the isotype control reagent are presumably due to an immune response against different epitopes. including the idiotype. After the first treatment cycle, 0.36 mg/liter (26% of total reactivity against the 16H5 antibody) of 16H5-specific HAMAs developed. and after the second treatment cycle the amounts detectable (0.39 mgiliter 20%) were comparable. In an additional experiment, sera were absorbed by immobilized murine IgG. By this procedure the increase of the response to the subclass control was diminished. With regard to the response to the 16H5 monoclonal antibody, 24%~of the total amount of HAMAs were still present after absorption. So, a demonstrable amount of HAMAs directed against the epitopes specific for the 16H5 monoclonal antibody (including the idiotype) developed. Overall, these data indicate that treatment with the anti-CD4 monoclonal antibody 16H5 leads to the development of only low amounts of anti-mouse antibodies which allow for at least one additional and possibly more treatment cycles without an apparent loss of efficacy. Concomitant or profound previous immunosuppressive therapy may be able to completely inhibit the development of anti-mouse immunoglobulins. Therefore, the human anti-mouse response does not appear to be a major problem in the initial studies using anti-CD4 monoclonal antibodies as a promising new approach to treat diseases with autoimmune features. REFERENCES 1. Schroff, R. W., Foon. K. A.. Beatty. S. M.. Oldham, R. K., and Morgan, A. C., Human antimurine immunoglobulin response in patients receiving monoclonal antibody therapy. Cancer Res. 45, 879-885, 1985. 2. Courtenay-Luck, N. S.. Epentos, A. A., Moore, R., Larche M., Pectasides, D., Dhokia, B.. and Ritter. M.. Development of primary and secondary immune response to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasm. Cancer Res. 46, 64896493. 1986. 3. Thistlethwaite, J. R.. Stuart. J. K.. Mayes. J. T., Gaber, A. O., Woodle, S., Buckingham, M. R., and Stuart, F. P.. Complications and monitoring of OKT3 therapy. Amer. J. Kidney Dis. 11, 112-I 19. 1988. 4. Ortho Multicenter Transplant Study Group, A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplant. N. Eng. J. Med. 313, 337-342, 1985. 5. Horneff. G., Burmester. G. R.. Emmrich, F.. and Kalden. J. R., Treatment of rheumatoid arthritis with an anti-CD4 monoclonal antibody. Arthritis Rheum.. in press. 6. Herzog, C.. Walker C., Pichler W.. Aeschhmann, A.. Wassmer, P., Stockinger, H., Knapp, W.. Rieber, P., and Miiller. W., Monoclonal anati-CD4 in arthritis. Lancer 2, 1461-1462, 1987. 7. Hafler, D. A., and Weiner, H. L.. Immunosupression with monoclonal antibodies in multiple sclerosis. Neurology 38, Suppl. 2. 4247, 1988. 8. Zweig, M. H., Csako, G., Benson. C. C.. Weintraub, B. D.. and Kahn. B. B., Interference by anti-immunoglobulin G antibodies in immunoradiometric assays of thyreotropin involving mouse monoclonal antibodies. Clin. Chem. 33, 840-844, 1987. 9. Primus. F. J., Kelley, E. A.. Hansen, H. J., and Goldenberg, D. M., Sandwich-type immunoassay of carcinoembryonic antigen in patients receiving murine monoclonal antibodies for diagnosis and therapy. C/in. Chem. 34, 261-264. 1988. 10. Benjamin, R. J.. Qin, S. X., Wise, M. P., Cobbold. S. P., and Waldmann, H., Mechanism of monoclonal antibody-facilitated tolerance induction: A possible role for the CD4 (L3T4) and CDlla (LFAI) molecules in self-non-self discrimination. Eur. J. Immune/. 18, 1079-1088, 1988. 11. Goronzy. J., Weyand, C. M., and Fathman, C. G., Long-term humoral unresponsiveness in vivo induced by treatment with monoclonal antibody against L3T4. J. Exp. Med. 164, 91 l-925, 1986.
