Anti-CD20 therapy of treatment-resistant Wegener's granulomatosis: favourable but temporary response.

Rituximab is a genetically engineered chimeric monoclonal immunoglobulin (Ig)G1 antibody. It binds the CD20 trans-membrane surface antigen expressed by mature B cells but not by antibody secreting plasma cells, and removes the cells by activating complement, inducing cell-mediated lysis, and by apoptosis. Mainly used for the treatment of non-Hodgkin's lymphomas, rituximab has recently been tried with favourable responses in rheumatoid arthritis, systemic lupus erythematosus, and other chronic immunological diseases. Wegener's granulomatosis (WG) is a granulomatous vasculitis with high morbidity and mortality. It is thought that anti-neutrophil cytoplasmatic antibodies (ANCA) with specificity for proteinase 3 (PR3) are possibly involved in the pathogenesis of the disease. Conventional therapy with cyclophosphamide and corticosteroids generally succeeds in inducing remission, but relapses frequently follow. Among the biological agents, tumour necrosis factor-alpha (TNF-alpha) inhibitors have been tried with some success. Based on a case report we recently treated three refractory WG patients with rituximab and achieved almost complete but temporary remission. CD20+ cells disappeared rapidly in peripheral blood, only to rise prior to subsequent disease flares occurring at 34, 63, and 54 weeks, respectively (Figure 1). A new flare occurred in one patient at 86 weeks. At the end of the observation periods (54, 102, and 120 weeks), only one patient had proteinuria. Chest radiographs became normal in two patients, while infiltrates remained unchanged in the third. Granulomatous retro-orbital or sinus masses in two patients seemed unresponsive to therapy.

Anti-idiotypic immune responses against adjuvant-free isologous IgM monoclonal antibodies and their augmentation by complex formation between IgM and albumin in bovine serum.

In previous studies we demonstrated that the hypermutated isologous myeloma protein MOPC 315 (isotype IgA; lambda 2) is recognized by T helper cells like an ordinary foreign protein antigen. To what extent can an immune system recognize and respond to V domains from the primary (pre-immune) repertoire? To study this question we made 21 BALB/c hybridoma anti-2,4,6 trinitrophenyl monoclonal antibodies (mAb) of IgM; lambda isotype. All mAb purified from supernatants containing fetal bovine serum had formed spontaneous complexes with bovine serum albumin possibly by way of disulfide interchange. Twenty of twenty-one mAb from this source elicited IgG1 anti-idiotypic (Id) Ab when given as a single adjuvant-free dose of 200 micrograms. For 12 of them even 10 micrograms was sufficient. This indicated that BSA augmented the anti-Id responses by a carrier effect. Three of the mAb were therefore purified from ascites fluid and from serum-free medium. Only one of them then induced humoral anti-Id responses in BALB/c mice when given as a single adjuvant-free dose of 100 micrograms. The other two became immunogenic when emulsified in Freund's complete adjuvant. The results indicate that some IgM mAb exist whose Id determinants alone can elicit substantial anti-Id responses because they are recognized like ordinary foreign protein antigens. Since albumin in fetal bovine serum forms complexes with IgM and greatly augments its immunogenicity, serum-free medium should be used for production of human or humanized therapeutic IgM mAb. A possible role for Id for IgM Ab as cardinal autoantigens is discussed.

Immune responses to an adjuvant-free native syngeneic myeloma protein (M315).

Myeloma protein 315 (M315; isotype IgA, lambda 2) is used in this report as a model to explore the immunogenicity of a syngeneic Ig under nearly physiological conditions. We have previously shown that a synthetic peptide spanning the mutated HV3 loop of the L-315 chain, when emulsified in complete Freund's adjuvant, elicits T helper cells (Th) that respond to a boost with L-315 or M315, indicating that M315 is recognized as a processed protein antigen. We now show that the adjuvant-free 7S monomer of native or of mildly reduced and alkylated M315, given in divided doses totalling 300 or 800 micrograms to BALB/c mice, induced persistent anti-M315 antibodies (Ab), a large part of which was IgG1 directed mainly to idiotypes (Id) associated with M315's hapten-binding site. Polymers of M315 IgA (800 micrograms) failed to induce Ab, due probably to their rapid clearance into bile. Short-term treatment with anti-CD4 monoclonal Ab GK1.5 at the time of priming with 7S M315 inhibited the responses almost completely. The spleens of M315-immune mice contained Th that recognized the L-chain subunit of M315 as a carrier indicating that these Th did not require an assembled (VH-VL) pair of 315 V regions to be activated. We also observed low amounts of Ab specific for epitopes of the C alpha region. This evidence opens the possibility that a distinct autoimmune pathway exists for elicitation of rheumatoid factor (RF; autoAb to Fc gamma) that involves help to RF-producing B cells by Id-specific Th. We suggest that these Th recognize V-region peptides from IgG that have been captured, processed and presented by these B cells.

Are idiotypic peptides from variable domains critical for T-dependent production of rheumatoid factors?

