Hilmar Lemke

High Plasma Levels of the Soluble Form of CD30 Activation Molecule Reflect Disease Activity in Patients with Wegener's Granulomatosis

PURPOSE To determine the plasma levels of soluble CD30 (sCD30) in Wegeners granulomatosis (WG) patients, and to investigate the possible correlation of sCD30 with disease extent and activity. PATIENTS AND METHODS sCD30 was determined by radioimmunoassay in 57 WG patients, 25 patients with rheumatoid arthritis (RA), 23 patients with bacterial infections and 21 healthy controls (HC). The extent and activity of WG disease were assayed according to disease extent index (DEI) and standard laboratory parameters. RESULTS Plasma sCD30 levels in generalized WG (22.5 +/- 1.5 U/mL), but not in initial phase WG (12.1 +/- 4.0 U/mL), were significantly increased compared with HC (8.8 +/- 0.9 U/mL, P < 0.0001). Furthermore, of 11 generalized WG patients who received long-term follow-up, sCD30 levels declined when the disease activity changed from active disease to remission (29.1 +/- 1.9 U/mL to 15.9 +/- 1.8 U/mL, P = 0.0001). Similar results were observed in the whole group of generalized WG, eg, sCD30 levels in active disease (29.4 +/- 1.4 U/mL) were significantly higher than in partial remission (17.9 +/- 1.9 U/mL, P < 0.001) and in complete remission (13.7 +/- 3.3 U/mL, P < 0.001). No significant difference was noted between complete remission and HC. In addition, sCD30 levels were correlated with other parameters of disease extent and activity such as DEI, plasma levels of sIL-2R, PR3-ANCA, ESR and CRP. The sCD30 levels were increased in RA patients compared with HC (15.2 +/- 2.1 U/mL, P < 0.05), but no correlation was found between disease activity parameters and sCD30 levels. In contrast, in patients with bacterial infections sCD30 levels (6.9 +/- 0.9 U/mL) were not significantly different compared with HC. CONCLUSION Plasma levels of sCD30 are not only significantly increased but also correlate with disease extent and activity in generalized WG. These findings suggest that sCD30 can act as a useful marker for evaluation of disease extent and activity, and that generalized WG may be associated with Th2-type immune response.

An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line

SummaryThe human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin’s lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETA’). The resulting immunotoxin Ki-4(scFv)-ETA’ specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin’s lymphoma or other CD30+ malignancies.

Antigen‐independent suppression of the IgE immune response to bee venom phospholipase A2 by maternally derived monoclonal IgG antibodies

The IgE immune response to ovalbumin in rats can be suppressed by prior immunization of the dams. The results reported in this paper extend this observation to include a different antigen and another species, namely the IgE immune response to bee venom phospholipase A2 (PLA2 ) in CBA/J mice. The degree of suppression seemed to depend on the amount of IgG antibodies transferred to the offspring. Moreover, we found that the maternally mediated suppression of the IgE response could be achieved in a completely antigen‐free system in which exogenous monoclonal anti‐PLA2 IgG antibodies were transferred from the dams to the offspring. The following results were obtained: (i) The IgE suppression by monoclonal IgG antibodies was induced as efficiently with one single anti‐PLA2 IgG1 antibody as with a mixture of ten antibodies (nine IgG1, one IgG2b). (ii) Even after several immunizations up to an age of 6 months with a dose of PLA2 that normally induces IgE production, none of the F1 mice developed an IgE response. (iii) This long‐lasting suppression was observed in mice which were first immunized at an age of 4 weeks ( i.e. when low amounts of maternally derived monoclonal IgG were still present), as well as in mice which were first immunized at an age of 8 weeks, when no such maternal antibodies could be detected in their sera. The corresponding IgG responses showed, compared to normal mice, a transient enhancement in the maternally influenced mice. It is concluded that the immunological experience of the mother is of particular importance for the isotype regulation in the newborns, especially with respect to the ability to elicit an IgE response. The possible implications for the development of allergic diseases in humans are discussed.

