- Email: [email protected]
Genetically engineered lipid-tagged antibody; use in liposome-based fluoroimmunoassay
Abstracts results in the generation of anti-idiotype antibodies, a resultant increased rate o f h M A b elimination and potential anaphylactoid responses (J. Immunol. 147, 1352, 1991). In the preclinical safety assessment of a humanized antibody in cynomolgus macaques, it was found that the monkeys raised primarily anti-isotype antibodies following single intravenous or intramuscular administrations. Following repeat administration, a stronger anti-idiotype response was detected, but the anti-iso- type response remained immunodominant. Monkey antibodies also recognized non-related mono- and polyclonal human IgGs. Analysis of the monkey antibodies revealed that the epitopes(s) were in the Fab fragment. Within this fragment, the IgG light and heavy chain constant regions were immunodominant. All monkey anti-hMAbs neutralized binding of hMAb to antigen, but only very high titer anti-idiotype antibodies expressed following repeat administration were neutralizing of the antiviral effect of the hMAb. Plasma clearance of hMab was much more rapid in the presence of monkey antibodies. These studies demonstrate that single doses of hMAb in monkeys can be antigenic, and that strong anti-isotype responses to a more conserved immunoglobulin (vs. murine sequences) are possible.
In vivo expression of recombinant anti-tumor immunoglobulinIL-2 fusion protein. S.V.S. Kashmiri, G.L. Prasad, H-S. Lee, M. Iwahashi, D. Milenic, J. Schlom, L TIB, National Cancer Institute, Bethesda, MD 20892, USA. The murine monoclonal antibody (MAb) CC49, which reacts with tumor associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancers. To target IL-2 selectively to the tumor site and develop a reagent for CC49/IL-2 combination therapy, we developed a single-gene encoded single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein derived from the chimeric CC49. Transfection of murine myeloma cells with the single-gene expression construct pLNCS23-IL-2-expressed a single-chain protein of approximately 70 kD, which was secreted into the tissue culture fluid as a homodimer of approximately 140 kD. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently joined through a (GGGGS) 3 linker peptide. The carboxyl end of the variable domain was linked to the amino terminus of the human IL-2 through a G G G S G G G linker peptide. In this study, the possibility of in vivo expression of the single-gene construct by direct plasmid injection into the mouse quadriceps muscle has been examined. Plasmid DNA was delivered by surgically opening the skin of the mouse and injecting the DNA into the quadriceps muscle. Following a single injection of the expression plasmid, the SCIg-IL-2 was readily detected in animal sera. In sera obtained 28 days after injection, the level of expression was approximately 39 ng/ml. The in vivo expressed SCIg-IL-2 was shown to persist in plasma for as long as 90 days. Cellular immune responses to the in vivo produced SCIg-IL-2 were measured by lymphoproliferative assay. Lymphocytes from mice injected with the expression construct showed dose depen-
301
dent proliferative responses when stimulated with human lgGl. Sera of the inoculated animals were also analyzed by HPLC for the presence of antibodies against the CC49 variable as well as the human 71 Fc regions. The HPLC analyses of the sera collected 70 days after the injection of the plasmid showed formation of the immune complexes with radio labeled MAb CC49, CC49 sFv and goat anti human IgG. Combining recombinant antibody methodology with the in vivo gene inoculation technique should open new avenues for gene therapy. In vivo inoculation of the gene encoding SCIg-IL-2 and its sustained expression at the inoculation site and in plasma demonstrate the feasibility of this approach for gene therapy in the future.