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TREATMENT
103
12. Goldstein,G., Fuccello,A. J., Norman,D. J., et al., OKT3 monoclonalantibodyplasmalevels duringtherapyandthe subsequent development of hostantibodies to OKT3.Transplantafion 42, SO7-510. 1986. 13. Jaffers,G., Fuller, T. C., Cosimi,A. B.. et al., Monoclonalantibodytherapy:Antiidiotypic and nonantiidiotypicantibodiesto OKT3arisedespiteintenseimmunosuppression. Transplanfation 41. 572-582. 1986. 14. Amett. F. C., Edworthy. S. M.. Bloch. D. A.. McShane. D. J., Fries, J. F., Cooper, N. S.. Healey. L. A., Kaplan et ul.. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315-324. 1988. I.(. Moebius, U., Cluster Report CD4. In “White Cell Differentiation Antigens: Leukocyte Typing IV.” pp. 314-316, Oxford Univ. Press, London/New York, 1989. 16. Szer. I. S.. Irani. A.. Burmester, G. R.. and Winchester, R. J.. Four new surface antigens on T lymphocytes. In “Leukocyte Typing,” pp. 718-719, Springer-Verlag. New York/Berlin, 1981. 17. Carson. D. A., Rheumatoid factor. In “Textbook of Rheumatology” (W. N. Kelley, E. D. Harris. S. Ruddy, and C. B. Sledge, Eds.). 2nd ed., pp. 664679, Saunders, Philadelphia, 1985. 18. Casali, P., and Notkins. A. L., Probing the human B-cell repertoire with EBV: Polyreactive antibodies and CDS + B lymphocytes. Annu. Rev. Immunol. 7, 213-535. 1989. 19. Dillman, R. O., Human antimouse and antiglobulin responses to monoclonal antibodies. Antibody Inlmuno~onjuRates Radiopharm. 1, I-15. 1990. Received May 30. 1990; accepted with revision November 8, 1990
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
59, 89-103 (1991)
Human Anti-mouse Antibody Response Induced by Anti-CD4 Monoclonal Antibody Therapy in Patients with Rheumatoid Arthritis’ GERD HORNEFF, THOMAS WINKLER, JOACHIM R. KALDEN, FRANK EMMRICH,* AND GERD R. BURMESTER *Max Planck Clinical Research Institute of Clinical Immunology and Rheumatology, Department of Medicine Ill, Krankenhausstr. 12. University of Erlangen-NCrnberg, Federal Republic of Germany
Group,
The development of human anti-mouse monoclonal antibodies (HAMAs) was investigated in 10 patients with rheumatoid arthritis (RA) who had undergone an experimental therapeutic trial with an anti-CD4 monoclonal antibody. In this patient group, the antibody 16H5 of the IgGl isotype had been administered in a median total dosage of 140 mg per treatment cycle. Four patients took part in a second treatment regimen 6-8 weeks later. After the first treatment cycle, detectable HAMAs developed in 5 out of 10 patients. In 4 individuals undergoing a second course of therapy, increases of HAMAs were evident only in the 3 patients with previous HAMA responses. HAMAs were primarily of the IgG isotype, while the presence of rheumatoid factors usually interfered with the detectability of IgM HAMAs. However, using isolated Ftab), fragments of the monoclonal reagent used for therapy, HAMAs of the IgM isotype were also detectable. HAMAs of the IgG isotype did not exceed levels of 2.0 mg/hter after a single treatment cycle and 2.2 mg/hter after a repeated cycle. No IgE responses were detectable. Absorption experiments indicated that approximately 25% of the HAMA activity was directed against specific determinants of the 16HS monoclonal antibody, presumably including anti-idiotypic reactivities. These data demonstrate that HAMAs developed only in a proportion of RA patients treated with the anti-CD4 monoclonal antibody 16H5. However, the amounts were rather low compared to other monoclonal reagents used in cancer patients and were therefore allowed for repeated applications without an apparent loss
Of efficacy.
0 1991 Academic
PKS~, Inc.
INTRODUCTION Monoclonal antibody treatment and diagnostic procedures are increasingly used in cancer patients (1, 2), organ transplantation (3, 4), and certain diseases with autoimmune features, including rheumatoid arthritis (5-7). The development of human anti-mouse monoclonal antibodies (HAMAs) is of crucial importance with regard to repeated diagnostic and therapeutic procedures. Moreover, HAMAs interfere with in vitro assays using murine monoclonal antibodies in ELISA or IRMA systems (8, 9). Monoclonal antibody treatment has initially been used for cancer treatment administering reagents directed against tumor-associated antigens (1). Since these ’ Supported by the Federal Ministry of Research and Technology (grant “Lymphokine” 01 ZU 8601, No. 41) and the European Concerted Action on Immunopathogenesis and Immunotherapy of Chronic Arthritis. Dr. Homeff is a postdoctoral fellow of the Deutsche Forschungsgemeinschaft, Grant Ho143/1-2. 89 0090-1229191 $1.50 Copyright All tights
Q 1991 by Academic Press. Inc. of reproduction in any fan reserved.
