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[Cancer Research 60, 1921-1926, April 1, 2000]
© 2000 American Association for Cancer Research

Immunology

The Human Antimouse Immunoglobulin Response and the Anti-idiotypic Network Have No Influence on Clinical Outcome in Patients with Minimal Residual Colorectal Cancer Treated with Monoclonal Antibody CO17–1A

Rudolf Gruber1, Leonardus J. M. van Haarlem, Sven O. Warnaar, Elena Holz and Gert Riethmüller

Institut für Immunologie der Ludwig-Maximilians-Universität München, 80336 Munich, Germany [R. G., G. R.]; University Hospital Leiden, 2300 RC Leiden, the Netherlands [L. J. M. v. H., S. O. W.]; and Amgen GmbH, 80992 Munich Germany [E. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Murine monoclonal antibodies (mAbs), when administered to patients, induce a human antimouse immunoglobulin immune response, especially when multiple infusions are required to obtain therapeutic efficacy. In a randomized Phase II clinical study, 83 patients with colorectal carcinoma of stage Dukes C were treated with the murine IgG2a mAb 17–1A (ab1) after curative surgery. The regimen consisted of a single infusion of 500mg of 17–1A within 2 weeks after surgery, followed by 100mg of mAbs four times every 4 weeks. Sera were taken every 2–3 weeks and screened for human antimouse antibodies (HAMA). HAMA were measured by a capture ELISA and an indirect antihuman immunoglobulin ELISA for the analysis of IgG and IgM isotypes. Anti-idiotypic antibodies (ab2) were detected by an inhibition ELISA, and anti-anti-idiotypic antibodies (ab3), recognizing the original antigen, were determined by flow cytometric analysis. About 20% of patients failed to develop HAMA; in the other patients, antibody titers were initially low after the first two infusions and reached their maximum only after a fifth infusion at 18–20 weeks after surgery. An analysis that differentiated between patients who developed recurrences and those who remained tumor-free did not show any difference in antibody titers between the two groups, neither for total HAMA nor for IgG, IgM, or ab2. The formation of ab3 was analyzed in eight patients and proved to be negative in all of them. HAMA remained detectable up to 2 years after the last treatment. In patients who experienced adverse events associated with therapy, HAMA titers tended to rise earlier; this difference, however, was not statistically significant. Thus, neither a beneficial nor a detrimental effect of HAMA formation could be determined for the clinical response to antibody therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently the interest in the therapeutic applications of mAbs2 have been revived, particularly of those directed at tumor-associated antigens. Although single infusions may be adequate for tumor imaging applications, effective tumor therapy with mAbs will require multiple infusions (1 , 2) . Because these mAbs were until now derived from murine or rat hybridomas, their immunogenicity in patients is not surprising. Serological analysis of sera from patients in clinical trials with mAbs have revealed the HAMA response that was characterized as quantitative amounts of total HAMA and specifically ab2 and ab3 (3, 4, 5, 6) . An attempt to correlate HAMA formation and therapeutic efficacy has yielded equivocal results thus far. On the one hand, HAMA may neutralize the injected mAb directly by immune complex formation, which could lead to rapid clearance or to hypersensitivity reactions (7, 8, 9) . On the other hand, HAMA may also be associated with a positive clinical outcome. Apart from direct cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent tumor cell lysis, and apoptosis induction, ab2 and ab3 have been postulated according to the network hypothesis of Jerne to induce tumor cell rejection (5 , 10, 11, 12) . Evidence has been proposed for a specific T-cell response to the mouse immunoglobulin that could be correlated to the induction of the anti-idiotypic cascade (ab1 -> ab2 -> ab3; Refs. 13, 14, 15 ). These later results have even stimulated vaccination trials in which ab2, either monoclonal or polyclonal—produced against the internal image of a therapeutic mAb—were used to raise ab3 antibodies against the tumor antigen. In experimental tumor systems, ab2 have been shown to induce antigen-specific humoral and cellular immune responses that result in the suppression of tumor growth. Clinical trials with polyclonal antibodies and mAbs (ab2), mimicking the colorectal-carcinoma-associated antigen 17–1A, have been carried out that also report an increased survival of patients (16 , 17) . Most of these trials have been carried out on only a few patients who had terminal tumor disease and who were immunosuppressed by the tumor burden or by chemo- and radiotherapy. Moreover, the patient populations in these reports were rather heterogeneous, and the response pattern to mAb therapy has been barely subjected to a rigorous statistical analysis. Therefore, we here present data of total HAMA, ab2, and ab3 from patients with resected Dukes C colorectal carcinoma randomized to a prospective adjuvant antibody therapy trial (18 , 19) .


