[CANCER RESEARCH 61, 8737–8742, December 15, 2001] Human Angiogenin Fused to Human CD30 Ligand (Ang-CD30L) Exhibits Specific Cytotoxicity against CD30-positive Lymphoma1 Michael Huhn, Stephanie Sasse, Mehmet K. Tur, Ba¨rbel Matthey, Timo Schinko¨the, Susanna M. Rybak, Stefan Barth,2 and Andreas Engert Fraunhofer IME, Department of Pharmaceutical Product Development, 52074 Aachen, Germany [M. K. T., M. H., S. B.]; Laboratory of Immunotherapy, Department I of Internal Medicine, University Hospital Cologne, 50931 Cologne, Germany [S. S., B. M., T. S., A. E.]; Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702 [S. M. R.] ABSTRACT first and then to administer ITs to kill residual H-RS cells. One of the most promising target antigens for immunotherapy of malignant lym- A number of different immunotoxins composed of cell-specific target- phoma such as Hodgkin’s lymphoma or anaplastic large cell lym- ing structures coupled to plant or bacterial toxins have increasingly been phoma is the CD30 receptor. This antigen was originally discovered evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based on cultured H-RS cells using the moab Ki-1 (1). The gene encoding fusion toxin (Ang-CD30L) using the human RNase angiogenin. The com- the CD30 receptor molecule (2) is located on chromosome 1p36. The pletely human fusion gene was inserted into a pET-based expression naturally occurring CD30 ligand has also been identified and cloned plasmid. Transformed Escherichia coli BL21(DE3) were grown under (3). The CD30/CD30 ligand system triggers cytolytic cell death in osmotic stress conditions in the presence of compatible solutes. After malignant lymphoma cell lines and induces proliferation and cytokine ␤ isopropyl -D-thiogalactoside induction, the Mr 37,000 His10-tagged Ang- production in T cells or neutrophils (4). Moabs against CD30 have CD30L was directed into the periplasmic space and functionally purified been explored as vehicles for cytostatic drugs (5) or plant toxins (6). by a combination of metal ion affinity followed by enterokinase cleavage ITs constructed with anti-CD30 moabs chemically linked to catalyt- of the His -Tag and molecular size chromatography. The characteristics 10 ically active toxins demonstrated specificity and potent antitumor of the recombinant protein were assessed by ELISA, flow cytometry, and ؉ activity against Hodgkin’s lymphoma cells in vitro and in mouse toxicity assays showing specific activity against CD30 Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition models (7, 8). A total of 12 patients with refractory relapsed with anti-CD30 monoclonal antibody Ki-4 and commercially available Hodgkin’s lymphoma were treated with an IT constructed by conju- CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in gating the anti-CD30 moab BerH2 to Saporin-6 (Ber-H2-S6; Ref. 9). vitro. The human recombinant immunotoxin showed significant toxicity Rapid regression of tumor masses ranging from 50% to Ͼ 75% toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and (lasting 2–4 months) were observed in ϳ50% of patients underlining ؍ L540Cy) and exhibited highest cytotoxicity against L540 cells (IC50 8 the validity of CD30 as a target antigen in HD (10, 11). The major ng/ml) as determined by cell proliferation assays. CD30 specificity was obstacles observed in this and other trials are unspecific toxicities, confirmed by competitive toxicity assays. This is the first report on the mainly related to the vascular leak syndrome, and the immunogenicity specific cytotoxicity of a recombinant completely human fusion toxin with of the foreign proteins resulting in only a limited number of applica- possibly largely reduced immunogenicity for the treatment of CD30- tions (12, 13). positive malignancies. By using phage display technology, we generated several Hodgkin’s lymphoma-specific scFvs, which were then genetically INTRODUCTION fused to ETA’ (14). The rITs recovered from periplasmic space of E. coli grown under osmotic stress conditions in the presence of Hodgkin’s lymphoma is one of the best suited malignancies for compatible solutes were highly functional in terms of specific in vitro targeted immunotherapy for several reasons: (a) the lymphocyte ac- 3 binding and in vivo cytotoxic activity. tivation marker CD30 is expressed in high copy numbers on H-RS Humanized or human antibody fragments have been used to reduce cells; (b) the number of malignant cells that needs to be killed is small the immune response against xenogeneic proteins and indeed attenu- because the majority of cells in Hodgkin’s lymphoma are nonmalig- ated immunogenicity in patients (15). We reported recently on a nant reactive cells; (c) Hodgkin tumors are usually well vascularized, recombinant fusion protein consisting of recombinant human CD30 suggesting sufficient access of an immunotherapeutic agent like an IT ligand fused to ETA’ and showed the CD30-specific