Antibodies Induced By Cytokine Therapy

Induction of anticytokine Ab has been reported in patients treated with human recombinant cytokines such as IFN-a, IFN-P, IFN-y, IL-2, and GM-CSF (3,6,7,123-127). ELISA and Western blotting have been used most frequently for the detection of cytokine-binding Ab, but this does not necessarily predict a neutralizing capacity of these Ab in vitro or in vivo (see below). Furthermore, these Ab are in many cases not autoreactive and therefore cannot be considered as autoantibodies, because they fail to react with the native cytokines. For example, patients with response failure to human recombinant IFN-a due to Ab development may respond to natural IFN-a (6). On the other hand, patients with preexisting Ab, at least to IFN-a, appear to be particularly at risk of developing high levels of neutralizing Ab and response failure (C. Ross et al. unpublished data).

Interferons

Since the introduction of pharmaceutical preparations of recombinant IFN-a, IFN-P, and IFN-y, it is being increasingly recognized that antibodies to these cytokines develop in many patients, particularly during prolonged therapy (6). There is some controversy, however, as to whether the development of such antibodies always leads to response failure. Some investigators distinguish between ''binding'' and ''neutralizing'' Ab and argue that only the latter have clinical importance. In this regard, it is appropriate to realize that immunohis-tochemical binding analyses and biological assays often vary significantly in sensitivity, specificity, and precision. For example, misinterpretations of antiviral neutralization bioassays are likely if they are carried out without controls for sample toxicity and endogenous antiviral activity. In our experience, high-titered ''binding'' IFN Ab always neutralize their respective cytokine in vitro. It is therefore advisable to monitor patients on IFN therapy for IFN Ab using validated analyses. Should Ab develop, determinations of their specificity might allow for rescue treatment using alternative IFN preparations.

Antibodies to Recombinant and Native IFN-a

Because of its antiviral, antitumor, and immunoregulatory effects, IFN-a has been given to patients with a variety of diseases with varying clinical responses

(6,7,128-130). IFN-neutralizing Ab of IgG type were first demonstrated in a patient suffering from nasopharyngeal carcinoma treated with human fibroblast IFN (123). Later, IFN Ab were observed in three patients with metastatic cancers treated with human leukocyte IFN (15). Since then, many investigations have focused on the immunogenicity of different IFN species and the possible clinical significance of neutralizing Ab (131).

Therapeutic Importance. Response failure in patients with hairy cell leukemia and malignant carcinoid tumors treated with recombinant IFN-a2a has been associated with neutralizing IFN Ab (132,133). Clinical resistance to IFN of various degrees was present in 6 of 16 patients with neutralizing Ab. Furthermore the neutralizing Ab did not inhibit the antiviral effects of natural IFN-a 1a in vitro. At the same time, response failure was observed in a patient with Philadelphia chromosome-positive chronic myeloid leukemia correlating with the development of neutralizing Ab (134). No cross reactivity was observed against human leukocyte IFN-a, and switching of treatment to this product resulted in hematological remission.

The immunogenicity of two recombinant IFN-a subtypes and lympho-blastoid IFN-a was compared in a multicenter study employing 296 patients with different forms of chronic hepatitis (128). IFN-a2a was significantly more immunogenic (20% seroconversion) compared with IFN-a2b (7%) or lymphoblastoid IFN-a (1%). Recently, neutralizing IFN-a Ab were reported in 39% of patients with breakthrough hepatitis during treatment with recombinant IFN-a2a and IFN-a2b compared with 3% in complete responders, suggesting that the development of neutralizing Ab may cause response failure at least in some patients (135).

The vast majority of therapy-induced IFN-a Ab are of IgG type, indicating a secondary immune response. Epitopes recognized by neutralizing IFN-a2 Ab have been localized within the N-terminal functional domain of the molecule (136).

Generally, recombinant IFN-a seems to be more immunogenic than pharmaceutic preparations of native IFN. For example, in patients with an initial response to recombinant IFN-a therapy and a subsequent response failure associated with development of neutralizing Ab, a clinical effect has been regained after change of therapy to natural leukocyte-derived IFN-a (137,138). This difference may be due to the lack of glycosylation in the E. coli-derived recombinant cytokines, the many subtypes in natural IFN preparations, or to different formulations of the cytokine preparations. That the latter is important is supported by the finding that protein aggregates are crucial for the antigenicity of IFN-a in normal and transgenic mice (139). This has recently lead to a change in the formulation of recombinant IFN-a2a to avoid immunogenicity.

Patients suffering from cutaneous T-cell lymphoma with preexisting IFN-a Ab have been shown to be particularly at risk of developing high levels of neutralizing Ab during therapy with recombinant IFN-a2 (140). It appears therefore that some patients develop specific Ab to the IFN species used for therapy and not cross reacting with natural IFN, whereas others appear to have a boost of production of preexisting Ab to native IFN.

Antibodies to Recombinant IFN-P

IFN-P-neutralizing Ab were first demonstrated in high-risk malignant melanoma patients receiving combination therapies of recombinant IFN-P and IFN-Y (141). Although no Ab to IFN-y were found, 56% developed IFN-P-neutralizing Ab; notably in patients receiving IFN-P subcutaneously. In 1993, IFN-P was shown to reduce the exacerbation frequency and decrease lesion formation visualized by magnetic resonance (MR) scans in patients with multiple sclerosis (MS) (142). On this basis, prolonged therapy with two recombinant IFN-P products has been approved for the treatment of MS: a glycosylated form with the predicted natural amino acid sequence (IFN-P 1a) and a nonglycosylated form with a Met-1 deletion and a Cys-17 to Ser mutation (IFNP1b) (143).

Therapeutic Importance. Many studies have focused on the immu-nogenicity of IFN-P preparations in relapsing-remitting MS (144-146). The reported frequencies and titers of these Ab vary depending on the IFN-P preparations and assays for antibody detection. The clinical importance of the Ab has also been difficult to evaluate owing to the natural relapsing-remitting course of the disease. However, the relapse rates and MR scan results have been at placebo levels in patients with neutralizing Ab (144). An effect on the final clinical outcome has yet to be demonstrated.

We have found that a significant number of MS patients develop neutralizing IFN-P Ab within 6 months of IFN-P therapy (Ross et al., unpublished data). IFNP1P appears to be more immunogenic than IFNP1a, and high-dose IFN-P 1a therapy induced a faster appearance of IFN-P Ab. The clinical significance of these neutralizing Ab is not known.

Antibodies to Recombinant IFN-y

Because of its ability to activate M0 and NK cells, and its immunoregulatory effects, INF-y has been used to treat patients with various diseases, including cancers, chronic infections, and systemic mastocytosis. Although INF-y Ab seem to occur rarely in cancer patients, case reports mention the appearance of INF-y Ab after 4-6 months of therapy in patients with systemic mastocytosis

(147,148). Secondary treatment failure could be associated with the appearance of Ab that neutralized the antiviral activity of recombinant human INF-Y in vitro. In one patient, the Ab were of polyclonal origin with a predominance of IgG1 and IgG2 and small amounts of IgA and IgM (148).

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