A monoclonal antibody that blocks HIV-I gpl20 glycoprotein binding to neurons. Norman Latov, Grace Lee, Department of Neurology, Columbia University, New York City, New York 10032, USA. The gpl20 glycoprotein of HIV-I binds to rat and human dorsal root ganglia neurons. The bound gpl20 causes lysis of the neurons by complement dependent mechanism. The gpl20 binding may thereby mediate the neuropathy which is associated with HIV-I infection. The neuronal gpl20 receptor appears to be a protein rather than a lipid as gpl20 binding to neurons is removed by pre-treating the cells with trypsin but not with organic solvents. A mouse anti-neuronal monoclonal IgM antibody, designated 01, was found to specifically inhibit binding of gpl20 glycoprotein to neurons. The monoclonal antibody bound to several glycolipids that contain terminal fl-1 linked galactosyl groups including to galactocerebroside, monogalactosyl diglyceride, psychosine, and to asialo-GM1, GMI, and G D I b ganliosides. It also bound to an, as yet, unidentified 220-kD neuronal glycoprotein, which presumedly has a similar terminal oligosaccharide determinant. The cDNAs encoding the 01 antibody were cloned and sequenced. The heavy chain was encoded by a germline member of the V n 558 gene family, which was also used to encode an anti-cytochrome C antibody. The kappa light chain was encoded by a germline member of the VL gene gamily, which was also used to encode an anti-Sin antibody. Single chain antibody reagents are being generated using the 01 antibody genes. These will be used in experiments investigating the role of gpl20 in the development of AIDS neuropathy, and could be used as therapeutic agents to prevent the disease. Genetically engineered lipid-tagged antibody; use in liposomebased fluoroimmunoassay. Marja-Leena Laukkanen, gari Kein~inen, VTT Biotechnology and Food Research, P.O Box 1500, FIN-02044 VTT (Espoo) Finland. In order to obtain the stable and oriented immobilization of antibodies to liposomes, the biosynthetical lipid-tagging of antibody fragments inbacterial cells was developed. The resulting single-chain antibody of an anti-2-phenyloxazolone IgG1 contains a bacterial lipoprotein-specific covalently-linked glycerolipid at the N-terminus which anchors the antibody to the outer membrane of E. coli. Upon detergent removal by dialysis, the purified lipid-tagged antibody was efficiently incorporated to spontaneously formed
302
Abstracts
liposomes. By electron microcopy these immunoliposomes appeared as a relatively homogeneous population of 100-200 nm vesicles and in ELISA they showed specific binding to immobilized hapten. The binding of the immunoliposomes was specifically inhibited in the presence of soluble hapten as demonstrated by the real-time surface plasmon resonance (SPR) measurements which also revealed the practically irreversible binding of these immunoliposomes. Alternatively, the specific hapten-binding activity of immunoliposomes was measured by time-resolved fluorometry (TRF). In these experiments, the lipid-tagged antibody was immobilized to europium (Eu 3 +)-chelate (DTPA)-loaded liposomes either by detegent dialysis or by direct adsorption to premade liposomes. The resulting Eu-immunoliposomes were stable after a 2-month storage at + 4°C and showed specific binding to hapten leading to substantial signal amplification as compared with Eu-labeled free antibody. Our results indicate that by genetic engineering a soluble protein can be converted into a functional membranebound form without tedious chemical treatments. We have demonstrated the potential use of the Eu-immunoliposomes harboring the lipid-tagged antibody as a specific and stable reagent in immunoassay. Other potential applications for lipidtagged antibody/protein containing liposomes include targetspecific drug delivery, development of vaccines and biosensors.
Selection of bivalent anti-PSA antibody clones from a phage display library. John Link, Ralph Abraham, Rodger G. Smith, Colleen Venti, Michael Darsley, IGEN Inc., 16020 Industrial Dr., Gaithersburg, MD 20877, USA. Accurate quantitation of serum levels of free prostate specific antigen (PSA) versus PSA complexed to ~l-antichymotrypsin (PSA/ACT) may provide a clinically relevant assay for differentiating prostate cancer from benign prostatic hyperplasia (BPH). We used phage display technology as a method for generating antibody reagents for creating a diagnostically useful PSA assay. The phage library was constructed from VH and VK antibody fragments amplified by PCR from spleen RNA of a mouse immunized with PSA/ACT complex. These fragments were linked together with a two amino acid linker fragment which allows the fragments to form a bivalent sFv (diabody) when expressed on the phage surface. The randomly combined fragment repertoires were ligated into a modified pCANTAB-5 phagemid vector. The vector modifications allow c-terminal additions of a FLAG epitope for immunological detection and His 6 sequence for IMAC purification to the expressed diabody molecules. A l0 7 member phage display library was generated and specific phage selected by panning against either PSA or PSA/ACT complex. After several rounds of panning, clones were screened for binding to PSA, ACT and PSA/ACT complex using a rapid screening assay based on ORIGEN~ electrochemi-luminescent technology. The diversity of the positive binding clones was determined by BstN1 digests and clones with unique patterns were sequenced. The binding properties of selected clones were analyzed further using BIAcore. The result of this approach was the identification of specific phage clones expressing diabodies with nanomolar affinity for all three
components of the starting immtmogen; PSA, ACT and PSA/ ACT complex. We arc currently focusing on developing ORIGEN ® based diagnostic assays with these diabody reagents for quantitating free and total PSA in human serum.