antibodies are of xenogenic origin, the development of a strong immune respunsc against mouse reagents is not surprising. Ilnfortunately. this response inhibited further applications of mouse monoclonal antibodies in this patient group (2). This situation may be completely different in therapeutical trials where antibodies with highly immunosuppressive potential are used (IO, I I). The rational in these settings is that the immune system is severely suppressed by the antibody itself, thereby not allowing for a vigorous anti-mouse monoclonal antibody response. Especially, application of anti-CD4 and foreign antigens in parallel resulted in a persistant tolerance to the antigen in animal models (I I). However, studies using the OKT3 monoclonal reagent directed against all mature T cells for the treatment of organ rejection episodes have shown a considerable reactivity toward this reagent (12, 13). Therefore, the objective of the present study was to investigate the response against another immunomodulatory reagent directed against the CD4 antigen characteristic of the T helper cell subset. The data were derived from a therapeutic trial in patients with rheumatoid arthritis refractory to conventional therapy (5) treated with an anti-CD4 antibody in an attempt to induce a beneficial immunomodulation. MATERIALS
AND METHODS
Patients Patients undergoing the clinical trial with the anti-CD4 monoclonal antibody 16H5 suffered from severe and active rheumatoid arthritis according to the ACR criteria (14). The study protocol was designed according to the guide lines of the “Gesellschaft fur Immunologie” (Society for Immunology) and the Paul Martini Endowment. It was reviewed and approved by the ethical committee of the University of Erlangen-Ntirnberg. Written and informed consent was obtained from all patients after information about the possible risks of treatment. Effects seen in this patient group have been reported previously (5) and consisted of a persistent decrease of CD4 + cells, decreased CD4/CD8 ratios for 3 to more than 8 weeks, reduced lymphocyte proliferations induced either by mitogens (phythohemagglutinin, pokeweed mitogen, and concanavalin A) or antigens (tetanus toxoid, diphteria toxoid, and tuberculin), and reduced skin test responses along with significant clinical benefits. Side effects were mild elevations of body temperature in three cases, presumably due to cytokine release and an apparently allergic skin reaction in one individual. Concommitant therapy consisted of only nonsteroidal anti-inflammatory drugs (NSAIDs) in three patients, and NSAIDs and prednisone (less than 10 mg/day) in six patients. One patient was on a low dosage of cyclophosphamide (50 mg/day) in addition to 10 mg of prednisone due to severe vasculitis. Another individual had received a total nodal irradiation because of severe RA 5 years prior to this therapeutic trial. The antibody was administered intravenously in an approximate dosage of 0.3 mg/kg body weight during an infusion time of 30 min in the morning on 7 subsequent days. The total amount of antibodies applicated varied from 105 to 175 mg
HAMAs
AND
ANTI-CD4
TREATMENT
91
(median, 140 mg). Repeated treatment cycles were performed using the same dosage in three patients and a higher dose in one individual (total amount: 140 mg vs 10s mg). Sera from 10 patients (9 rheumatoid factor positive) treated with reagent 16H5, 18 additional RA patients (13 seropositive in the Waaler Rose test, range 50 to 800 IE) without antibody treatment, and 8 normal donors without a history of joint diseases or allergy were tested. All sera were separated immediately from peripheral blood and stored at - 20°C until used. Rheumatoid factors were determined using standard Waaler Rose and Latex agglutination assays. In addition, rheumatoid factors were measured by the ELISA technique. Absorption
of Sera
To determine the anti-idiotypic proportion of the anti-mouse response, sera were diluted 1:20 in phosphate-buffered saline (PBS), and absorption was performed using immobilized murine IgG (Pierce). Sera were incubated for 1 hr and subsequently separated by centrifugation. In parallel, absorbed sera and nonabsorbed sera were tested on plates coated with the monoclonal antibody 16H5 and the subclass control antibody as described below. Preparation
of Antibodies
The murine monoclonal antibody 16H5 (IgGl K) directed against the CD4 antigen present on the T-helper/inducer cell subset (15) had been purified for treatment according to the regimens of the European Community Committee for Proprietary Medicinal Products (June 1986,111/859/86EN). F(ab), fragments of this antibody were prepared by incubation of the antibody with immobilized Pepsin (Pierce) in 0.02 M sodiumacetate buffer (pH 4.2) for 4 hr. After neutralization with Tris-HCl and dialysis against PBS, F(ab), fragments were isolated from undigested IgG by affinity chromatography using a protein G column (Pharmacia, Uppsala, Sweden). Subsequently, dialysis against PBS with a membrane cutoff point of 30 kDa was performed to eliminate contaminating protein fragments. The presence of F(ab), fragments was verified by SDS gel electrophoresis. The purity of this preparation was tested by the ELISA technique which demonstrated a good reactivity with F(ab),-specific antisera, while there was no reactivity with an Fc-specific goat anti-mouse antiserum. The monoclonal anti-CD3 reagent 89bl (IgGl K) was used as an isotype control (16). After an ip injection of the mouse hybridoma 89b1, ascites was obtained from BALB/c mice. After precipitation with half-saturated ammonium sulfate solution and resuspension in PBS, the antibody was purified using a protein G fast flow column (Pharmacia). ELISA
The respective antigens were coated to standard ELISA plates (NUNC, Roskilde, Denmark) in a concentration of 10 kg/ml in 0.05 sodium carbonate (pH 9.6) for 18 hr at +4”C. Testing for coating efficacy using an alkaline phosphataseconjugated goat anti-mouse antiserum revealed equivalent amounts of murine IgG coated to the plates. The plates were washed three times with PBS containing 0.05% Tween (Sigma Chemical Co., Munich, FRG). Excess binding sites were
