Survival benefit associated with human anti-mouse antibody (HAMA) in patients with B-cell malignancies
Background: About one-third of patients with relapsed B-cell malignancies develop human anti-mouse antibody (HAMA) following mouse antibody treatment. The purpose of this study was to assess the relationship between HAMA and survival in patients given a mouse anti-lymphoma monoclonal antibody (mAb), Lym-1, directed against a unique epitope of HLA-DR antigen that is up-regulated on malignant B-cells. Methods: ELISA was used to quantify HAMA in 51 patients with B-cell malignancies treated with iodine-131 (131I) labeled Lym-1. Sera were collected prior to and following radioimmunotherapy (RIT) with 131I-Lym-1 until documented to be HAMA negative or throughout lifetime. Univariate, then multivariate analyses including other risk factors, were used to analyze the relationship of HAMA to survival. The relationships of HAMA to prior chemotherapies and to absolute lymphocyte counts prior to RIT were also assessed. Results: Eighteen of 51 patients (35%) developed HAMA following RIT (range of ultimate maximum titers, 6.6–1,802 μg/ml). Using the time dependent Cox proportional hazards model, maximum HAMA titers were associated with survival (P=0.02). HAMA continued to be significant for survival in multivariate analyses that included known risk factors. In Landmark analysis of 39 patients that survived at least 16 weeks, median survival of patients with HAMA less than 5 μg/ml was 61 versus 103 weeks for patients with HAMA equal or greater than 5 μg/ml at 16 weeks (P=0.02). The median survival of the five patients with highest maximum HAMA titers was 244 weeks. At 16 weeks, there was an inverse correlation between the maximum HAMA titer and the number of previous chemotherapies (P<0.003). Absolute lymphocyte counts prior to 131I-Lym-1 treatment for patients that seroconverted were higher than those for patients that did not seroconvert (P=0.01). Conclusions: Patients with B-cell malignancies that developed high HAMA titers had longer survival that was not explained by risk factors or histologic grade, suggesting the importance of the immune system.
KeywordsNon-Hodgkin’s lymphomaRadioimmunotherapySurvivalHuman anti-mouse antibodyMonoclonal antibodyHLA-DR
Immunotherapy and radioimmunotherapy (RIT) using mAbs have favorable efficacy–toxicity profiles in B-cell malignancies, such as non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL) [1–5]. A polyclonal human antibody response, either anti-isotypic or anti-idiotypic in nature, can occur whether the administered mAb is mouse or has been “humanized”, albeit the frequency and titer are higher in the former circumstance . Although the clinical sequelae of human anti-mouse antibody (HAMA) are usually minor [7–9], subsequent treatment may be affected by rapid clearance of the mAb from the blood . Seroconversion frequencies range from 1 to 35% in patients with relapsed B-cell malignancies and from 40 to 100% in patients with solid tumors [1, 11–15], in part reflecting known differences in the immunocompetence of these patients.
A survival benefit, linked to the development of anti-idiotypic antibodies, has been reported for HAMA [13, 16–18]. The purpose of this study was to assess the relationship between HAMA and survival in patients with B-cell malignancies given multiple doses of a mouse anti-lymphoma mAb, Lym-1, directed against an HLA-DR antigen up-regulated on malignant B-cells . HLA-DR binds exogenously-derived peptides providing a repertoire of peptides on the surface of antigen-presenting cells for inspection by CD4 positive T cells. HLA-DR residues critical for Lym-1 binding are in the immediate area of the peptide presenting pit. HAMA was mentioned briefly in an earlier examination of 27 radiolabeled Lym-1 treatment factors . Because of the potential importance of an association of HAMA and survival to the future clinical management of patients with B-cell malignancies, the data herein represent an in depth survival analysis and discussion. Additionally, biostatistical evaluations to assess whether the association between HAMA and survival could be explained by known risk factors for these malignancies or factors known to be related to immune competence have been conducted.