Detecting novel cell surface antigens using phage antibody display. Ton Logtenberg, Anne Rene van Vuurst, Lucia Cilenti, John. de kruif, Dept. Immunology, University Hospital, Utrecht, The Netherlands. The construction of large-sized libraries of recombinant antibody molecules that are expressed on the surface of filamentous phage and the selection of specific phages by binding to antigens offers a powerful means of generating monoclonal antibodies. We constructed a very large synthetic phage library of human antibodies [1,2] and developed selection strategies using cells in suspension, whole tissues and tissue sections as targets to establish a large collection of high affinity recombinant antibodies against cell surface antigens. We have thus established a collection of > 80 different recombinant antibodies recognizing tumor cells and cells of various hematopoietic and stromal lineages and differentiation stages. Some of these antibodies appear to recognize hitherto unknown antigens. We designed a series of constructs that allow the conversion of selected antibodies to derivatives with desired properties. Thus, we have produced derivatized antibodies with a variety of peptide tags for purification and detection, bi-valent and bi-specific antibodies using fof-fos and fos-jun leucine zipper domains, whole 'conventional' human antibody molecules of every isotype and in vivo biosynthetically lipid-modified antibodies for incorporation in liposomes. These experiments demonstrate that our synthetic phage display library represents a virtually unlimited source of versatile, recombinant antibodies that can be effectively used to detect novel surface antigens and that can be derivatized to yield molecules with desired properties. [1] de Kruif, J., Boel, E. and Logtenberg, T. (1995) Selection and application of human scFv antibody fragments from a semi-synthetic phage antibody display library with 'designed' CDR3 regions. J. Mol. Biol. 248, 97. [2] de Kruif, J., Terstappen, L., Boel, E. and Logtenberg, T. (1995) Rapid selection of cell population-specific human monoclonal antibodies from a synthetic phage antibody library. Proc. Natl. Acad. Scl. USA. 92, 3938. Nuclear localization of autoantibodies: novel insights into protein translocation and cellular function. M.P. Madaio, K. Yanase, M.H. Foster, R.M. Smith, T. Kieber Emmons L. Jarett, M. Fabbi, A. Puccetti, Univ. Pennsylvania, Phils. PA. and Adv. Biotechnolgy Ctr. Genoa, Italy. In the investigation of pathogenic autoantibodies, we identified monoclonal anti-DNA antibodies derived from lupusprone mice, that cross both cellular and nuclear membranes to deposit in nuclei of cells in whole animals. In the kidney, this was associated with glomerular hypercellularity and proteinuria. Upon further investigation, it was discovered that these same Ig localize within nuclei of cultured cells. After direct interaction with cell surface membranes, energy-dependent cellular entry of Ig was mediated through the antigen binding
In vivo expression of recombinant anti-tumor immunoglobulinIL-2 fusion protein. S.V.S. Kashmiri, G.L. Prasad, H-S. Lee, M. Iwahashi, D. Milenic, J. Schlom, L TIB, National Cancer Institute, Bethesda, MD 20892, USA. The murine monoclonal antibody (MAb) CC49, which reacts with tumor associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancers. To target IL-2 selectively to the tumor site and develop a reagent for CC49/IL-2 combination therapy, we developed a single-gene encoded single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein derived from the chimeric CC49. Transfection of murine myeloma cells with the single-gene expression construct pLNCS23-IL-2-expressed a single-chain protein of approximately 70 kD, which was secreted into the tissue culture fluid as a homodimer of approximately 140 kD. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently joined through a (GGGGS) 3 linker peptide. The carboxyl end of the variable domain was linked to the amino terminus of the human IL-2 through a G G G S G G G linker peptide. In this study, the possibility of in vivo expression of the single-gene construct by direct plasmid injection into the mouse quadriceps muscle has been examined. Plasmid DNA was delivered by surgically opening the skin of the mouse and injecting the DNA into the quadriceps muscle. Following a single injection of the expression plasmid, the SCIg-IL-2 was readily detected in animal sera. In sera obtained 28 days after injection, the level of expression was approximately 39 ng/ml. The in vivo expressed SCIg-IL-2 was shown to persist in plasma for as long as 90 days. Cellular immune responses to the in vivo produced SCIg-IL-2 were measured by lymphoproliferative assay. Lymphocytes from mice injected with the expression construct showed dose depen-
301
dent proliferative responses when stimulated with human lgGl. Sera of the inoculated animals were also analyzed by HPLC for the presence of antibodies against the CC49 variable as well as the human 71 Fc regions. The HPLC analyses of the sera collected 70 days after the injection of the plasmid showed formation of the immune complexes with radio labeled MAb CC49, CC49 sFv and goat anti human IgG. Combining recombinant antibody methodology with the in vivo gene inoculation technique should open new avenues for gene therapy. In vivo inoculation of the gene encoding SCIg-IL-2 and its sustained expression at the inoculation site and in plasma demonstrate the feasibility of this approach for gene therapy in the future.