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
All of the sera analyzed in this study had been taken from patients participating in a prospective randomized multicenter clinical trial with mAb 17–1A. Informed consent was obtained from each subject. One hundred eighty-nine patients with colorectal carcinoma stage Dukes C were entered into the trial. After surgery, the patients were randomized to a control arm, i.e., observation only, and to a treatment group. The treatment group received 500 mg of mAb 17–1A within 2 weeks of surgery followed by four subsequent monthly infusions of 100 mg of the antibody. According to the most recent follow-up of 7 years, therapy with the antibody was found to decrease the overall death rate by 32% and reduced the recurrence rate by 23%. Details of the treatment protocol, patient characteristics, response criteria, and responses have been published previously (19) . From each patient, 17 serum samples (1 pretreatment sample and 16 samples obtained during and after the mAb treatment) were intended to be analyzed for the occurrence of HAMA. This report includes 60 patients with more than three serum samples available for HAMA testing, one patient with three serum samples available, and five patients with one serum sample. From 12 treated patients, no sera were available. All of the sera were tested for the presence of total HAMA, but only the 60 patients with more than three serum samples were included in the statistical analysis, and, because of the clinical outcome, the patients were allocated into the two groups "tumor-free" and "recurrence." Furthermore, 25 sera from 5 patients in the control group and 25 healthy controls were also tested for the occurrence of HAMA. All of the sera were stored at -20°C until use. For the analysis of ab2, 110 sera from 23 patients and, for the analysis of ab3, 64 sera from 8 patients were tested.

mAbs.
Mouse mAb 17–1A (IgG2a), raised against the tumor-associated antigen CO17–1A/GA733/Ep-CAM, was used both for treatment of patients and in vitro tests. Ep-CAM is a Mr 37,000–40,000 surface glycoprotein that is expressed on malignant and normal epithelial cells and is probably involved in homotypic intercellular adhesion and adhesion to extracellular matrix (20) .

Human Antibodies against mAb 17–1A.
Two different ELISA systems were applied for the detection of total HAMA. In the first assay, mAb 17–1A diluted in carbonate buffer (pH 9.6) at a concentration of 10 µg/ml was coated in 100 µl at 4°C overnight onto flat-bottomed microtiter ELISA plates (Greiner, Nürtingen, Germany). After washing and blocking with PBS/2% MP plates were incubated for 2 h with serum samples diluted 1:3 and 1:9 in PBS/2% MP. Bound HAMA were detected by the binding of biotinylated 17–1A diluted at 5 µg/ml in PBS/2% MP followed by incubation with an avidin-peroxidase conjugate, diluted 1:2500 in PBS/2% MP (Dako Corp., Hamburg, Germany). After extensive washing, ABTS [2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (Roche, Mannheim, Germany) was added as substrate, and the extinction was measured in an ELISA-reader (SLT-Labinstruments, Nürtingen, Germany) at 405 nm. A standard curve was established and run in each ELISA-plate. The standard consisted of rabbit antimouse immunoglobulin (Dako Corp.) serially diluted from 1:100 to 1:3200. The antimouse-immunoglobulin content of this standard was 400 µg/ml; therefore, corresponding concentrations of the standard curve were 4 µg/ml to 62.5 ng/ml. Absorbance (A) values of samples below the A of the 1:3200 dilution of the standard were considered as negative. The HAMA concentrations of the samples were quantified according to the estimated rabbit antimouse activity of the standard curve. A commercially distributed HAMA-ELISA (Medac, Hamburg, Germany) was used for validation and comparison of this HAMA-ELISA. The test principle of that ELISA was identical, with the exception of using mouse immunoglobulin and biotinylated mouse immunoglobulin instead of mAb 17–1A and 17–1A-bio, respectively. The assay was done according to the manufacturer’s instructions. A second ELISA assay was used to measure IgG and IgM HAMA, respectively. mAb 17–1A was coated on microtiter plates at 200 µg/ml diluted in PBS (pH 7.0) and was incubated overnight at 4°C. After washing and blocking the wells with PBS/1%BSA, serial 1:2 dilutions of patients sera starting at 1:50 were incubated for 2 h at 37°C. After washing, bound human immunoglobulin was detected either by a goat antihuman IgG or IgM antiserum conjugated to alkaline phosphates. A chromogen solution was added and measured on an ELISA reader. The reciprocal dilution of the serum that yields an absorbance reading of 1.0 in the assay is referred to as the HAMA titer.