Similarity of the characteristics of the patients entered in the two trials
Trial no. 1
Trial no. 2
No. of patients
Mean age (range)
Multiple dose trial design
Mean KPS (range)
Ann Arbor stage 4/3
Mean prior therapies (range)
Overall response rate
Mean total Lym-1 mg (range)
Of the 53 patients entered in the trials, 51 were evaluable for HAMA response. Two patients were excluded because of death on days 2 and 9 after initial exposure to Lym-1; in these two patients there was insufficient time to develop HAMA . The patient population included 31 males and 20 females. Median KPS was 70 (range 40–90), and median age was 54 years (range 29–74). Histologic grade included 8 patients with high-grade, 27 intermediate-grade and 16 low-grade NHL. Five patients with CLL and adenopathy are included in the latter group. Forty-seven patients were Ann Arbor stage IV NHL and four were stage III.
Human anti-mouse antibody was titered before, during and after RIT until death . Because these trials were designed for administration of multiple doses of 131I-Lym-1, they provided an opportunity to characterize HAMA and survival over time. The date of first treatment with 131I-Lym-1 was uniformly used for all time dependent analyses HAMA seroconverters fell into either of two categories differing in seroconversion time and titer. HAMA titer was considered in the analyses in addition to simple seroconversion status. Possible relationships between maximum HAMA titer, survival and known risk factors were assessed. In an earlier multivariate analysis of factors prognostic for RIT outcomes in B-cell malignancies, pretherapy serum LDH and KPS best predicted survival, complete and partial remission and time to progression after 131I-Lym-1 treatment . A risk factor index based on LDH and KPS, and the IPI (that includes these and three other factors and has been reported to be predictive of chemotherapy outcomes ) were used to test the significance of HAMA titer in time dependent, multivariate proportional hazards analyses of survival. The relationships of HAMA to prior chemotherapies and to absolute lymphocyte counts prior to RIT were also assessed.
Lym-1 (Damon Biotechnology, Needham Heights, MA, USA or Techniclone, Inc., Tustin, CA, USA) is an IgG2a mouse mAb with high affinity against a discontinuous epitope of the beta chain of the HLA-DR10 antigen up-regulated on the surface membrane of malignant B-lymphocytes . The Lym-1 antigen is not modulated, and Lym-1 activates antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and direct cytotoxicity on target Raji human NHL cells in vitro [24, 25].
Lym-1 was labeled with 131I using chloramine-T at a mass ratio of 1–10 μg of Lym-1, and high specific activity 131I sodium iodide in 0.05N sodium hydroxide. Radioiodination provided radiolabeling efficiencies of 97±2% and 131I-Lym-1 specific activities of 10 mCi/mg of Lym-1.
Serum samples were obtained from each patient prior to exposure to 131I-Lym-1 (baseline), prior to each subsequent 131I-Lym-1 treatment, 1 month and biannually after the last 131I-Lym-1 treatment. HAMA titers were quantified using a two-step detection method in an ELISA format and 96-well plates (PRO-BIND™’ Becton Dickinson, Lincoln Park, NJ, USA), as described . Briefly, Lym-1 coated plates with non-specific binding sites blocked were used to capture HAMA. The captured IgG was detected and amplified in a second step using biotinylated goat anti-human IgG (Amersham, Arlington Heights, IL, USA) or biotinylated mouse anti-human IgG (Sigma, St. Louis, MO, USA) followed by streptavidin-horseradish peroxidase (Amersham) and ABTS substrate (Sigma). The optical density of each well was read at 405 nm in an ELISA reader (Dynatech MR-300, Chantilly, VA, USA). Serial twofold dilutions of serum containing a known amount of HAMA were used to generate a reference standard curve for each ELISA assay to quantify each patient’s HAMA titer. The HAMA titer was defined as the amount in the patient’s least-dilute sample that fell within the linear portion of the standard curve. For treatment purposes a positive HAMA was defined as a value greater than 5 μg/ml. This value was selected because it was four standard deviations above the mean of the baseline titer for a group of 15 volunteers. The maximum HAMA titer was defined as the highest HAMA titer observed for a patient among all follow-up samples, and may not have represented the true maximum in the patients that died. For time dependent analyses, the maximum HAMA titer up to that point in time was used.