92
HORNEFF
El
Al
blocked by postcoating the wells with 10% (v/v) fetal calf serum in PBS-Tween for I hr at room temperature. Washing procedures were repeated. For comparison, on each plate standard curves were generated using a defined primate anti-mouse immunoglobulin solution (Medac, Hamburg, FRG) containing 220 rig/ml of antimouse antibodies. Experiments had been carried out to investigate the quality of this standard preparation. In these assays, dilutions of a human IgG preparation (Medac) were coated to an ELISA plate in concentrations from 500 down to 3.9 rig/ml). In parallel, the murine monoclonal l6H5 was coated in a concentration of 10 pg/ml to the same plate. In a second step, a serial dilution of the primate anti-mouse antibody preparation was added to the well coated with the murine monoclonal reagent. In a third step, both serial dilutions (the human IgG coated directly to the plastic, the primate IgG binding to the murine antibody) were incubated with a goat anti-human IgG alkaline phosphatase conjugate. Bound primate anti-mouse antibodies of the standard preparation used in the assays revealed equivalent optical densities in a range from 3 to 200 rig/ml compared to defined amounts of a human IgG preparation directly coated to the wells. Sera were diluted I :20 with PBS-Tween and incubated at room temperature for 90 min. After consecutive washing procedures, a 1: 1000 dilution of an alkaline phosphate-conjugated goat anti-human immunoglobulin specific for y or p, chains was added (Medac). After incubation for 90 min and consecutive washing procedures, staining was performed using p-nitrophenyl-phosphate (Sigma) in a concentration of 0.5 mg/ml in 0.05 M sodium bicarbonate solution containing 0.01 M magnesium chlorid. Substrate turnover was stopped after 10 min and subsequently determined at 405 nm versus 630 nm with an ELISA reader (Titertek multiskan, EFLAB, Finland). Optical densities were calculated to nanograms per milliliter using standard curves derived from each plate. For the detection of HAMAs of the IgE class, a three-step ELISA system including a biotin/streptavidine step (biotinylized goat anti-human IgE and a horseradish peroxidase-conjugated streptavidin, all Medac) with increased sensitivity was used. For the detection of rheumatoid factors by the ELISA technique, rabbit IgG was coated as described above. Sera were diluted 1: 10 for determining IgG rheumatoid factor and 1:50 for IgM rheumatoid factor. After incubation for 90 min and subsequent washing procedures, goat anti-human IgG and IgM were added, respectively. The quality of this assay was tested using sera of 10 normal donors, 14 seronegative, and 25 seropositive (according to the Waaler Rose and Latex assay) RA patients. There was a correlation coefficient to the Latex assay of 0.53 (P < 0.005) for the IgG and 0.68 (P < 0.0001) for the IgM rheumatoid factor determined by the ELISA technique. In the IgG assay, normal donors revealed extinctions of 535 t 113 versus 397 & 188 in seronegative RA patients and 971 ‘-t- 142 of seropositive RA patients. In the IgM assay, these values were 260 ‘-’ 40, 275 * 108, and 609 + 82, respectively. Differences of seropositive RA patients vs seronegative and normal donors were highly significant (P < 0.0001). Statistical Methods Correlations between quantitative
variables were tested by means of simple
HAMAs
AND ANTI-CD4
93
TREATMENT
linear regression analysis. The differences seen in normal donors and RA patients were compared using the Student t test after testing for normal distribution using the Kolmogoroff Smirnov test. RESULTS Preexisting HAMA Responses in RA Patients and Normal Donors
The detection of a newly formed human antibody response against xenogenic antibodies is influenced by preexisting anti-immunoglobulin antibodies, especially of the rheumatoid factor type which is a human anti-IgG antibody primarily detectable in the IgM isotype fraction. Figure 1 demonstrates the reactivity of sera from normal donors and untreated patients with rheumatoid arthritis. Anti-murine immunoglobulin antibodies were detected in the ELISA system using plates coated with either the whole antibody 16H5 or the F(ab), fragment. Using the whole antibody as antigen coupled to the plates, there was a significant correlation between the IgM responses toward 16H5 and the titers of rheumatoid factor in the Waaler Rose test (r = 0.76, P < 0.001). In contrast, there were no significant correlations between titers of rheumatoid factors and the IgM responses against the F(ab), fragment (r = 0.06, P = 0.81) or the IgG responses against both the whole antibody (Y = 0.19, P = 0.71) or the F(ab), fragment (r = -0.16, P = 0.53). Therefore, in all subsequent experiments, IgG responses were tested against both the whole antibody and the F(ab), fragment and IgM responses were investigated against the F(ab), fragment only. The rheumatoid factors determined by the Latex agglutination test or the
IgG
‘gM
”
16H5 Normal
F(ab)Z Donors
16H5
F(ab)2 RA
Patients
16H5 Normal
F(ab)Z Donors
16H5
F(ab)P
RA
Patients
FIG. 1. Human anti-mouse response measured in the sera of 9 normal donors and 18 untreated RA patients (IgG in mg/liter, IgM in OD). A considerable HAMA activity of the IgM class may be due to the presence of rheumatoid factors as indicated by the marked reduction of HAMA activity against the F(ab), fragment as compared to the whole antibody. There was a significant difference of HAMA responses between the normal donors and RA patients, both against the whole antibody and the F(ab), fragment (P < 0.01, asterisks). Preexisting HAMA activity of the IgG class did not correlate to rheumatoid factors and was found in normal donors as well as in RA patients without a significant difference (P > 0.10).