A monoclonal antibody that blocks HIV-I gpl20 glycoprotein binding to neurons. Norman Latov, Grace Lee, Department of Neurology, Columbia University, New York City, New York 10032, USA. The gpl20 glycoprotein of HIV-I binds to rat and human dorsal root ganglia neurons. The bound gpl20 causes lysis of the neurons by complement dependent mechanism. The gpl20 binding may thereby mediate the neuropathy which is associated with HIV-I infection. The neuronal gpl20 receptor appears to be a protein rather than a lipid as gpl20 binding to neurons is removed by pre-treating the cells with trypsin but not with organic solvents. A mouse anti-neuronal monoclonal IgM antibody, designated 01, was found to specifically inhibit binding of gpl20 glycoprotein to neurons. The monoclonal antibody bound to several glycolipids that contain terminal fl-1 linked galactosyl groups including to galactocerebroside, monogalactosyl diglyceride, psychosine, and to asialo-GM1, GMI, and G D I b ganliosides. It also bound to an, as yet, unidentified 220-kD neuronal glycoprotein, which presumedly has a similar terminal oligosaccharide determinant. The cDNAs encoding the 01 antibody were cloned and sequenced. The heavy chain was encoded by a germline member of the V n 558 gene family, which was also used to encode an anti-cytochrome C antibody. The kappa light chain was encoded by a germline member of the VL gene gamily, which was also used to encode an anti-Sin antibody. Single chain antibody reagents are being generated using the 01 antibody genes. These will be used in experiments investigating the role of gpl20 in the development of AIDS neuropathy, and could be used as therapeutic agents to prevent the disease. Genetically engineered lipid-tagged antibody; use in liposomebased fluoroimmunoassay. Marja-Leena Laukkanen, gari Kein~inen, VTT Biotechnology and Food Research, P.O Box 1500, FIN-02044 VTT (Espoo) Finland. In order to obtain the stable and oriented immobilization of antibodies to liposomes, the biosynthetical lipid-tagging of antibody fragments inbacterial cells was developed. The resulting single-chain antibody of an anti-2-phenyloxazolone IgG1 contains a bacterial lipoprotein-specific covalently-linked glycerolipid at the N-terminus which anchors the antibody to the outer membrane of E. coli. Upon detergent removal by dialysis, the purified lipid-tagged antibody was efficiently incorporated to spontaneously formed
302
Abstracts
liposomes. By electron microcopy these immunoliposomes appeared as a relatively homogeneous population of 100-200 nm vesicles and in ELISA they showed specific binding to immobilized hapten. The binding of the immunoliposomes was specifically inhibited in the presence of soluble hapten as demonstrated by the real-time surface plasmon resonance (SPR) measurements which also revealed the practically irreversible binding of these immunoliposomes. Alternatively, the specific hapten-binding activity of immunoliposomes was measured by time-resolved fluorometry (TRF). In these experiments, the lipid-tagged antibody was immobilized to europium (Eu 3 +)-chelate (DTPA)-loaded liposomes either by detegent dialysis or by direct adsorption to premade liposomes. The resulting Eu-immunoliposomes were stable after a 2-month storage at + 4°C and showed specific binding to hapten leading to substantial signal amplification as compared with Eu-labeled free antibody. Our results indicate that by genetic engineering a soluble protein can be converted into a functional membranebound form without tedious chemical treatments. We have demonstrated the potential use of the Eu-immunoliposomes harboring the lipid-tagged antibody as a specific and stable reagent in immunoassay. Other potential applications for lipidtagged antibody/protein containing liposomes include targetspecific drug delivery, development of vaccines and biosensors.