Number of initial 51 patients seroconverting and surviving at each time point following first 131I-Lym-1 treatment
Survival curves were estimated based on the method of Kaplan–Meier . Survival was measured from the date of first treatment with 131I-Lym-1, to death or to the date of this analysis; one patient was alive at this time. The relationship of HAMA seroconversion at 16 weeks and absolute lymphocyte count prior to 131I-Lym-1 treatment was assessed using the Wilcoxon rank sum test; seven patients with large numbers of circulating malignant lymphocytes were not included in this analysis. The association of maximum HAMA by 16 weeks with the number of prior chemotherapies  was analyzed using the Spearman rank correlation coefficient.
Relationship of seroconversion to protocol, histologic grade and risk factors
Risk level (LDH, KPS)
From time of first 131I-Lym-1 treatment, the median survival of the 51 patients was 45 weeks. The median survival of the 5 patients with highest maximum HAMA titers (all greater than 1,000 μg/ml) was 244 (range 61–579) weeks.
Association of maximum HAMA titer at 16 weeks (5, 20, or 80 μg/ml) with survival using the Landmark model on 39 patients alive at 16 weeks
Maximum HAMA (μg/ml)
Survival at 2 years (%)b
Patients with B-cell malignancies have been documented to be immunodeficient . This is the first detailed report showing a survival benefit for a group of patients that seroconverted following mAb treatment for B-cell malignancies. Using the time dependent Cox proportional hazards method and the maximum HAMA titer up to each point in time for all 51 patients, HAMA was significantly and directly related to patient survival. In order to further assure that there was no bias in the assessment, the Landmark method of analysis was also used at 16 weeks after initial 131I-Lym-1 treatment. This time point was chosen because 39 of 51 patients remained alive and 15 of the 18 seroconverting patients had done so, 11 of these having reached their ultimate maximum by that time. For patients still alive at 16 weeks, the presence of HAMA remained significantly related to survival. For patients with positive HAMA at 16 weeks, the estimated 2-year survival was twice that for the patients failing to seroconvert. If the analysis threshold for HAMA titer was increased, survival advantage was retained. HAMA seroconversion and titer were not related to the number of doses/exposures and amounts of Lym-1 received by these patients . Additionally, no relationship between HAMA and risk categories for these malignancies was observed; HAMA remained significantly related to survival in multivariate analyses that considered known risk factors.
“Humanized” antibodies have been engineered for use in immunotherapy and radioimmunotherapy. One of the most successful of this class of therapeutics, rituximab (Rituxan®) has achieved widespread use in the treatment of NHL because of its attractive efficacy and adverse event profiles . B-cell depletion occurs because this chimerized anti-CD20 mAb has potent cytocidal effects on both normal and malignant B-lymphocytes. Highly durable remissions have been reported for subsets of patients treated with two other anti-CD20 mAbs, 131I-tositumomab (Bexxar™) and 90Y-ibritumomab tiuxetan (Zevalin™) [30–32]. Currently, intense study is underway to define the nature and extent of these extended remissions. However, they are likely unrelated to HAMA for the following reasons. With either of these drugs as a pretreatment, a large amount of unconjugated mAb, that induces B-cell depletion, is given. Induction of HAMA is rare (less than 1%) following 90Y-ibritumomab tiuxetan with rituximab pretreatment . HAMA occurs more frequently with tositumomab (up to 8% in patients having previously received chemotherapy) likely because both pretreatment and treatment mAbs are mouse in origin . When used as first line therapy for follicular lymphoma, tositumomab had a higher HAMA rate . There may be another relevant difference between the anti-CD20 mAbs and Lym-1. Lym-1 binds to a unique epitope that includes the same HLA-DR beta chain residue, R71, that binds antigenic peptides displayed on HLA-DR . Furthermore, Lym-1 does not reduce the peripheral blood lymphocyte count but does decrease the number of circulating malignant lymphocytes .