BENEFITS AND BURDEN OF THE MATERNALLY-MEDIATED IMMUNOLOGICAL IMPRINTING

The ontogenetic development of both the immune and the nervous system entirely depend on external environmental signals that induce a lifelong learning process. The resulting collective immunological knowledge about the external world is transmitted in an epi-genetic fashion to the offspring, but only from the maternal and not the paternal side, with maternal IgG as the main transgenerational vector. As products of thymus-dependent responses, maternal IgG have undergone immune maturation by somatic hypermutations and are, therefore, acquired immunological phenotypes representing a great deal of the mothers immunological experience. During a limited neonatal imprinting period, maternal antibodies induce T cell-dependent idiotypic responses. These exert up to life-long determinative influences which may even be dominant over seemingly genetic predispositions. Such long-term immunological imprinting effects can be detected as (a) selection of the adult T and B cell repertoires, (b) anti-microbial protection by antigen-reactive antibodies (idiotypes) and anti-idiotypes, (c) allergen-specific suppression of IgE responsiveness by allergen-reactive IgG idiotype or corresponding anti-idiotype and (d) induction of autoimmune diseases by maternally-derived autoantibodies. Hence, immunological imprinting by maternal IgG antibodies will mostly be beneficial, but in case of autoantibodies can also be a burden for the initial development of the nascent immune system.

Non-genetic inheritable potential of maternal antibodies

Maternal antibodies (IgG and IgA) not only provide passive protection against microbial infections, but also exert a variety of equally important active, idiotypically-mediated immunoregulatory functions. Since the generation of maternal antibodies depends entirely on the stimulation of the mothers immune system by external mainly thymus-dependent antigens, with long-lived antigen independent plasma cells in the bone marrow, maternal antibodies represent the mothers collective ontogenetic immunological experience. Although their stimulatory potential in mice is restricted to the neonatal imprinting period, maternal antibodies exert a life-long determinative influence which is even dominant over seemingly genetic predispositions. Therefore, the functional impact of maternal IgG antibodies appears phenotypically as a non-genetic inheritance.

Reversal of the adult IgE high responder phenotype in mice by maternally transferred allergen‐specific monoclonal IgG antibodies during a sensitive period in early ontogeny

IgE is an important trigger in allergy and asthma, diseases whose development is suggested to depend on an initial sensitization in early life. While induction of murine IgE responses requiresboth a genetically based IgE high responder phenotype and defined experimental conditions, maternally transferred IgG can override these prerequisites and suppress IgE formation in an allergen‐specific manner. Here, we show that maternally transferred monoclonal IgG, irrespective of their subclass and recognized epitopes, induce IgE unresponsiveness, which is effective for parenteral immunization with bee venom phospholipase A2 as well as for airway‐immunization with nebulized ovomucoid‐containing ovalbumin. This IgE suppression is detectable in the offspring during the first 4 months of life, but not thereafter and not in the dams. However, when the initial immunization at an age of 3 or 4 months was followed by further application of both allergens via their respective routes, IgE suppression persisted up to an age of more than one year. If applicable to man, these findings may allow the development of a new strategy for the prevention of allergy and asthma by maternally transferred or neonatally injected allergen‐specific mAb in combination with natural or prophylactic exposure to the respective allergens during early childhood.

A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene

A new phagemid cloning vector for positive selection of recombinants, pBa‐7, was constructed which contains an active barnase gene encoding the cytotoxic ribonuclease from Bacillus amyloliquefaciens, under control of the lac promoter. PBa‐7 is a derivative of the high‐copy number pBluescript II KS+ phagemid in which the modified barnase killer gene has been fused downstream from the lac promoter of the pBluescript II KS+ multiple restriction site. When a lacIq‐negative Escherichia coli strain is transformed by this vector, the active barnase blocks bacterial growth by massive RNA destruction [1]. However, if barnase is inactivated by insertion of a foreign DNA fragment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are considerably simplified. When E. coli transformants are plated out on rich medium with ampicillin, only cells containing recombinant plasmids give rise to colonies. In a lacIq‐positive host, the positive selection is IPTG‐dependent. Therefore, pBa‐7 phagemid can be amplified and prepared in large quantities from lacIq‐positive E. coli hosts. Hence, pBa‐7 seems to be suitable for most genetic engineering manipulations.