94
HORNkFl,
1.1
‘\I
Waaler Rose test were decreased in 7 out of 9 rheumatoid factor-positive patients immediately at the end of the treatment period, while in three individuals titers of rheumatoid factors were slightly increased (one dilution step) during the whole observation period. Thus. there was no evidence of increasing levels of rheumatoid factor resulting in positive HAMA levels. Especially. no correlation was found between the levels of rheumatoid factor determined by Waaler Rose or Latex agglutination and the IgG or IgM HAMAs against F(ab), fragments in the sera used in this investigation. In addition, rheumatoid factors were evaluated by the ELISA technique as described above. In Fig. 2, levels of HAMAs of the IgG (A) and 1gM (B) isotype were compared to rheumatoid factors determined by the ELISA technique in the sera of a patient who had undergone a repeated treatment regimen. There was no increase of either the IgG or the IgM response to rabbit IgG in contrast to a demonstrable anti-mouse response. HAMA
Responses
in RA Patients
after Treutnrent
a*ith an Anti-CD4
Antibody
Figure 3 demonstrates the IgG responses of two individuals after a single treatment (A) and a second treatment cycle (B) against the therapeutical antibody l6H5, the isotype control, and the F(ab)? fragment of the 16H5 reagent. There was a low preexisting reactivity against both complete antibodies. The patient in Fig. 3A was rheumatoid factor negative as determined by the Waaler Rose and the Latex agglutination assays, while the patient in Fig. 3B had a high titer (800 IU). Concommitant therapy consisted of NSAIDs and 9 mg (patient A) or 8 mg (patient B) prednisone. In both patients, HAMAs developed 2-3 weeks after the treatment had been started. HAMAs were detectable for IO-12 weeks after a single treatment. There was a parallel development of HAMAs against the whole antibody and the F(ab)? fragment with a higher response against the antibody used for therapy compared to the isotype control antibody. A second treatment cycle (Fig. 3B) resulted in enhanced levels of HAMAs which included reactivities against the epitopes present on the F(ab), fragment. Figure 4 shows the HAMA responses against the F(ab), fragment of 16H5 of all patients treated. There was a demonstrable elevation in 5 out of IO individuals after a single treatment (A and B), but in three cases only did HAMAs reach levels higher than the untreated healthy control population (mean t SD was 0.23 ? 0. I I mg/liter). A maximum level of 0.8 mg/liter developed above baseline, and elevations of HAMAs were detectable for l&12 weeks. In 3 out or 4 patients, a rise in the HAMA titer was developed after the second treatment cycle (Fig. 4B, arrows). HAMAs reached levels of less than I.7 mg/Iiter after the booster. The I individual without a demonstrable HAMA response (upper curve) had a remarkable preexisting activity without a booster reaction. In contrast, in this patient the level of HAMA activity decreased after each treatment cycle, possibly due to a consumption of preexisting anti-mouse antibodies upon therapy. The HAMA response of the IgM isotype directed against the F(ab), determinants obtained in four patients treated twice is shown in Fig. 5. The development of this type of IgM HAMAs was comparable to the IgG isotype in this group and was the most pronounced after the second treatment cycle (B,D). In six patients
HAMAs
AND
ANTI-CD4
95
TREATMENT
0.6
0
a
0 0
20
40
60
60
100
120
80
100
120
timefdays) 6)
1200: 4 1000
8
,,I
600
piiq
C
400
-
0
20
40
60
time(dsys)
of IgG (A) and IgM (B) HAMAs and rheumatoid factor activity in a patient with rheumatoid arthritis who had undergone a repeated anti-CD4 treatment. HAMA responses to the whole antibody (a)+ HAMA response to the F(ab), fragment (m), and response to rabbit IgG (A) (0) 0% (0). FIG. 2. Kinetics
who received a single treatment cycle, IgM HAMAs directed against the F(ab), fragment were not demonstrable (data not shown). IgE responses were not detectable in the patients treated twice nor in the one patient who had developed skin urticaria (the limit of sensitivity of the assay was 1.5 x&ml). Also, this patient did not show a preexisting IgG or IgM response to the monoclonal antibody and did even not develop demonstrable HAMAs.
96
HORNEFF
A)
ET AL.
IgG HAMA
000
20
40
60
80
100
120
80
100
120
time(days)
B)
0
20
40
60 time(days)
FIG. 3. IgG HAMA responses after a single (A) or repeated (B) treatment regimen against the whole antibody 16H5, the F(ab), fragment, and an isotype control antibody. The figure illustrates the two individuals with the highest amounts of IgG HAMAs that developed after treatment. The first 7-day treatment was started at t = 0 days, the repeated treatment regimen was started at t = 32 days (B) in this patient. There was a preexisting elevated IgG HAMA activity against both whole antibodies as compared to the F(ab), fragment. The patient in A was rheumatoid factor negative while the patient in B was highly positive (800 W/ml).