It has been reported that cancer patients developing a HAMA after mAb exposure can have unexpectedly prolonged survival [11, 13, 34–37], but the relationship between HAMA and survival is controversial [11, 13, 18, 35, 37, 38]. To date, the largest trial using unconjugated mAb for randomized adjuvant therapy in colon cancer failed to show a clear survival benefit related to HAMA , although an earlier, randomized trial by the same group did show that the mouse mAb “extend(ed) life and prolong(ed) remission” . These discrepancies may be explained by failure of the analyses to consider the requirement for a threshold HAMA titer . Miotti et al.  showed that 88% of the patients treated with a bispecific antibody for ovarian cancer had a HAMA but only patients with HAMA titers greater than 150 μg/ml (56% of the HAMA positive patients) showed a survival advantage. Similarly, higher HAMA titers were associated with longer survivals in our patients treated with 131I-Lym-1. Evidence supporting the hypothesis that the survival benefit incident to HAMA is related to an idiotypic antibody cascade that includes the development of anti-idiotypic antibodies (Ab2) and anti-anti-idiotypic antibodies (Ab3), reflecting mirror images of the cancer antigen and the administered mAb, respectively, has been described [16, 17, 40, 41]. In one of our patients, direct evidence for initiation of a multilevel idiotypic cascade that was associated with prolonged disease-free survival was documented . This patient with aggressive, Ann Arbor stage IV NHL developed within 1 month of initial 131I-Lym-1 treatment a high HAMA titer that was sustained over her 9-year survival. The HAMA reflected an idiotypic cascade that consisted mostly of anti-idiotypic antibodies that included anti-Lym-1 specific antibodies (Ab2) and antibodies (Ab3) potent in a cytotoxicity assay (ADCC) against Raji human Burkitt’s NHL cells [16, 42]. The patient’s prolonged survival despite aggressive, advanced stage NHL may, thus, be understood in the context of the generation induced by 131I-Lym-1 treatment of HAMA and endogenous, self-perpetuating tumor-specific antibodies.
Although these observations remain to be confirmed and their exact implications for immunotherapy and radioimmunotherapy, and mAb choices therein, remain to be determined, their clinical significance for the management of cancer, specifically NHL, seems important because the observations underscore the need to consider the immune implications of treatment. Certainly, the choice of mAb seems relevant despite appreciation that complex factors are involved. Specific to the Lym-1 mAb, it binds poorly to normal lymphocytes and its HLA-DR epitope is up-regulated on malignant lymphocytes . HLA-DR is the most highly represented HLA class II subset. Furthermore, Lym-1 binds the same HLA-DR beta chain residue that binds exogenously-derived antigenic peptides displayed on HLA-DR . These factors are worthy of consideration in view of the presumed generation of an idiotypic antibody cascade in an immune deficient population of patients.
Among the patients alive at 16 weeks, there was an inverse relationship between the maximum HAMA titer to that time and the number of previous chemotherapies. This observation is not surprising given the effect of the aforementioned therapies on the responsiveness of the human immune system. Further evidence in support of an immune basis for the survival advantage associated with seroconversion was provided by the significantly higher absolute number of lymphocytes prior to 131I-Lym-1 treatment in patients that seroconverted.
The pivotal role of B and T cells in immune surveillance, and the depleting effect of cytotoxic therapies on these cells, has been recognized, documented and expanded upon for several decades. One scenario for a revised cancer treatment strategy is that proposed by Timmerman  who suggested that immunosuppressive drugs be deferred until later (unless curative). This would allow for initial activation of the antibody repertoire before treatment-induced abrogation of the host’s ability to mount an immune response.
In summary, elevated HAMA titer was associated with survival benefit in patients with B-cell malignancies, putatively due to induction of an idiotypic antibody cascade. HAMA titer remained significant in multivariate analyses, correlated negatively with the number of previous treatments, and seroconversion was directly related to pretreatment absolute lymphocyte counts. These observations have potential significance for the management of patients with cancer, specifically B-cell malignancies. It is to be hoped that they will be confirmed and elaborated upon by others in prospective studies.
Conflict of interest
The authors declare that no conflicts of interest exist.
This research was supported by grants from the National Cancer Institute (PHS CA 47829) and the Department of Energy (DE FG03-84ER60233). I.A. was supported by a grant from the Hospital San Jaime, Torrevieja, Alicante (Spain).