Studies from this laboratory have indicated that isologous myeloma protein 315 (M315, isotype IgA, lambda 2) elicits T helper cells which recognize processed forms (peptides) of its V-domains and that recognition is controlled by H-2 linked immune-response (Ir) genes (J Exp Med 1982; 155:1587-96; Eur J Immunol 1986; 16:889-93). We now report that adjuvant-free soluble M315, particularly the mildly reduced and alkylated form, stimulates a T-dependent antibody response mainly specific for M315's paired V-domains. A small subset of the antibodies appeared to recognize the C-region of IgA, thus being analogous to rheumatoid factors (RF). On the basis of these observations, we propose that one pathway to RF production depends on T helper cells that interact directly with RF-producing B cells across peptide-MHC bridges. These peptides are envisaged to be derived from hypervariable regions of IgG V-domains, and they are therefore called "idiotypic peptides". This hypothesis assumes that the number of different idiotypic peptides is so large that the immune system has not developed tolerance to all of them.

Two M315 idiotopes defined by isologous monoclonal antibodies: one depends on germline and the other on mutated murine lambda 2 light chain sequences.

We have previously reported that T helper cells of BALB/c mice recognize the unique mutated sequence Phe94, Arg95, Asn96 of the lambda 2 L chain of isologous (BALB/c) myeloma protein 315. Here we study two Id (Id-315.1.4 and Id-315.TH) of the DNP-Lys binding M315 defined by two monoclonal isologous anti-Id Ab (Ab 2-1.4 and Ab 2-TH). Both Id were (1) totally expressed by Fv-315, but not by free unpaired V domains, (2) specifically dependent on VH-315, since lambda 2-315 recombined with four other H chains did not express the Id, (3) related to the hapten-binding site because their expression was blocked by the haptens DNP-Lys and DNP-Gly, and (4) topographically related because Ab 2-1.4 and Ab 2-TH competed with each other for binding to M315. The contribution of lambda chain V regions was studied with the aid of reconstituted Ig molecules of H-315 paired with lambda 1, lambda 2, and lambda 3 L chains. Id-315.TH was expressed equally well by reconstituted Ig containing three different lambda 2 chains (lambda 2-5-7, lambda 2-T952, and lambda 2-315), but its expression was profoundly reduced when H-315 was associated with lambda 3-SAPC15 or lambda 1-J558 L chains; it therefore depended upon amino acids encoded by germline lambda 2 genes. By contrast, Id-315.1.4 was only restored by the lambda 2-315 chain paired with H-315. Since lambda 2-5-7 and lambda 2-T952 differ from lambda 2-315 at positions 38, 94, 95, 96, and 98 or 99, respectively, Id-315.1.4 probably requires the unique mutated amino acids Phe94, Arg95, Asn96 of lambda 2-315. This resembles the effects on Id expression of previously reported unique amino acids of the D region. We failed to confirm that hyperimmunization of BALB/c mice with Ab 2-1.4 cross-linked to KLH induced M315-like Ab. The results are discussed in terms of the contribution of the third hypervariable loop of lambda chains to Id and the immunogenicity of isologous Ig.

The T lymphocyte response to syngeneic lambda 2 light chain idiotopes. Significance of individual amino acids revealed by variant lambda 2 chains and idiotope-mimicking chemically synthesized peptides.

In the present study we have investigated the structure of the helper T cell (Th)-defined idiotope (Id) of myeloma protein 315 lambda 2 light chain (lambda 2(315] in BALB/c (H-2d) mice which carry a high-responder immune response gene for this Id. Three peptides were synthesized which spanned the third hypervariable region (HV3) of lambda 2(315): peptides 88-99, 94-108 and 91-108. Only peptide 91-108 was capable of eliciting carrier-specific Th that recognized M315 or free lambda 2(315). These Th did not recognize lambda 2(5-7) chain which differs from lambda 2(315) at 4 positions in this region; these are Tyr94, Ser95, Thr96, Tyr98 for lambda 2(5-7) and Phe94, Arg95, Asn96, Phe98 for lambda 2(315). Immunization with peptide analogues revealed that substitution of Tyr for Phe94 was compatible with Id-lambda 2(315) mimicry, but substitution of Ser for Arg95 or Thr for Asn96 destroyed the Th-recognized Id. Furthermore, Th primed with lambda 2(5-7) chain did not cross-react with lambda 2T952; these lambda 2 chains only differ from each other at positions 98 and 99 at the V lambda 2-J lambda 2 junction. The data indicate that individual amino acids of short peptide segments are critical for Th-recognized Id of the lambda 2 HV3 loop and V lambda 2-J lambda 2 junction. Furthermore, the immunogenicity of a small peptide suggests that the carrier (lambda 2)-specific Th recognize Id that have been processed by antigen-presenting cells (APC). This implies the existence of two categories of "internal images" of foreign or of self antigens: (a) serologically defined and (b) T lymphocyte defined. We propose that as a rule, Id processing by APC, including B cells, destroys the first and reveals the second category. The possible physiological function of these Id-specific T cells in network interactions with B cells is discussed.