Protein kinase activity of the intracellular but not of the membrane-associated form of the KI-1 antigen (CD30)

The Hodgkin-associated Ki-1 antigen (CD30) consists of a 120-kDa membrane-associated glycosylated phosphoprotein (Ki-1/120) and a 57-kDa non-glycosylated phosphoprotein (Ki-1/57) which only occurs intracellularly. Both molecules are phosphorylated at serine residues. An analysis of the peptide fragments resulting from staphylococcal V8-protease digestion of the Ki-1/57 molecule revealed identical bands irrespective of the cell source. Only a few bands of the Ki-1/57 digests appeared among the peptide fragments of the Ki-1/120 membrane antigen. The protein kinase activity was tested for both forms of the Ki-1 antigen. The Ki-1/120, devoid of the Ki-1/57 molecule, was immunoprecipitated from cell lysates of Hodgkin-analogous cell lines L428 or L540, which had been loaded with the Ki-1 or the Ki-1-analogous antibodies Ber-H2, HSR-1, -2 and -3 (method 1). These other antibodies reacted with the Ki-1/120, but not with the Ki-1/57 antigen. The latter, devoid of the Ki-1/120, was isolated from L540 cells after removal of the membrane form by method 1 or from U266/Bl myeloma or Raji Burkitt lymphoma cells which only contain the smaller form. Effects of non-specific adsorption were eliminated by various control precipitates. The Ki-1/57 intracellular antigen showed autophosphorylation and could phosphorylate histones as well. In contrast, a protein kinase activity of the membrane-associated Ki-1/120 could not be demonstrated.

Inhibition of metalloproteinases enhances the internalization of anti-CD30 antibody Ki-3 and the cytotoxic activity of Ki-3 immunotoxin.

CD30 is selectively expressed on the tumor cells of a variety of malignant disorders of the immune system and can therefore be used as a target for an anti‐CD30 antibody‐based immunotherapy. However, CD30 is cleaved at the cell surface by tumor necrosis factor‐α converting enzyme (TACE). This metalloproteinase releases the soluble ectodomain of CD30 (sCD30), which is able to neutralize immunotherapeutic agents before these reach their target cells. Such constitutive CD30 cleavage is enhanced after binding of most anti‐CD30 antibodies, leading to a downregulation of CD30 and an increase of sCD30 in the cell environment. Here, we demonstrate that CD30 shedding from the cell line Karpas 299 could effectively be blocked by the hydroxamic acid‐based metalloproteinase inhibitors BB‐3644 (IC50 = 180 nM), BB‐2116 (IC50 = 230 nM), BB‐94 (batimastat, IC50 = 230 nM) and BB‐2516 (marimastat, IC50 = 1 μM). This inhibition reduced the concentration of sCD30 in the cell environment to the background level, prolonged the persistence of the anti‐CD30 antibody Ki‐3 on Karpas 299 cells and favored its internalization. Moreover, a nontoxic concentration of the inhibitor BB‐3644 significantly increased the cytotoxic activity of the anti‐CD30 ricin A‐chain immunotoxin Ki‐3.dgA towards the CD30+ Hodgkin‐derived cell line L540. Hence, the metalloproteinase inhibitor BB‐3644 may be a promising compound to improve the immunotherapy of CD30+ malignancies.

ADAM17-overexpressing breast cancer cells selectively targeted by antibody–toxin conjugates

A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery.