Advertisement

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins*

  • Elisa Ventura
    Footnotes
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Francesca Sassi
    Footnotes
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Sara Fossati
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy

    Unit of Innovative Therapies, Istituto G. Gaslini, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Arianna Parodi
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy

    Unit of Innovative Therapies, Istituto G. Gaslini, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • William Blalock
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy

    Unit of Innovative Therapies, Istituto G. Gaslini, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Enrica Balza
    Affiliations
    Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Patrizia Castellani
    Affiliations
    Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Laura Borsi
    Affiliations
    Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Barbara Carnemolla
    Affiliations
    Laboratory of Immunology, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Luciano Zardi
    Correspondence
    To whom correspondence should be addressed: Laboratory of Recombinant Therapeutic Proteins, Centro Biotecnologie Avanzate, Largo Rosanna Benzi, 10, 16132, Genoa, Italy. Fax: 39-010-5299074
    Affiliations
    Laboratory of Recombinant Therapeutic Proteins, Advanced Biotechnology Centre, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy

    Unit of Innovative Therapies, Istituto G. Gaslini, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Comitato Interministeriale Programmazione Economica, Rome, Regione Liguria, Genoa (to L. Z.), European Union Grants FP6 LSHC-CT-2003-503233-STROMA and FP6 LSHC-CT-2006-037489-ImmunoPDT (to L. Z.), Istituto Superiore di Sanità, Rome, Grant ISS2006, rare diseases (to B. C. and L. B.), Italian Ministry of Health, Rome, Grant RF-IST-2006-384590 (to L. B.), and Alleanza Contro il Cancro Grant ACC2007 (to L. B.).
    1 Both authors contributed equally to this work.
Open AccessPublished:July 24, 2009DOI:https://doi.org/10.1074/jbc.M109.025924
      We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-α, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.
      The generation of recombinant polyvalent and/or polyspecific fusion proteins for use as components of novel drugs is still hindered by factors that limit their production, storage, and use, chief of which are issues related to instability and/or inadequate solubility. Here we describe a novel approach based on the use of uteroglobin (UG)
      The abbreviations used are: UG
      uteroglobin
      FN
      fibronectin
      B-FN
      extra domain B containing fibronectin
      scFv
      single chain variable fragment
      SIP
      small immunoprotein
      CHO
      Chinese hamster ovary
      TNF-α
      tumor necrosis factor-α
      hTNF-α
      human TNF-α
      IL
      interleukin
      PBS
      phosphate-buffered saline
      ELISA
      enzyme-linked immunosorbent assay.
      3The abbreviations used are: UG
      uteroglobin
      FN
      fibronectin
      B-FN
      extra domain B containing fibronectin
      scFv
      single chain variable fragment
      SIP
      small immunoprotein
      CHO
      Chinese hamster ovary
      TNF-α
      tumor necrosis factor-α
      hTNF-α
      human TNF-α
      IL
      interleukin
      PBS
      phosphate-buffered saline
      ELISA
      enzyme-linked immunosorbent assay.
      as a skeleton for the generation of polyvalent/polyspecific recombinant proteins. Human UG is a small (15.8 kDa) globular, nonglycosylated, and homodimeric secreted protein that was discovered independently by two groups in the 1960s in rabbit uterus (
      • Krishnan R.S.
      • Daniel Jr., J.C.
      ,
      • Beier H.M.
      ), and it is the first member of a new superfamily of proteins, the so-called Secretoglobins (Scgb) (
      • Klug J.
      • Beier H.M.
      • Bernard A.
      • Chilton B.S.
      • Fleming T.P.
      • Lehrer R.I.
      • Miele L.
      • Pattabiraman N.
      • Singh G.
      ). UG is present in the blood at a concentration of about 15 μg/ml and is found in urine and in other body fluids. The UG monomer is composed of about 70 amino acids, depending on the species, and is organized in a four α-helix secondary structure; the two subunits are joined in an anti-parallel fashion by disulfide bridges established between two highly conserved cysteine residues in amino- and carboxyl-terminal positions (
      • Morize I.
      • Surcouf E.
      • Vaney M.C.
      • Epelboin Y.
      • Buehner M.
      • Fridlansky F.
      • Milgrom E.
      • Mornon J.P.
      ) (see Fig. 1). The exact functions of UG are not yet clear, but the protein has been reported to have anti-inflammatory properties due to its ability to inhibit the soluble phospholipase A2. Moreover, UG contains a central hydrophobic cavity able to accommodate hydrophobic molecules such as progesterone, retinol, and prostaglandin D2. Theoretically, this cavity could be loaded with different types of therapeutic hydrophobic substances and delivered to targets (for exhaustive reviews on UG, see Refs.
      • Mukherjee A.B.
      • Chilton B.S.
      The Uterogloglobin/Clara Cell Protein Family.
      ,
      • Mukherjee A.B.
      • Zhang Z.
      • Chilton B.S.
      and references therein).
      Figure thumbnail gr1
      FIGURE 1Central part of the figure depicts the ribbon structure of the oxidized homodimer of UG (adapted with permission from Ref.
      • Morize I.
      • Surcouf E.
      • Vaney M.C.
      • Epelboin Y.
      • Buehner M.
      • Fridlansky F.
      • Milgrom E.
      • Mornon J.P.
      ). A–E show the schemes of the various fusion proteins produced using UG as a central core. L19 is an scFv specific for the angiogenesis-associated FN isoform, and D2E7 is an scFv able to neutralize TNF-α.
      The high solubility and stability of UG to pH and temperature variations, its resistance to proteases, and its homodimeric structure prompted us to consider the protein as a candidate linker for the generation of polyvalent and polyspecific recombinant proteins. We demonstrate here that the use of UG as a linker could provide a general method for the generation of covalently linked bivalent and tetravalent antibodies, either monospecific or bispecific, as well as of different kinds of fusion proteins, which, compared with similar fusion proteins without UG, possess generally enhanced properties of solubility and stability, factors that expedite their storage and clinical use.
      We describe the use of UG for the production of a bivalent and tetravalent format of L19, an scFv specific for the angiogenesis-associated extra domain B (ED-B) of fibronectin (FN) (
      • Pini A.
      • Viti F.
      • Santucci A.
      • Carnemolla B.
      • Zardi L.
      • Neri P.
      • Neri D.
      ), of an immunocytokine composed of IL2 and L19, and of a tetravalent dual specificity antibody composed of L19 and the scFv D2E7, a human antibody able to neutralize TNF-α activity (
      • Tracey D.
      • Klareskog L.
      • Sasso E.H.
      • Salfeld J.G.
      • Tak P.P.
      ). We report and discuss the characterization, properties, and the biological activity, both in vitro and in vivo, of these molecules.

      RESULTS

      Fig. 1 shows the ribbon representation of the molecular structure of oxidized UG (
      • Morize I.
      • Surcouf E.
      • Vaney M.C.
      • Epelboin Y.
      • Buehner M.
      • Fridlansky F.
      • Milgrom E.
      • Mornon J.P.
      ). The UG monomer structure is composed of four α-helices. The two monomers of human UG are held together in anti-parallel fashion by two disulfide bonds between Cys-3 and Cys-69′ and the other between Cys-3′ and Cys-69. Fig. 1, A–E, depicts the hypothetical domain structures of the various fusion proteins containing UG that we describe here as follows: dimeric (Fig. 1A) and tetrameric (Fig. 1B) formats of the scFv L19 (specific for the ED-B of FN, a marker of angiogenesis); an immunocytokine composed of L19 and IL2 (Fig. 1C); the dimeric format of the scFv D2E7 (a human scFv able to neutralize TNF-α activity) (Fig. 1D); and a tetravalent dual specificity antibody composed of L19 and D2E7 (Fig. 1E).

      L19-UG and L19-UG-L19

      To produce the divalent L19, we prepared a cDNA construct composed of the scFv L19 cDNA connected at the 3′ end to the 5′ end of the UG cDNA, and to produce the tetravalent L19 format, we appended L19 cDNA at both 3′ and 5′ ends of the UG cDNA. The resulting L19-UG and L19-UG-L19 constructs (Fig. 2a) were then cloned into the vector pcDNA3.1 and used to transfect CHO cells grown in ProCHO5 animal protein-free media (Lonza, Verviers, Belgium) to produce 5–10 mg/liter recombinant protein that can be efficiently purified either on ED-B (the antigen of L19) or protein-A affinity chromatography because the variable heavy region of immunoglobulin chain of L19 belongs to the subgroup III and thus contains protein A-binding sites (
      • Pini A.
      • Viti F.
      • Santucci A.
      • Carnemolla B.
      • Zardi L.
      • Neri P.
      • Neri D.
      ,
      • Hillson J.L.
      • Karr N.S.
      • Oppliger I.R.
      • Mannik M.
      • Sasso E.H.
      ).
      Figure thumbnail gr2
      FIGURE 2a, scheme of the cDNA constructs of L19-UG (left) and L19-UG-L19 (right). Sp, signal peptide sequence. b, size exclusion chromatography profiles (Superdex 200) and SDS-PAGE analysis of the purified fusion proteins L19-UG (left) and L19-UG-L19 (right). Nonreducing (NR) and reducing (R) conditions, respectively. ST, molecular mass standard. c, size exclusion chromatography profiles of L19-UG and L19-UG-L19 reconstituted in distilled water after vacuum drying. d, comparative biodistribution experiments in F9 teratocarcinoma-bearing mice of three radioiodinated L19 formats: L19-UG, L19-UG-L19, and L19-SIP. The % ID/g in the tumor at the indicated times after intravenous injection is shown on the left (mean ± S.D.). To the right the tumor to blood ratio of the % ID/g at different times after injection of the radioiodinated proteins are plotted. VL, variable light region of immunoglobulin; CMV, cytomegalovirus; mAu, milli-absorbance units.
      In SDS-PAGE both purified proteins migrate as homodimers in nonreducing conditions and as monomers in reducing conditions, showing apparent sizes of about 63 and 35 kDa, respectively, for the divalent format and of 124 and 62 kDa, respectively, for the tetravalent format. The apparent size of the nonreduced divalent format was lower compared with the expected size of 70 kDa, very likely due to the compact conformation of the nonreduced molecule. In nonreducing conditions both molecules were more than 95% covalently linked dimers (Fig. 2b). The size exclusion chromatography (SEC) profiles of both fusion proteins showed a single peak with a retention volume corresponding to the molecular mass of the homodimers and the absence of aggregates (Fig. 2b). Both proteins were soluble in PBS at concentrations over 2 mg/ml, and after vacuum drying, these proteins could be reconstituted without any loss and without the formation of aggregates (Fig. 2c). These fusion proteins were generated using both mouse and human UG, and all the proteins presented identical properties. Our group has previously reported on a different covalently linked L19 homodimer obtained using the domain 4 of the constant heavy region of human IgE secretory isoform (small immune protein (SIP)) (
      • Borsi L.
      • Balza E.
      • Bestagno M.
      • Castellani P.
      • Carnemolla B.
      • Biro A.
      • Leprini A.
      • Sepulveda J.
      • Burrone O.
      • Neri D.
      • Zardi L.
      ). This protein radiolabeled with 131I is now extensively used in phase I/II radioimmunotherapy trials (
      • Santimaria M.
      • Moscatelli G.
      • Viale G.L.
      • Giovannoni L.
      • Neri G.
      • Viti F.
      • Leprini A.
      • Borsi L.
      • Castellani P.
      • Zardi L.
      • Neri D.
      • Riva P.
      ,
      • Sauer S.
      • Erba P.A.
      • Petrini M.
      • Menrad A.
      • Giovannoni L.
      • Grana C.
      • Hirsch B.
      • Zardi L.
      • Paganelli G.
      • Mariani G.
      • Neri D.
      • Dürkop H.
      • Menssen H.D.
      ,
      • Del Conte G.
      • Tosi D.
      • Fasolo A.
      • Chiesa C.
      • Erba P.A.
      • Grana C.M.
      • Menssen H.D.
      • Mariani G.
      • Bombardieri E.
      • Gianni L.
      ). However, L19-SIP, as well as the scFv per se, presents a much lower solubility than L19-UG fusion proteins, and it cannot be reconstituted after vacuum drying without aggregation and precipitation. To obtain a stable solution of SIP, it is necessary to keep it at a concentration not exceeding 0.5 mg per ml and at a temperature of −80 °C in the presence of stabilizers such as sucrose or Tween to avoid the formation of precipitates during thawing. By contrast, the UG constructs can be kept in solution in PBS at either −20 or −80 °C and thawed without the formation of any aggregates. These properties, together with the possibility to keep the molecules in the dry state, are of noteworthy importance for the storage of agents to be used in therapies in hospitals. We compared the tumor-targeting performance of the three radioiodinated L19 homodimers (L19-SIP, L19-UG. and L19-UG-L19) (Fig. 2d) in 129/SvHsd mice bearing the syngeneic F9 teratocarcinoma. As reported previously, the vasculature of the teratocarcinoma F9 presents the accumulation of B-FN (
      • Borsi L.
      • Balza E.
      • Carnemolla B.
      • Sassi F.
      • Castellani P.
      • Berndt A.
      • Kosmehl H.
      • Biro A.
      • Siri A.
      • Orecchia P.
      • Grassi J.
      • Neri D.
      • Zardi L.
      ). Fig. 2d (left panel) shows the percentage of injected dose per g of tissue (% ID/g) in the tumor at different times after injection of the three radioiodinated L19 formats. Considering the area under the curves, L19-UG-L19 performed best, although the L19-UG and the L19-SIP shared a similar area. Fig. 2d (right panel) shows the ratio of the % ID/g of tumor and of blood for the three L19 formats as follows: in this case the tumor/blood ratio was more than two times higher for the two UG formats than for the SIP format, because of the faster blood clearance profile of the former. The rapid clearance of molecules used for immunoradiotherapy is quite important because it limits the exposure of other organs to radiation.
      The ratios of the % ID/g in the tumor versus other organs were in all cases, at 48 h from injection of the radioiodinated proteins, greater than 10 (data not shown). The biodistribution of these L19 formats in other experimental tumor models was also studied, and in all cases the UG formats performed better than the SIP format.
      L. Zardi, manuscript in preparation.

      L19-UG-IL2

      To demonstrate that UG can also be used to generate active immunocytokines in the format of stable covalently linked homodimers, we expressed the immunocytokine L19-UG-IL2. The choice of this immunocytokine was prompted by the fact that we had previously produced L19-IL2 without UG (
      • Carnemolla B.
      • Borsi L.
      • Balza E.
      • Castellani P.
      • Meazza R.
      • Berndt A.
      • Ferrini S.
      • Kosmehl H.
      • Neri D.
      • Zardi L.
      ); therefore, this allowed us the opportunity to compare the properties and the biological activity of the two molecules and to validate the use of UG for the generation of immunocytokines. As is shown in Fig. 3A, the cDNA construct of L19-UG-IL2 was engineered by recombinant DNA technology by ligating the cDNA of the scFv L19 at the 5′ end and the cDNA of IL2 at the 3′ end of the UG cDNA. After cloning in the vector pcDNA3.1, CHO cells were transfected, and the clones produced roughly 3 mg/liter recombinant protein. In SDS-PAGE the purified L19-UG-IL2 migrated as a homodimer (more than 95%) in nonreducing conditions and as a monomer in reducing conditions at the expected sizes of about 104 and 52 kDa, respectively (Fig. 3B). The SEC profile also showed a main peak with a retention volume corresponding to the apparent molecular mass of the homodimer (Fig. 3C). In this case too, it was possible to vacuum dry and reconstitute the fusion protein without any loss and without the formation of aggregates (data not shown). On the contrary, the fusion protein without UG could not be vacuum-dried and had to be stored at −80 °C with stabilizers to prevent the formation of precipitates during thawing; the fusion protein containing UG, on the other hand, could be kept at −80 °C in PBS without any stabilizer and thawed without aggregation and/or formation of precipitates.
      Figure thumbnail gr3
      FIGURE 3A, scheme of the cDNA construct of L19-UG-IL2. Sp, signal peptide sequence. B, SDS-PAGE analysis (nonreducing (NR) and reducing conditions, respectively); C, size exclusion chromatography profile (Superdex 200) of purified L19-UG-IL2. D, proliferation assay on CTLL cells using [3H]thymidine. Equimolar amounts of L19-IL2 and L19-UG-IL2 induced identical thymidine incorporation into cells, indicating that the two proteins share identical IL2 activity. Abs, antibodies. E, tumor growth inhibition of equimolar amounts of L19-IL2 and L19-UG-IL2 or only saline. Each group consisted of four mice. Therapy started 7 days after tumor implantation. Arrows indicate the days of treatment. Tumor size was determined as reported under “Experimental Procedures.” S.E. are indicated. VL, variable light region of immunoglobulin; CMV, cytomegalovirus; mAu, milli-absorbance units.
      We compared the in vitro biological activity of IL2 by CTLL proliferation assay (
      • Meazza R.
      • Marciano S.
      • Sforzini S.
      • Orengo A.M.
      • Coppolecchia M.
      • Musiani P.
      • Ardizzoni A.
      • Santi L.
      • Azzarone B.
      • Ferrini S.
      ). Equimolar amounts of L19-IL2 and L19-UG-IL2 showed identical IL2 activity (Fig. 3D). We also compared the in vivo ability of L19-UG-IL2 and L19-IL2 to inhibit the growth of F9 teratocarcinoma in syngeneic mice. When the tumors reached a volume of nearly 0.3 cm3, groups of animals were treated for 6 days with daily intravenous injections of 250,000 units of IL2 as L19-IL2, L19-UG-IL2, or saline alone. The results depicted in Fig. 3E show that L19-UG-IL2 had an anti-tumor activity identical to that previously reported for L19-IL2 (
      • Carnemolla B.
      • Borsi L.
      • Balza E.
      • Castellani P.
      • Meazza R.
      • Berndt A.
      • Ferrini S.
      • Kosmehl H.
      • Neri D.
      • Zardi L.
      ).

      L19-UG-D2E7

      We generated a dual specificity tetravalent molecule using the scFv L19 (anti-ED-B) and the scFv D2E7 (inhibiting TNF-α) with UG as a central skeleton. As is shown in Fig. 4A, the cDNA construct of L19-UG-D2E7 was prepared by ligating the cDNA of the scFv L19 and the cDNA of the scFv D2E7 at the 5′ and 3′ ends, respectively, of the UG cDNA. This construct was inserted in the vector pcDNA3.1, and CHO cells were transfected; the resulting clones produced about 3 mg/liter recombinant protein. As a control, the fusion protein D2E7-mUG was obtained with a similar approach (data not shown). Fig. 4, B–F, shows the characterization of the purified dual specificity tetravalent molecule L19-UG-D2E7. In SDS-PAGE (Fig. 4B), the purified protein migrated as a homodimer (more than 95%) in nonreducing conditions, showing the expected mass of about 124 kDa, and as a monomer with a mass of 62 kDa in reducing conditions. The SEC profile (Fig. 4C) showed a main peak with a retention volume corresponding to the molecular mass of the homodimer. In this case, too, it was possible to vacuum dry and reconstitute the fusion protein without any loss and without the formation of aggregates (Fig. 4C). The immunoreactivity of L19-UG-D2E7 was tested by ELISA against the two antigens, the ED-B and TNF-α. The results, shown in Fig. 4, D and E, show that L19-UG and L19-UG-D2E7 reacted equally well with the ED-B (Fig. 4D), and that D2E7-UG and L19-UG-D2E7 reacted equally well with TNF-α (Fig. 4E), thereby demonstrating that the two scFvs within the L19-UG-D2E7 molecule do not interfere with each other. The results of an experiment of TNF-α cytotoxicity inhibition on LM cells using L19-UG-D2E7 and D2E7-UG show (Fig. 4F) that the two formats share a similar neutralizing ability, again demonstrating that the two antibodies on the same molecule do not interfere with each other.
      We also demonstrated that in L19-UG-D2E7 each antibody can properly function in either solution or solid phase. Addition of an excess amount of ED-B (100 nm) to L19-UG-D2E7 in PBS containing 2% of bovine serum albumin, although abolishing the reactivity with the immobilized ED-B, revealed no interference with the reactivity of the D2E7 moiety with TNF-α (Fig. 5A). To demonstrate that each binding domain could also function independently in solid phase, ELISA wells were coated with TNF-α and incubated with L19-UG-D2E7. The excess antibody was washed out, and the FN fragment composed of the type III repeat 7B89 was then added to the well (Fig. 5, B and C). This fragment bound to the L19 moiety and was then detected using a monoclonal antibody specific for the FN repeat 9. The results showed that even when an scFv is bound to the antigen in solid phase, the other is still free to react with its antigen.
      Not only was the specific binding ability preserved but the biological activity of the antibodies was as well, as revealed by the inhibition of the TNF-α-mediated cytotoxicity experiments on LM fibroblasts (Fig. 5D). To mimic the targeted delivery of D2E7 in tissues containing B-FN, the inhibitory activity of L19-mUG-D2E7 on hTNF-α cytotoxicity was evaluated on LM cells on culture plates pre-coated with the type III repeat 7B89. D2E7-UG was used as a nontargeted control. After incubation of cells with the fusion proteins and washing out the excess, hTNF-α was added. In this condition only the L19-UG-D2E7 should have been present on the plates, because it binds to the FN fragment with which the plates were coated. The results obtained (Fig. 5D) demonstrate that even when L19 is bound to the extra domain B, the D2E7 moiety is still able to neutralize hTNF-α.