Anti-idiotypic
Response
Compared to the response against the isotype control antibody investigated in parallel, the HAMA reactivity developing against the 16H5 antibody was higher in each case. Absorption of the sera by immobilized murine IgG resulted in an unresponsiveness to the subclass control while the response to 16H5 was reduced to 24% at the time point of the maximal HAMA response as shown in Fig. 6. In Table 1, the maximal values of HAMAs of the IgG isotype against both complete antibodies are shown. The preexisting reactivities of all 6 patients’ sera outlined
HAMAs
4
AND
Single
ANTI-CD4
treatment
97
TREATMENT
cycle
0.8
0.6
-,
0.4
F
-
mean +2SD
-
+lSD
0.2
-
0 20
40
60
80
100
time(days)
Two
8)
treatment
cycles
1.8
1.5
0.6
0
20
40
60
80
100
120
140
tlme(days)
FIG. 4. IgG HAMA development after a single or repeated treatment regimens against the F(ab), fragment. Seven-day treatment cycles are indicated by arrows. There was a remarkable HAMA development above the means -+ I SD of 18 untreated RA patients in 2 out of 6 patients after a single treatment (A) and in 3 or 4 patients who had undergone a repeated treatment cycle (B).
in Table I were comparable (0.66 +- 0.12 mg/liter for the 16H5 antibody and 0.68 5 0.15 mgiliter for the isotype control antibody). After treatment, HAMAs of the IgG isotype were detectable in 5 out of 10 individuals as judged by values higher than the means 5 SD of untreated RA patients and the healthy control population.
98
HORNEFf
El Al
‘loo B
+ 30” ff I i
FIG. 5. IgM HAMA development against the F(ab)2 fragment in four patients who had undergone a repeated treatment regimen. Seven-day treatment cycles are indicated by arrows. There was a detectable HAMA response of the IgM class in three out of the four patients who had undergone a repeated treatment cycle.
In one additional individual (No. I), low levels of HAMAs were detectable, not exceeding the mean baseline of untreated RA patients. In all of these 6 individuals, the difference in response to either the monoclonal reagent used or to the subclass control was 0.36 k 0.06 mg/liter (26 k 5% of the total response). In 3 out of 4 individuals who underwent a repeated trial, a rise in HAMA levels was detectable, and the difference in response was 0.39 +- 0.08 mg/liter (20 + 6%) of the total reactivity. DISCUSSION
The investigation of the development of human anti-mouse antibodies in patients with rheumatoid arthritis treated with an anti-CD4 monoclonal antibody led to the following principle observations: 1. In RA patients, preexisting antibodies of the IgM class with an anti-mouse antibody activity showed strong correlations to the presence of IgM rheumatoid factors. 2. In contrast, IgG antibodies with HAMA activity did not show correlations to rheumatoid factors. 3. Specific HAMA activity was primarily of the IgG isotype, indicating a secondary immune response. Interestingly, increased levels of specific HAMA activity of the IgM-isotype against the F(ab)* part were the most pronounced after the second treatment cycle. 4. Treatment with an anti-CD4 monoclonal antibody resulted in only low
HAMAs
0
AND ANTI-CD4
1 0
20
I
40
.
1
60
.
1
80
99
TREATMENT
.
I
100
I
120
.
140
I
I
160
180
time(days)
0
] 0
,
I 20
40
'
I. 60
I 80
*
I. 100
I 120
-
I 140
'
I. 160
I 180
time(days)
FIG. 6. Response to the whole antibody 16H5 (A) and the subclass control (B) before (0) and after absorption (0) on immobilized murine IgG in a patient who had undergone a repeated treatment cycle (arrows).
amounts of HAMAs, even after a second treatment course, and these were demonstrable only in 5 out of 10 subjects. 5. Approximately 25% of HAMA activities are directed against specific determinants of the 16H5 antibody, including anti-idiotypic reactivities. The study of human anti-mouse immunoglobulin antibodies appears to be a particular problem in individuals with rheumatoid arthritis, a patient group that is characterized by the presence of high titers of antibodies toward the Fc portion of immunoglobulins, i.e., the rheumatoid factors. These antibodies are not only directed toward antigenic determinants of the human Fc part, but also react with antigens present on rodent antibodies, as has been thoroughly studied with rabbit sera (for review see Ref. (17)). Indeed, the Waaler Rose test, commonly used to identify rheumatoid factors, uses rabbit IgG bound to sheep erythrocytes in the
100
HORNEFF El AL. ‘TABLE
DIFFERENCES
IN HAMA
I
ACTIVITY AGAINST THE ANTIBODY ISOTYPE CONTROL ANTIBODY
USED
FOR TREATMENT
AND
AN
I6h5 (mg/liter)
Control tmgiliter)
Difference tmg/liter)
‘$,
A. IgG-HAMA activity (mg/liter) after a single treatment cycle I” 2 3 4 5 6 Mean SEM
o.88h 1.25 1.61 1.66 1.56 I .98 1.49 0.14
0.46 0.87 1.48 1.17 1.18 1.61 1.11 0.15
0.42 0.38 0.13 0.49 0.38 0.37 0.36 0.06
48 30 I 30 24 19 26 5
B. IgG-HAMA activity (mgkter) after the second treatment 4” 5 6
1.69 2.16 2.22
1.12 1.80 1.99
0.57 0.36 0.23
34 17 10
2.02 0.14
1.64 0.22
0.39 0.08
20 6
Mean
SEM
(1Only patients with detectable HAMA development are outlined above. ’ Maximal values which developed against the whole antibodies are shown. Patients 1, 2, and 3 were treated once, and patients 4, 5. and 6 were retreated.