Selection of bivalent anti-PSA antibody clones from a phage display library. John Link, Ralph Abraham, Rodger G. Smith, Colleen Venti, Michael Darsley, IGEN Inc., 16020 Industrial Dr., Gaithersburg, MD 20877, USA. Accurate quantitation of serum levels of free prostate specific antigen (PSA) versus PSA complexed to ~l-antichymotrypsin (PSA/ACT) may provide a clinically relevant assay for differentiating prostate cancer from benign prostatic hyperplasia (BPH). We used phage display technology as a method for generating antibody reagents for creating a diagnostically useful PSA assay. The phage library was constructed from VH and VK antibody fragments amplified by PCR from spleen RNA of a mouse immunized with PSA/ACT complex. These fragments were linked together with a two amino acid linker fragment which allows the fragments to form a bivalent sFv (diabody) when expressed on the phage surface. The randomly combined fragment repertoires were ligated into a modified pCANTAB-5 phagemid vector. The vector modifications allow c-terminal additions of a FLAG epitope for immunological detection and His 6 sequence for IMAC purification to the expressed diabody molecules. A l0 7 member phage display library was generated and specific phage selected by panning against either PSA or PSA/ACT complex. After several rounds of panning, clones were screened for binding to PSA, ACT and PSA/ACT complex using a rapid screening assay based on ORIGEN~ electrochemi-luminescent technology. The diversity of the positive binding clones was determined by BstN1 digests and clones with unique patterns were sequenced. The binding properties of selected clones were analyzed further using BIAcore. The result of this approach was the identification of specific phage clones expressing diabodies with nanomolar affinity for all three
components of the starting immtmogen; PSA, ACT and PSA/ ACT complex. We arc currently focusing on developing ORIGEN ® based diagnostic assays with these diabody reagents for quantitating free and total PSA in human serum.
Detecting novel cell surface antigens using phage antibody display. Ton Logtenberg, Anne Rene van Vuurst, Lucia Cilenti, John. de kruif, Dept. Immunology, University Hospital, Utrecht, The Netherlands. The construction of large-sized libraries of recombinant antibody molecules that are expressed on the surface of filamentous phage and the selection of specific phages by binding to antigens offers a powerful means of generating monoclonal antibodies. We constructed a very large synthetic phage library of human antibodies [1,2] and developed selection strategies using cells in suspension, whole tissues and tissue sections as targets to establish a large collection of high affinity recombinant antibodies against cell surface antigens. We have thus established a collection of > 80 different recombinant antibodies recognizing tumor cells and cells of various hematopoietic and stromal lineages and differentiation stages. Some of these antibodies appear to recognize hitherto unknown antigens. We designed a series of constructs that allow the conversion of selected antibodies to derivatives with desired properties. Thus, we have produced derivatized antibodies with a variety of peptide tags for purification and detection, bi-valent and bi-specific antibodies using fof-fos and fos-jun leucine zipper domains, whole 'conventional' human antibody molecules of every isotype and in vivo biosynthetically lipid-modified antibodies for incorporation in liposomes. These experiments demonstrate that our synthetic phage display library represents a virtually unlimited source of versatile, recombinant antibodies that can be effectively used to detect novel surface antigens and that can be derivatized to yield molecules with desired properties. [1] de Kruif, J., Boel, E. and Logtenberg, T. (1995) Selection and application of human scFv antibody fragments from a semi-synthetic phage antibody display library with 'designed' CDR3 regions. J. Mol. Biol. 248, 97. [2] de Kruif, J., Terstappen, L., Boel, E. and Logtenberg, T. (1995) Rapid selection of cell population-specific human monoclonal antibodies from a synthetic phage antibody library. Proc. Natl. Acad. Scl. USA. 92, 3938. Nuclear localization of autoantibodies: novel insights into protein translocation and cellular function. M.P. Madaio, K. Yanase, M.H. Foster, R.M. Smith, T. Kieber Emmons L. Jarett, M. Fabbi, A. Puccetti, Univ. Pennsylvania, Phils. PA. and Adv. Biotechnolgy Ctr. Genoa, Italy. In the investigation of pathogenic autoantibodies, we identified monoclonal anti-DNA antibodies derived from lupusprone mice, that cross both cellular and nuclear membranes to deposit in nuclei of cells in whole animals. In the kidney, this was associated with glomerular hypercellularity and proteinuria. Upon further investigation, it was discovered that these same Ig localize within nuclei of cultured cells. After direct interaction with cell surface membranes, energy-dependent cellular entry of Ig was mediated through the antigen binding