      DISCUSSION

      The generation of effective proteins, particularly antibody derivatives, is beset by a number of problems, chief of which are the complex production processes and aggregation and stability issues arising during storage. Despite various attempts to overcome difficulties (
      • Wu C.
      • Ying H.
      • Grinnell C.
      • Bryant S.
      • Miller R.
      • Clabbers A.
      • Bose S.
      • McCarthy D.
      • Zhu R.R.
      • Santora L.
      • Davis-Taber R.
      • Kunes Y.
      • Fung E.
      • Schwartz A.
      • Sakorafas P.
      • Gu J.
      • Tarcsa E.
      • Murtaza A.
      • Ghayur T.
      ,
      • Holliger P.
      • Hudson P.J.
      ,
      • Kriangkum J.
      • Xu B.
      • Nagata L.P.
      • Fulton R.E.
      • Suresh M.R.
      ,
      • Marvin J.S.
      • Zhu Z.
      ), these obstacles remain.
      Here we describe a novel and generally applicable approach to generate tetravalent, dual specificity fusion proteins using UG as a central core of the molecules. We found that, compared with formats in which UG was not used, the use of UG as the scaffold enhanced stability and solubility of the fusion proteins. Such improvements in these properties open the possibility to store them at 4 °C in a vacuum-dried state. Indeed, using UG as a central core, we have demonstrated the possibility to produce various immunologically and biologically active, covalently linked fusion proteins, such as divalent and tetravalent scFvs, immunocytokines composed of scFvs and a cytokine, as well as tetravalent dual specificity antibodies.
      We generated a divalent and tetravalent format of the human scFv L19, specific for the ED-B containing isoform of FN (
      • Zardi L.
      • Carnemolla B.
      • Siri A.
      • Petersen T.E.
      • Paolella G.
      • Sebastio G.
      • Baralle F.E.
      ). FN is a large glycoprotein that is present both in plasma and tissues. The ED-B is a 91-amino acid type III homology repeat that is inserted in the FN molecule under tissue-remodeling conditions by preferential alternative splicing of the primary transcript (
      • Zardi L.
      • Carnemolla B.
      • Siri A.
      • Petersen T.E.
      • Paolella G.
      • Sebastio G.
      • Baralle F.E.
      ). The ED-B is undetectable in tissues from healthy adult individuals, but B-FN is abundant in many aggressive cancers (
      • Carnemolla B.
      • Balza E.
      • Siri A.
      • Zardi L.
      • Nicotra M.R.
      • Bigotti A.
      • Natali P.G.
      ,
      • Kaczmarek J.
      • Castellani P.
      • Nicolo G.
      • Spina B.
      • Allemanni G.
      • Zardi L.
      ,
      • Castellani P.
      • Viale G.
      • Dorcaratto A.
      • Nicolo G.
      • Kaczmarek J.
      • Querze G.
      • Zardi L.
      ,
      • Kosmehl H.
      • Berndt A.
      • Katenkamp D.
      ,
      • Castellani P.
      • Borsi L.
      • Carnemolla B.
      • Birò A.
      • Dorcaratto A.
      • Viale G.L.
      • Neri D.
      • Zardi L.
      ,
      • Birchler M.T.
      • Milisavlijevic D.
      • Pfaltz M.
      • Neri D.
      • Odermatt B.
      • Schmid S.
      • Stoeckli S.J.
      ). The scFv L19 has been shown to efficiently and selectively localize in tumor blood vessels in animal models and in patients with cancer following intravenous injection (
      • Borsi L.
      • Balza E.
      • Bestagno M.
      • Castellani P.
      • Carnemolla B.
      • Biro A.
      • Leprini A.
      • Sepulveda J.
      • Burrone O.
      • Neri D.
      • Zardi L.
      ,
      • Tarli L.
      • Balza E.
      • Viti F.
      • Borsi L.
      • Castellani P.
      • Berndorff D.
      • Dinkelborg L.
      • Neri D.
      • Zardi L.
      ,
      • Santimaria M.
      • Moscatelli G.
      • Viale G.L.
      • Giovannoni L.
      • Neri G.
      • Viti F.
      • Leprini A.
      • Borsi L.
      • Castellani P.
      • Zardi L.
      • Neri D.
      • Riva P.
      ,
      • Viti F.
      • Tarli L.
      • Giovannoni L.
      • Zardi L.
      • Neri D.
      ,
      • Demartis S.
      • Huber A.
      • Viti F.
      • Lozzi L.
      • Giovannoni L.
      • Neri P.
      • Winter G.
      • Neri D.
      ). Thanks to its ability to selectively accumulate in neoplastic tissues, L19 is currently undergoing extensive testing in clinical trials for the radioimmunotherapy of various forms of tumors, being administered as the divalent format of SIP radiolabeled with 131I (
      • Borsi L.
      • Balza E.
      • Bestagno M.
      • Castellani P.
      • Carnemolla B.
      • Biro A.
      • Leprini A.
      • Sepulveda J.
      • Burrone O.
      • Neri D.
      • Zardi L.
      ,
      • Sauer S.
      • Erba P.A.
      • Petrini M.
      • Menrad A.
      • Giovannoni L.
      • Grana C.
      • Hirsch B.
      • Zardi L.
      • Paganelli G.
      • Mariani G.
      • Neri D.
      • Dürkop H.
      • Menssen H.D.
      ,
      • Del Conte G.
      • Tosi D.
      • Fasolo A.
      • Chiesa C.
      • Erba P.A.
      • Grana C.M.
      • Menssen H.D.
      • Mariani G.
      • Bombardieri E.
      • Gianni L.
      ,
      • Menrad A.
      • Menssen H.D.
      ,
      • Neri D.
      • Bicknell R.
      ).
      Here we present two novel formats of L19 that seem particularly suitable for immunoradiotherapy and that present considerable advantages compared with the L19 in the scFv and SIP formats previously used. In fact, these novel L19-UG formats performed better than L19-SIP in biodistribution experiments in tumor-bearing mice (Fig. 2d), showing a higher tumor blood ratio of the % ID/g of tissue. In addition, considering that UG is an inhibitor of inflammation and fibrosis (
      • Pilon A.L.
      ), two important side effects of radiotherapy, its presence in the radiolabeled protein might help to reduce these unwanted effects. Moreover, both the divalent and tetravalent UG are much more soluble than previously used formats; in fact, in PBS, a concentration of 2 mg/ml without any additive can be achieved without observing any precipitation or aggregation, qualities that would facilitate the radioiodination process. We conducted vacuum drying experiments using both of the L19-UG formats in PBS and found that it was possible to reconstitute the proteins using distilled water without any loss of protein, precipitation, or formation of aggregates. On the contrary, the SIP did not possess these properties, making its clinical use less easy.
      We also generated an immunocytokine composed of the scFv L19 and IL2, and again we demonstrated that this molecule is active in both the antibody and the cytokine moieties. Our group previously prepared a UG-free immunocytokine composed of IL2 and L19 and showed that its fusion with L19 enhanced the therapeutic index of the cytokine (
      • Carnemolla B.
      • Borsi L.
      • Balza E.
      • Castellani P.
      • Meazza R.
      • Berndt A.
      • Ferrini S.
      • Kosmehl H.
      • Neri D.
      • Zardi L.
      ). This fusion protein is currently being investigated in a multicenter phase II clinical study in Italy and Germany (
      • Curigliano G.
      • Spitalieri G.
      • De Pas T.
      • Noberasco C.
      • Giovannoni L.
      • Menssen H.D.
      • Zardi L.
      • Milani A.
      • Neri D.
      • De Braud F.
      ). Here we compared the biological activity of L19-UG-IL2 and L19-IL2 both in vitro on T-cells and in vivo on an experimental animal model and obtained identical results (Fig. 3), thereby demonstrating that UG could be used as a general approach for the generation of biologically active immunocytokines in the form of stable, covalently linked homodimers. In addition, in this case also the inclusion of UG as a scaffold permitted the vacuum drying of the construct, which was not possible with the immunocytokine without UG. Moreover, given its anti-inflammatory properties, UG could be used for the generation of immunocytokines with anti-inflammatory activity, such as IL10. We are currently investigating whether the presence of UG is able to enhance the anti-inflammatory properties of similar cytokines. In fact, it has recently been shown that an immunocytokine made up of the scFv L19 and IL10 selectively accumulates at sites of arthritis in collagen-induced arthritis experimental animal models and that it inhibits disease progression (
      • Trachsel E.
      • Bootz F.
      • Silacci M.
      • Kaspar M.
      • Kosmehl H.
      • Neri D.
      ). The activity of this anti-inflammatory immunocytokine could be further enhanced using UG, which, again, per se possesses anti-inflammatory properties and, in addition, yields a stable covalently linked homodimer.
      To further demonstrate the versatility of this procedure, we generated a dual specificity tetravalent antibody using the scFvs L19 and D2E7. D2E7 is a TNF-α-neutralizing scFv marketed as a complete IgG under the brand name Humira, which has been successfully used for the treatment of rheumatoid arthritis. The use of the dual specificity, tetravalent L19-UG-D2E7 fusion protein could afford a novel therapeutic approach, namely the in situ inhibition of TNF-α, because the presence of L19 would prompt the selective accumulation of the fusion protein at the site of disease sites where D2E7 could explicate its neutralizing activity. This selective delivery would, in turn, diminish the side effects of anti-TNF-α antibody treatment. This approach and the in situ inhibition of TNF-α could, of course, be performed using the entire repertoire of proteins able to inhibit TNF-α, including the TNF-α receptor (see Ref.
      • Tracey D.
      • Klareskog L.
      • Sasso E.H.
      • Salfeld J.G.
      • Tak P.P.
      and references therein). Admittedly, all of these hypotheses must be tested in vivo, but we have demonstrated the possibility to generate a dual specificity tetravalent molecule composed of an scFv, L19, which is able to selectively deliver the molecule to the diseased tissue, and by a second scFv that inhibits TNF-α, even when L19 is engaged with its antigen. All of the fusion proteins we have generated displayed optimal solubility properties and the absence of aggregates. We also show that each binding and/or biological active moiety could function independently without interfering with each other in either solution or solid phase.
      In conclusion, we describe here a flexible and robust procedure for the generation of fusion proteins suitable for different therapeutic options, namely radioimmunotherapy, photodynamic, anti-inflammatory, and immunocytokine therapies, in a very broad range of angiogenesis-associated pathologies, including cancer and degenerative diseases. In addition, the UG homodimer contains a central hydrophobic cavity with a volume adequate to accommodate hydrophobic molecules such as progesterone, retinol, and prostaglandin D2. Theoretically, this cavity could be loaded with different kinds of hydrophobic therapeutic substances and delivered to targeted organs or tissues. We are currently investigating such possibilities (
      • Mukherjee A.B.
      • Zhang Z.
      • Chilton B.S.
      ).

      Acknowledgments

      We thank Sibel Sümer for editorial assistance in the preparation of the manuscript and Thomas Wiley for manuscript revision. We thank Dr. Silvano Ferrini for help in the evaluation of the IL2 activity.

      REFERENCES

        • Krishnan R.S.
        • Daniel Jr., J.C.
        Science. 1967; 158: 490-492
        • Beier H.M.
        Biochim. Biophys. Acta. 1968; 160: 289-291
        • Klug J.
        • Beier H.M.
        • Bernard A.
        • Chilton B.S.
        • Fleming T.P.
        • Lehrer R.I.
        • Miele L.
        • Pattabiraman N.
        • Singh G.
        Ann. N.Y. Acad. Sci. 2000; 923: 348-354
        • Morize I.
        • Surcouf E.
        • Vaney M.C.
        • Epelboin Y.
        • Buehner M.
        • Fridlansky F.
        • Milgrom E.
        • Mornon J.P.
        J. Mol. Biol. 1987; 194: 725-739
        • Mukherjee A.B.
        • Chilton B.S.
        The Uterogloglobin/Clara Cell Protein Family.
        The New York Academy of Sciences, New York2000
        • Mukherjee A.B.
        • Zhang Z.
        • Chilton B.S.
        Endocr. Rev. 2007; 28: 707-725
        • Pini A.
        • Viti F.
        • Santucci A.
        • Carnemolla B.
        • Zardi L.
        • Neri P.
        • Neri D.
        J. Biol. Chem. 1998; 273: 21769-21776
        • Tracey D.
        • Klareskog L.
        • Sasso E.H.
        • Salfeld J.G.
        • Tak P.P.
        Pharmacol. Ther. 2008; 117: 244-279
        • Borsi L.
        • Balza E.
        • Carnemolla B.
        • Sassi F.
        • Castellani P.
        • Berndt A.
        • Kosmehl H.
        • Biro A.
        • Siri A.
        • Orecchia P.
        • Grassi J.
        • Neri D.
        • Zardi L.
        Blood. 2003; 102: 4384-4392
        • Li E.
        • Pedraza A.
        • Bestagno M.
        • Mancardi S.
        • Sanchez R.
        • Burrone O.
        Protein Eng. 1997; 10: 731-736
        • Carnemolla B.
        • Borsi L.
        • Balza E.
        • Castellani P.
        • Meazza R.
        • Berndt A.
        • Ferrini S.
        • Kosmehl H.
        • Neri D.
        • Zardi L.
        Blood. 2002; 99: 1659-1665
        • Carnemolla B.
        • Neri D.
        • Castellani P.
        • Leprini A.
        • Neri G.
        • Pini A.
        • Winter G.
        • Zardi L.
        Int. J. Cancer. 1996; 68: 397-405
        • Borsi L.
        • Balza E.
        • Bestagno M.
        • Castellani P.
        • Carnemolla B.
        • Biro A.
        • Leprini A.
        • Sepulveda J.
        • Burrone O.
        • Neri D.
        • Zardi L.
        Int. J. Cancer. 2002; 102: 75-85
        • Meazza R.
        • Marciano S.
        • Sforzini S.
        • Orengo A.M.
        • Coppolecchia M.
        • Musiani P.
        • Ardizzoni A.
        • Santi L.
        • Azzarone B.
        • Ferrini S.
        Br. J. Cancer. 1996; 74: 788-795
        • Tarli L.
        • Balza E.
        • Viti F.
        • Borsi L.
        • Castellani P.
        • Berndorff D.
        • Dinkelborg L.
        • Neri D.
        • Zardi L.
        Blood. 1999; 94: 192-198
        • Corti A.
        • Poiesi C.
        • Merli S.
        • Cassani G.
        J. Immunol. Methods. 1994; 177: 191-198
        • Hillson J.L.
        • Karr N.S.
        • Oppliger I.R.
        • Mannik M.
        • Sasso E.H.
        J. Exp. Med. 1993; 178: 331-336
        • Santimaria M.
        • Moscatelli G.
        • Viale G.L.
        • Giovannoni L.
        • Neri G.
        • Viti F.
        • Leprini A.
        • Borsi L.
        • Castellani P.
        • Zardi L.
        • Neri D.