detection system. Therefore, it was not surprising in the present investigation that there was a strong correlation between the titers of IgM rheumatoid factors and a preexisting HAMA activity of the IgM class in RA patients. However, it was an unexpected observation that no such correlations existed between rheumatoid factors and the IgG HAMA activity since rheumatoid factors of the IgG isotype are a typical finding in RA patients. Previous reports on HAMA activities in normal donors and cancer patients have proposed that the IgG HAMA activity seen in these groups may be attributable either to “multireactive” antibodies (18) or to low levels of IgG rheumatoid factors (2). The present investigation lends little support to the latter hypothesis unless one assumes that rheumatoid factors of the IgG isotype have a different distribution or are directed against completely different epitopes on mouse immunoglobulins as compared to their IgM counterparts. Alternative explanations for the presence of preexisting HAMA-like activities may include the contact to rodents as pets-particularly known to elicit IgEmediated allergic reactions. Indeed the only patient with an allergic skin reaction in our treatment series had kept a squirrel as a pet. The failure to detect HAMAs of the IgE isotype in this individual may be due to the low sensitivity of the assay system used.
HAMAs
AND
ANTI-CD4
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One way to circumvent the problem of preexisting HAMA activities in monitoring patients undergoing therapy with monoclonal murine antibodies appears to be the utilization of F(ab), fragments in the ELBA system as shown in the present investigation. Even though the sensitivity of detection will decrease-since the Fc part represents an important immunogenic region-the specificity appears to be greatly enhanced, especially in patient groups with high levels of rheumatoid factor activity. This is suggested by the observation in the present study that the development of IgM HAMA activity could be shown in the patients treated with the monoclonal reagent (Fig. 5). Therefore, these data suggest that using F(ab), fragments of the therapeutic antibody will be of great advantage in monitoring specific responses to murine antibodies. The present investigation demonstrated that therapy with the anti-CD4 antibody 16H5 led to the development of small amounts of HAMAs in five out of eight patients without a concomitant additive immunosuppressive treatment (four of them received 4 to 9 mg of prednisonelday, one had none). Only one of the three patients without the development of HAMAs was treated with prednisone (5 mg/day). One additional patient treated simultaneously with 50 mg of cyclophosphamid per day and a second subject who had received a total nodal radiation 5 years prior to antibody therapy did not develop HAMAs either of the IgG or IgM isotype. In all patients the IgG HAMA response did not exceed levels higher than 1.012.0 mg/liter after the first and 1.7/2.2 mg/liter after the second course of therapy as measured against the F(ab), fragment and the complete monoclonal antibody 16H5, respectively. This HAMA response developed during a phase of decreased counts of CD4 + T cells and inversed CD4KDS ratios in the peripheral blood which generally persisted 3-4 weeks after the anti-CD4 treatment (5). With regard to the high HAMA response of more than 100 mg/liter in immunocompetent patients treated with monoclonal murine antibodies against cancer antigens (1, 2, 19), the amounts of HAMAs found in our study were very low. Parallel investigations of the cellular immune system in the RA patients treated demonstrated a drastic and persistent depletion of CD4 + cells, a decreased antigen and mitogen-induced lymphocyte proliferation, and a decreased delayed-type hyperreactivity during therapy. Therefore, the low amounts of HAMAs detected here may be explained by a significant immunosuppression due to the anti-CD4 treatment. In addition, cell-mediated immune responses to murine immunoglobulines were determined by lymphocyte proliferation assays. There was a moderate rise in the proliferation index compared to the pretreatment situation. However, responses to common antigens like tetanus toxoid and diphteria toxoid demonstrated significant changes following anti-CD4 treatment with paradoxically elevated levels of T cell response in certain patients (5). Therefore, it was not possible to assess T cell reactivity to mouse immunoglobulins in a simple way. This will require further analysis on a clonal level. The HAMA response detected was primarily of the IgG isotype. Newly developed HAMA reactivity of the IgM isotype primarily occurred after a repeated treatment cycle. In general, the kinetics of the anti-F(ab), IgM response were comparable to the IgG HAMA activity, with lower amounts. The differences in the reactivities developed against the complete antibody