Detection of ab2.
The ab2 were detected in an inhibition ELISA. The ELISA plates were coated with goat-anti-17–1A ab2 (kindly provided by D. Herlyn, Wistar Institute, Philadelphia, PA), and the patients’ sera were serially diluted and coincubated with mAb 17–1A. Thus, the inhibition of 17–1A antibody binding to the goat-anti-idiotype antibody because of ab2 in the patients’ sera was measured. The bound 17–1A antibody was detected by antimouse-immunoglobulin antibodies (Dako Corp.) conjugated to peroxidase. After extensive washing, ABTS was added as substrate and the extinction was measured in an ELISA reader at 405 nm.

ab3.
In eight patients, human anti-17–1A antibodies (ab3) were measured by flow cytometric analysis. 17–1A-positive tumor cells [50,000 (Kato, ATCC)] were incubated at 4°C for 30 min with patients’ sera, diluted 1:10 in PBS/2%MP or with mAb 17–1A at serial dilutions from 10 µg/ml to 10ng/ml. After washing, bound immunoglobulin was detected by rabbit antihuman immunoglobulin FITC or rabbit antimouse immunoglobulin FITC (Dako Corp., Hamburg, Germany), respectively, and was analyzed on a flow cytometer (FACScan, Becton Dickinson, Heidelberg, Germany). The mean fluorescence channel was calculated for the sera and the standard curve by histogram analysis. The specificity of ab3 binding to the 17–1A molecule was evaluated by inhibition experiments. Positive sera were serially diluted from 1:10 to 1:320 and incubated with Kato cells. After washing, mAb 17–1A (10µg/ml) was incubated with the cells and detected with rabbit-antimouse-FITC.

Statistical Analysis.
The correlations of the different HAMA assays were analyzed using Spearman rank order correlations. The differences of HAMA titer between the tumor-free and the relapse group and the patients with and without adverse events were calculated using the two-sided Student t test with the statistical program STATISTCA/Mac (StatSoft Inc., Tulsa, OK).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparing Total HAMA, IgG HAMA, and IgM HAMA.
We used two very different ELISA assays for the quantification of total HAMA and the classification of HAMA isotypes. The ELISA for total HAMA is based on the capture of biotin-conjugated 17–1A mAb by antiglobulins in the patients serum. Therefore, this assay cannot distinguish between different HAMA isotypes, because the bridge between the coated 17–1A and the biotinylated 17–1A can be made by all of the isotypes and also by soluble antigen. As for the 17–1A antigen, it is known that it is not soluble and is never found in the circulation. The rabbit antimouse immunoglobulin, used for establishing the standard curve, had a specific antimouse immunoglobulin content of 400 µg/ml. A values at dilution up to 1:3200 of this antiserum were clearly above the background and, thus, were used as sensitivity cutoff of this assay (62.5 ng/ml). In this assay, 12 patients (20%) of the 60 were negative for HAMA during the whole course of therapy, 7 patients developed a maximum of 250 ng/ml, and 2 patients had more than 6000 ng/ml HAMA. The remaining 39 patients developed HAMA between 250 and 6000 ng/ml (Fig. 1Citation ). All of the pretreatment sera of these patients, the 25 sera of the control group, and the 25 sera of healthy controls were negative for HAMA. For the detection of the HAMA isotypes, we used an indirect antihuman immunoglobulin detection system. As expected for an immune response to foreign protein, the IgM titers rose earlier than the IgG titers with a peak at weeks 4–9 after the first mAb infusion for IgM and at weeks 15–17 for IgG, respectively (Fig. 2Citation and 3Citation ). A quantification in absolute HAMA concentrations per ml of serum was not done for this assay. The relative antimouse activity of the sera was expressed as inverse values of the last dilutions yielding an A of 1.0 (see "Materials and Methods"). For IgG, titers less than 1:50 were regarded as negative. Here, 6 of the 60 patients had low pretreatment titers (<100). Six of the patients were negative during the whole course of therapy, and nine patients developed very low HAMA titers (<200). Seven patients showed very high titers (>20,000). As known for other immune responses, for IgM maximal titers remained relatively low. Titers less than 1:10 were regarded as negative. Twenty-five patients showed low pretreatment antimouse immunoglobulin activity. Eleven patients remained negative during the whole course of the therapy. A comparison of the peak HAMA titers of all of the patients was done. Despite the essential difference of the ELISA systems, a reasonable correlation was found between the capture ELISA and the indirect IgG ELISA (r, 0.69; P < 0.0001). However, three patients were negative or reached very low titers (<100) in the IgG ELISA but showed medium or high titers in the capture ELISA (>500ng/ml), and 8 patients reached titers of >100 in the IgG ELISA but were only low-positive (<250ng/ml) or negative in the capture ELISA.