        • Riva P.
        Clin. Cancer Res. 2003; 9: 571-579
        • Sauer S.
        • Erba P.A.
        • Petrini M.
        • Menrad A.
        • Giovannoni L.
        • Grana C.
        • Hirsch B.
        • Zardi L.
        • Paganelli G.
        • Mariani G.
        • Neri D.
        • Dürkop H.
        • Menssen H.D.
        Blood. 2009; 113: 2265-2274
        • Del Conte G.
        • Tosi D.
        • Fasolo A.
        • Chiesa C.
        • Erba P.A.
        • Grana C.M.
        • Menssen H.D.
        • Mariani G.
        • Bombardieri E.
        • Gianni L.
        J. Clin. Oncol. 2008; 26 (2575): 15S
        • Wu C.
        • Ying H.
        • Grinnell C.
        • Bryant S.
        • Miller R.
        • Clabbers A.
        • Bose S.
        • McCarthy D.
        • Zhu R.R.
        • Santora L.
        • Davis-Taber R.
        • Kunes Y.
        • Fung E.
        • Schwartz A.
        • Sakorafas P.
        • Gu J.
        • Tarcsa E.
        • Murtaza A.
        • Ghayur T.
        Nat. Biotechnol. 2007; 25: 1290-1297
        • Holliger P.
        • Hudson P.J.
        Nat. Biotechnol. 2005; 23: 1126-1136
        • Kriangkum J.
        • Xu B.
        • Nagata L.P.
        • Fulton R.E.
        • Suresh M.R.
        Biomol. Eng. 2001; 18: 31-40
        • Marvin J.S.
        • Zhu Z.
        Acta Pharmacol. Sin. 2005; 26: 649-658
        • Zardi L.
        • Carnemolla B.
        • Siri A.
        • Petersen T.E.
        • Paolella G.
        • Sebastio G.
        • Baralle F.E.
        EMBO J. 1987; 6: 2337-2342
        • Carnemolla B.
        • Balza E.
        • Siri A.
        • Zardi L.
        • Nicotra M.R.
        • Bigotti A.
        • Natali P.G.
        J. Cell Biol. 1989; 108: 1139-1148
        • Kaczmarek J.
        • Castellani P.
        • Nicolo G.
        • Spina B.
        • Allemanni G.
        • Zardi L.
        Int. J. Cancer. 1994; 59: 11-16
        • Castellani P.
        • Viale G.
        • Dorcaratto A.
        • Nicolo G.
        • Kaczmarek J.
        • Querze G.
        • Zardi L.
        Int. J. Cancer. 1994; 59: 612-618
        • Kosmehl H.
        • Berndt A.
        • Katenkamp D.
        Virchows Arch. 1996; 429: 311-322
        • Castellani P.
        • Borsi L.
        • Carnemolla B.
        • Birò A.
        • Dorcaratto A.
        • Viale G.L.
        • Neri D.
        • Zardi L.
        Am. J. Pathol. 2002; 161: 1695-1700
        • Birchler M.T.
        • Milisavlijevic D.
        • Pfaltz M.
        • Neri D.
        • Odermatt B.
        • Schmid S.
        • Stoeckli S.J.
        Laryngoscope. 2003; 113: 1231-1237
        • Viti F.
        • Tarli L.
        • Giovannoni L.
        • Zardi L.
        • Neri D.
        Cancer Res. 1999; 59: 347-352
        • Demartis S.
        • Huber A.
        • Viti F.
        • Lozzi L.
        • Giovannoni L.
        • Neri P.
        • Winter G.
        • Neri D.
        J. Mol. Biol. 1999; 286: 617-633
        • Menrad A.
        • Menssen H.D.
        Expert Opin. Ther. Targets. 2005; 9: 491-500
        • Neri D.
        • Bicknell R.
        Nat. Rev. Cancer. 2005; 5: 436-446
        • Pilon A.L.
        Ann. N.Y. Acad. Sci. 2000; 923: 280-299
        • Curigliano G.
        • Spitalieri G.
        • De Pas T.
        • Noberasco C.
        • Giovannoni L.
        • Menssen H.D.
        • Zardi L.
        • Milani A.
        • Neri D.
        • De Braud F.
        J. Clin. Oncol. 2007; 25 (3057): 18S
        • Trachsel E.
        • Bootz F.
        • Silacci M.
        • Kaspar M.
        • Kosmehl H.
        • Neri D.
        Arthritis Res. Ther. 2007; 9: R9