cytotoxicity of to the target cells; and (d) although Hodgkin’s lymphoma is known to this monomeric protein (16). Because human RNases are present in respond well to chemotherapy, residual tumor cells remaining after extracellular fluids, human plasma, and tissues, they might be less first-line treatment have been demonstrated to correlate with the immunogenic when used as toxic component of ITs. Human Ang, a probability of a later relapse. Because the selective elimination of human plasma protein with 65% homology to RNase A (17), was residual H-RS cells might enhance the number of patients being cured, documented as a potent inhibitor of protein synthesis in cell-free it seems feasible to eradicate bulky disease by conventional therapy extracts and when injected into Xenopus oocytes (18). The Mr 14,000 single chain polypeptide is not cytotoxic toward a wide range of Received 10/30/00; accepted 10/16/01. cultured cells but exhibits specific cytotoxic activity when fused to a 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 ligand, which is internalized on binding to the target cell (19, 20). 18 U.S.C. Section 1734 solely to indicate this fact. In this paper, we report our results with the human CD30L coding 1 Supported in part by the Deutsche Forschungsgemeinschaft Grant SFB502 (to A. E. region genetically fused to the human Ang gene. We demonstrate that and S. B.). 2 To whom requests for reprints should be addressed, at Fraunhofer IME, Department this new and completely human rIT Ang-CD30L exhibits specific and of Pharmaceutical Product Development, Worringer Weg 1, 52074 Aachen, Germany. effective destruction of CD30ϩ lymphoma cells. Phone: 49-241-80-28399; Fax: 49-241-871062; E-mail: [email protected]. 3 The abbreviations used are: H-RS, Hodgkin/Reed-Sternberg; IT, immunotoxin; HD, Hodgkin’s Disease; ETA’, deletion mutant of Pseudomonas exotoxin; MOPS, 3-[N- MATERIALS AND METHODS morpholino]propanesulfonic acid; Ang, angiogenin; IPTG, isopropyl ␤-D-thiogalactoside; moab, monoclonal antibody; EtBr, ethidium bromide; XTT, 2,3-bis[2-methoxy-4-nitro- 5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; rIT, recombinant immunotoxin; Bacterial Strains, Oligonucleotides, and Plasmids. E. coli XL1-blue ϩ scFv, single-chain variable fragment; SEC, size exclusion chromatography. {supE44 hsdR17 recA1 endA1 gyr A46 thi relA1 lacF’[pro AB lacIq 8737 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. HUMAN IT FOR CD30-POSITIVE LYMPHOMA TREATMENT ⌬ r lacZ M15 Tn10(tet )]} was used for propagation of plasmids and E. coli Enterokinase Cleavage. To remove the NH2-terminal His10-Tag, highly Ϫ Ϫ Ϫ BL21[DE3; F ompT hsSB(rB MB ) gal dcm DE3] as host for synthesis of specific cleavage of rIT (behind asp-asp-asp-asp-lys) by recombinant enteroki- rITs. Synthetic oligonucleotides were synthesized by Eurogentec (Seraing, nase (1 unit/50 ␮g rIT) was performed in duplicate using the enterokinase Belgium). Plasmids were prepared by the alkaline lysis method and purified cleavage kit (Novagen, Abingdon, United Kingdom) as described by the using plasmid kits from Qiagen (Hilden, Germany). Restriction fragments or manufacturer. Functional rIT was finally purified using SEC on the BioLogic PCR products were separated by horizontal agarose gel electrophoresis and workstation by separation in PBS (pH 7.4). Purified protein was analyzed by extracted with Qiaex II (Qiagen). Cloning into plasmid vectors was performed SDS-PAGE and quantified by densitometry (GS-700 Imaging Densitometer; by standard methods. Bio-Rad) after Coomassie staining in comparison with BSA standards and Plasmid Construction. CD30L cDNA gene was released from plasmid verified by Bradford assays (Bio-Rad). pDC202 (21) by PCR using the oligonucleotides CD30LBack (CAC-TTG- SDS-PAGE and Western Blotting. SDS-PAGE and Western blotting GAT-CAG-TCA-ATT-TTC-CGT-CGT-CCG-gaa-ttc-CAG-AGG-ACG-GAC- were performed as described (31). Ang-CD30L was detected by anti-Ang- TCC-ATT-CCC-AAC-TCA-CCT; underlined: EcoRI consensus; double un- biotin moab (Sigma Chemical Co.). Bound antibody was stained with an derlined: 3Ј-Ang region) and CD30LFor (cgg-cgg-ggt-acc-TTA-GTC-TGA- alkaline-phosphatase-conjugated antimouse-IgG moab (Sigma Chemical Co., ATT-ACT-GTA-TAA-GAA-GAT-GGA-CAA; underlined: KpnI consensus). Deisenhofen, Germany) and a solution of Tris-HCl (pH 8.0) and 0.2 mg/ml The Ang coding region was similarly amplified using the oligonucleotides naphtol-AS-Bi-phosphate (Sigma Chemical Co.) plus 1 mg/ml Fast-Red AngBack (tat-tat-aag-ctt-CAG-GAT-AAC-TCC-AGG-TAC-ACA-CAC- (Serva, Heidelberg, Germany). TTC-CTG; underlined: HindIII consensus) and AngFor (AGG-TGA-GTT- Sandwich-ELISA. The binding activity of Ang-CD30L was determined by GGG-AAT-GGA-GTC-CGT-CCT-CTG-gaa-ttc-CGG-ACG-ACG-GAA-AAT- CD30 receptor Sandwich-ELISA as documented previously (DAKO CD30 TGA-CTG-ATC-CAA-GTG; underlined: EcoRI consensus; double kit).