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  		Fig. 1. Average of total human antimouse immunoglobulins HAMA capture ELISA, based on the capture of biotin-conjugated 17–1A mAb by antiglobulins in the patients serum. Twelve (20%) of the 60 patients were negative for HAMA during the whole course of therapy. For statistical analysis, the patients were differentiated into those remaining tumor-free ({blacksquare}) and those who developed recurrences ({square}). There was no significant difference in antibody titers between the two groups (two-sided Student’s t test). Double arrow, the first mAb infusion (500 mg); single arrows, the following infusions (100 mg each).

 


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  		Fig. 2. Average IgG human antimouse immunoglobulins HAMA isotypes detected by an indirect antihuman immunoglobulin ELISA. Serial dilutions of patients’ sera starting at 1:50 were measured. The reciprocal dilution of the serum that yields an absorbance reading of 1.0 in the assay is referred to as the HAMA titer (Y axis). IgG titers rose slowly with a peak at weeks 15–17. Tumor-free ({blacksquare}); tumor recurrences ({square}). Double arrow, the first mAb infusion (500 mg); single arrows, the following infusions (100 mg each).

 


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  		Fig. 3. Average IgM human antimouse immunoglobulins. Same assay as in Fig. 3Citation . Serial dilutions of sera started at 1:10. As expected for an immune response to foreign protein, the IgM titers rose earlier than the IgG titers, with a peak at weeks 4–9 after the first mAb infusion. Tumor-free ({blacksquare}); tumor recurrences ({square}). Double arrow, the first mAb infusion (500 mg), single arrows, the following infusions (100 mg each).

 
ab2 Titers.
An inhibition assay that detects ab2 from the ability to block the binding of the therapeutic mouse mAb 17–1A to a goat anti-idiotypic antiserum was used. We could detect blocking antibodies in 18 of 23 tested patients with titers up to four times that of the pretreatment value. The blocking antibodies increased slowly up to week 12 after surgery and remained elevated during the whole observation time. The absolute amounts of ab2 remained relatively low with a maximum of 5 µg/ml in one patient (Fig. 4Citation ).



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  		Fig. 4. Average of ab2. ab2 could be detected in 18 of 23 tested patients with titers up to four times that of the pretreatment value. The ab2 increased slowly up to week 12 after surgery and remained elevated during the whole observation time. The absolute amounts of ab2 remained relatively low with a maximum of 5 µg/ml in one patient. Tumor-free ({blacksquare}); tumor recurrences ({square}). Double arrow, the first mAb infusion (500 mg); single arrows, the following infusions (100 mg each).

 
ab3.
The induction of ab3 was tested by flow cytometric analysis with 17–1A-positive tumor cells. The specificity of binding to the 17–1A molecule could be shown by the inhibition of mAb 17–1A binding by positive sera diluted up to 1:80. Sixty-four sera from 8 patients were tested. Three patients had no detectable binding of human immunoglobulin to 17–1A-positive Kato cells, three patients had detectable reactivity (mean channel, >150), and two patients showed a strong reactivity (mean channel, >300). But in those latter patients, this reactivity was detectable before administration of the first mAb infusion in each patient, and no significant change in human anti-17–1A titers during therapy could be seen, which indicated that the binding detected was not specific for any ab3 (Fig. 5Citation ).



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  		Fig. 5. ab3. Sixty-four sera from eight patients were tested for the binding of human immunoglobulin to 17–1A-positive Kato cells. Three patients had no detectable binding, three patients had detectable reactivity (mean channel, >150), and two patients showed a strong reactivity (mean channel, >300). In the latter groups, this reactivity was detectable before the administration of the first mAb infusion, and no significant change in ab3 titers during therapy could be seen. Study centers in which the patients were treated: Han, Hannover; HH, Hamburg; Koe, Cologne; Mue, Munich.

 
HAMA Titer and Outcome of Therapy with 17–1A mAb.
A summary of patients treated with mAb 17–1A including the total dosage, adverse events during the mAb therapy, and the time point of relapse is shown (Table 1)Citation . A detailed description of the study and the clinical data are published elsewhere (18) . After the first 2 infusions, antibody titers were low and reached their maximum only after a fifth infusion, 18–20 weeks after surgery. 80% of treated patients developed a HAMA response after the second or third infusion. An analysis that differentiated between patients developing recurrences and those remaining tumor-free did not show a significant difference in antibody titers between the two groups. According to the total HAMA ELISA, 7 (23%) of the 30 analyzed tumor-free patients and 5 (17%) of patients with tumor relapse did not develop HAMA at all. Also for IgG HAMA, IgM HAMA, and ab2, no statistically significant difference between the two groups could be found (Fig. 1Citation 2Citation 3Citation ). There was a tendency that HAMA titers occurred earlier in the patients with adverse events, but these differences were not significant (Fig. 6Citation ). There was also no association of HAMA titers with relapse time after surgery or localization of relapse, i.e., local versus distant metastasis (data not shown).


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  		Table 1 Summary of all of the patients tested for the occurrence of HAMA

 


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  		Fig. 6. Comparison of HAMA titer in patients with and without adverse events associated with mAb therapy. ELISA assay as shown in Fig. 1Citation . Patients were divided into those with ({blacksquare}) or without ({square}) adverse events during mAb therapy. Double arrow, the first mAb infusion (500 mg); single arrows, the following infusions (100 mg each).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
mAbs offer abundant potential benefits to diagnosis and therapy of many conditions. However, with widespread use, comes greater concern regarding possible side effects and complications. The potential immunogenicity of murine mAbs, the type most commonly available, is of great concern. Regarding the prevalence of human antimurine immunoglobulin in the general population and the mechanism by which such antibodies are elicited, little—and often discrepant—data have been published. There is also no agreement for the optimal methods for assays of antimouse antibody response, and no data on the differences between antibodies detected in one or another assay procedure or on the predictive value of assays for the detection of HAMA are available. A question in this latter realm concerns the use of HAMA as a predictor of allergic responses or other adverse events of the mAb therapy. Furthermore, are strategies that aim at diminishing HAMA really necessary and effective clinically, or may HAMA, at least in some cases, even augment the desired effects (21) ?

In this study, we used two different principles for the detection of HAMA. The ELISA for total HAMA is based on the capture of biotin-conjugated mAb 17–1A by antimurine antiglobulins in the patient’s serum. This assay has some advantages over the indirect antihuman immunoglobulin detection system, particularly in that there is no potential for nonspecific cross-reactions between the patient’s serum and the detection reagent, or between the mAb bound to plastic and the antiglobulin. Moreover, backgrounds due to low-affinity rheumatoid factors are avoided because these would be unlikely to be captured effectively. There is an advantage in the quantification of the HAMA titer because hyperimmune serum from other than human species can be used in the same assay. However, this assay cannot distinguish between different HAMA isotypes, inasmuch as the bridge between the coated 17–1A and the biotinylated 17–1A can be made by all of the isotypes as well as by soluble antigen (22 , 23) . As for the 17–1A antigen, it is known that it is not soluble and is never found in the circulation (24) . For the detection of the IgM and IgG immune response, we used the indirect antihuman immunoglobulin detection system. Therefore, we can compare the HAMA results of these two different assay principles for 579 sera from 60 patients. There is a reasonable correlation between the total HAMA detected in the capture ELISA and the IgG values measured in the indirect antihuman immunoglobulin detection system. We conclude that the results of these two systems are comparable. However, three patients were negative or had very low titers in the IgG ELISA but showed medium or high titers in the capture ELISA, and five patients had medium-to-high titers in the IgG ELISA but were only low-positive or negative in the capture ELISA. Furthermore, one has to be careful about unspecific binding with the indirect antihuman immunoglobulin detection system as discussed in the literature (23) and shown in this study in the validation phase. Using acidic conditions for the coating of the mouse mAb hydrophobic sites on the antibody molecule that might become exposed can present antigenic sites that cross-react, for example, with IgG-type rheumatoid factor molecules. This may be responsible for the binding of many preimmune sera and false-positive HAMA reactions. Taken together, both of the HAMA ELISAs are very sensitive, and each is specific for the detection of an antimouse immunoglobulin response. Another possible way to overcome those methodological problems in the quantification of HAMA is the use of affinity chromatography (25) .