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16H5 and the isotype control reagent are presumably due to an immune response against different epitopes. including the idiotype. After the first treatment cycle, 0.36 mg/liter (26% of total reactivity against the 16H5 antibody) of 16H5-specific HAMAs developed. and after the second treatment cycle the amounts detectable (0.39 mgiliter 20%) were comparable. In an additional experiment, sera were absorbed by immobilized murine IgG. By this procedure the increase of the response to the subclass control was diminished. With regard to the response to the 16H5 monoclonal antibody, 24%~of the total amount of HAMAs were still present after absorption. So, a demonstrable amount of HAMAs directed against the epitopes specific for the 16H5 monoclonal antibody (including the idiotype) developed. Overall, these data indicate that treatment with the anti-CD4 monoclonal antibody 16H5 leads to the development of only low amounts of anti-mouse antibodies which allow for at least one additional and possibly more treatment cycles without an apparent loss of efficacy. Concomitant or profound previous immunosuppressive therapy may be able to completely inhibit the development of anti-mouse immunoglobulins. Therefore, the human anti-mouse response does not appear to be a major problem in the initial studies using anti-CD4 monoclonal antibodies as a promising new approach to treat diseases with autoimmune features. REFERENCES 1. Schroff, R. W., Foon. K. A.. Beatty. S. M.. Oldham, R. K., and Morgan, A. C., Human antimurine immunoglobulin response in patients receiving monoclonal antibody therapy. Cancer Res. 45, 879-885, 1985. 2. Courtenay-Luck, N. S.. Epentos, A. A., Moore, R., Larche M., Pectasides, D., Dhokia, B.. and Ritter. M.. Development of primary and secondary immune response to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasm. Cancer Res. 46, 64896493. 1986. 3. Thistlethwaite, J. R.. Stuart. J. K.. Mayes. J. T., Gaber, A. O., Woodle, S., Buckingham, M. R., and Stuart, F. P.. Complications and monitoring of OKT3 therapy. Amer. J. Kidney Dis. 11, 112-I 19. 1988. 4. Ortho Multicenter Transplant Study Group, A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplant. N. Eng. J. Med. 313, 337-342, 1985. 5. Horneff. G., Burmester. G. R.. Emmrich, F.. and Kalden. J. R., Treatment of rheumatoid arthritis with an anti-CD4 monoclonal antibody. Arthritis Rheum.. in press. 6. Herzog, C.. Walker C., Pichler W.. Aeschhmann, A.. Wassmer, P., Stockinger, H., Knapp, W.. Rieber, P., and Miiller. W., Monoclonal anati-CD4 in arthritis. Lancer 2, 1461-1462, 1987. 7. Hafler, D. A., and Weiner, H. L.. Immunosupression with monoclonal antibodies in multiple sclerosis. Neurology 38, Suppl. 2. 4247, 1988. 8. Zweig, M. H., Csako, G., Benson. C. C.. Weintraub, B. D.. and Kahn. B. B., Interference by anti-immunoglobulin G antibodies in immunoradiometric assays of thyreotropin involving mouse monoclonal antibodies. Clin. Chem. 33, 840-844, 1987. 9. Primus. F. J., Kelley, E. A.. Hansen, H. J., and Goldenberg, D. M., Sandwich-type immunoassay of carcinoembryonic antigen in patients receiving murine monoclonal antibodies for diagnosis and therapy. C/in. Chem. 34, 261-264. 1988. 10. Benjamin, R. J.. Qin, S. X., Wise, M. P., Cobbold. S. P., and Waldmann, H., Mechanism of monoclonal antibody-facilitated tolerance induction: A possible role for the CD4 (L3T4) and CDlla (LFAI) molecules in self-non-self discrimination. Eur. J. Immune/. 18, 1079-1088, 1988. 11. Goronzy. J., Weyand, C. M., and Fathman, C. G., Long-term humoral unresponsiveness in vivo induced by treatment with monoclonal antibody against L3T4. J. Exp. Med. 164, 91 l-925, 1986.
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12. Goldstein,G., Fuccello,A. J., Norman,D. J., et al., OKT3 monoclonalantibodyplasmalevels duringtherapyandthe subsequent development of hostantibodies to OKT3.Transplantafion 42, SO7-510. 1986. 13. Jaffers,G., Fuller, T. C., Cosimi,A. B.. et al., Monoclonalantibodytherapy:Antiidiotypic and nonantiidiotypicantibodiesto OKT3arisedespiteintenseimmunosuppression. Transplanfation 41. 572-582. 1986. 14. Amett. F. C., Edworthy. S. M.. Bloch. D. A.. McShane. D. J., Fries, J. F., Cooper, N. S.. Healey. L. A., Kaplan et ul.. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315-324. 1988. I.(. Moebius, U., Cluster Report CD4. In “White Cell Differentiation Antigens: Leukocyte Typing IV.” pp. 314-316, Oxford Univ. Press, London/New York, 1989. 16. Szer. I. S.. Irani. A.. Burmester, G. R.. and Winchester, R. J.. Four new surface antigens on T lymphocytes. In “Leukocyte Typing,” pp. 718-719, Springer-Verlag. New York/Berlin, 1981. 17. Carson. D. A., Rheumatoid factor. In “Textbook of Rheumatology” (W. N. Kelley, E. D. Harris. S. Ruddy, and C. B. Sledge, Eds.). 2nd ed., pp. 664679, Saunders, Philadelphia, 1985. 18. Casali, P., and Notkins. A. L., Probing the human B-cell repertoire with EBV: Polyreactive antibodies and CDS + B lymphocytes. Annu. Rev. Immunol. 7, 213-535. 1989. 19. Dillman, R. O., Human antimouse and antiglobulin responses to monoclonal antibodies. Antibody Inlmuno~onjuRates Radiopharm. 1, I-15. 1990. Received May 30. 1990; accepted with revision November 8, 1990