Our data demonstrate that the immune response of cancer patients treated with mouse mAbs is very heterogeneous, i.e., 20% of patients showed no detectable HAMA response at all, an additional 10% showed only very low titers of antibodies, and about 10% showed very high HAMA titers. The majority (60%) of patients showed medium levels of an antimouse immunoglobulin immune response, which were insufficient to neutralize the repeated doses of 100 mg of 17–1A. This is in agreement with previous therapeutic trials with mouse mAb against the 17–1A antigen in which the HAMA response is also quite heterogeneous, and 7–46% of the patients do not develop HAMA at all (26, 27, 28, 29, 30, 31) . The initial antibody dose (500 mg) was chosen because patients receiving 500 mg or more on the first infusion develop an antibody response less frequently than patients receiving smaller doses, as shown by previous studies (32) . Multiple injections of antibody were scheduled in the study design; therefore, a scheme of antibody administration that allowed tolerance induction seemed logical. For antimurine IgM antibodies, the highest titers were found only after the second or third injection, and titers remained relatively low. Because the IgM response usually takes place within the first 2 weeks after the first antigen contact, the patients in this study responded slowly to the 17–1A administered. HAMA IgG titers rose slowly too and reached only moderate maximal levels. These data confirm former studies of tolerance induction for mouse mAb infusions with high protein concentrations and support the use of this therapeutic scheme for additional studies (32) .

There is great concern about the potential induction of adverse events by HAMA. The inhibition of binding of the therapeutic mAb and blocking its effects, the immune complex formation with enhanced clearance of mAb from the circulation, and allergic reactions have all been described in the literature (7, 8, 9) . But, despite the presence of HAMA, experience has shown that the vast majority of HAMA-positive patients have no symptoms on additional mAb infusions (22) . In our study, adverse events were reported in 25 of the 60 patients analyzed for HAMA (33) . Comparing the HAMA response from these 25 patients versus the 35 patients without adverse events, there was a tendency of HAMA titers to occur earlier in the first group, but this difference was not significant.

On the other hand, there is great interest in the mechanisms of a potential positive effect of HAMA. Unconjugated mAbs inhibit growth of malignant tumor both in animal models (34) and in patients (35) . In addition to direct antitumor effector functions like antibody-dependent cell cytotoxicity complement-dependent cytolysis, and apoptosis (36 , 37) , the induction of an idiotypic network response has also been proposed to mediate tumor-cell killing, as an indirect effector mechanism (17 , 38) . ab2 and ab3 have been shown to be induced by mAb treatment in cancer patients and have been suggested to be of benefit for the patients (11 , 39 , 40) . Most of these studies were not controlled, and the case numbers were low. Here, we could compare HAMA from 60 patients in a controlled randomized mAb therapy trial. For statistical analysis, the patients were differentiated into those developing recurrences and those remaining tumor-free. The determination day for the grouping was the 7-year median follow-up date. At that time point, 28 patients were tumor-free, and 32 patients had had a local or distant relapse between 3 and 92 months after surgery. At no time point of our HAMA measurements was there any significant difference in the HAMA titers between the two groups in either assay (capture ELISA, IgG, IgM). Moreover, in the subgroup of patients analyzed for the ab2 immune response (10 patients in the tumor-free group, 13 patients in the relapse group), at no time point was there a significant difference for the ab2 titers. As already mentioned, we were not able to detect an induction of ab3. From eight patients tested for the presents of ab3, two had high "ab3-activity" and three had detectable and three had undetectable human anti-EpCAM/17–1A antibodies. However, those who were positive were already positive before treatment, which is probably due to thee formation of autoantibodies against 17–1A in these tumor patients, a phenomenon also described in other studies (41) .

The immune response of tumor patients to mouse mAbs is highly heterogeneous. In this study, this heterogeneity correlates neither with the response to therapy nor with adverse events. In conclusion, in our patients, there were no significant positive or negative effects of HAMA.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Institut für Immunologie der Ludwig-Maximilians-Universität, Goethestrasse 31, 80336 Munich, Germany. Phone: 89-5160-7678; Fax: 89-5160-4908; E-mail: Rudolf.Gruber{at}pk-i.med.uni-muenchen.de Back

2 The abbreviations used are: mAb, monoclonal antibody; HAMA, human antimouse antibody/antibodies; ab2, anti-idiotypic HAMA; ab3, anti-anti-idiotypic HAMA; MP, nonfatty dry milk powder. Back

Received 9/10/99. Accepted 2/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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