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WO1994014848A1 - Immunoglobulines produites par genie genetique - Google Patents

Immunoglobulines produites par genie genetique Download PDF

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Publication number
WO1994014848A1
WO1994014848A1 PCT/US1993/012356 US9312356W WO9414848A1 WO 1994014848 A1 WO1994014848 A1 WO 1994014848A1 US 9312356 W US9312356 W US 9312356W WO 9414848 A1 WO9414848 A1 WO 9414848A1
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Prior art keywords
rgd
immunoglobulin
cells
immunoglobulin molecule
heavy chain
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PCT/US1993/012356
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English (en)
Inventor
Maurizio Zanetti
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The Regents Of The University Of California
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Publication of WO1994014848A1 publication Critical patent/WO1994014848A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention may utilize in its preferred embodiments, the use of recombinant DNA technology to genetically engineer natural or synthetically-derived immunoglobulin molecules, imparting therein novel epitopes, so as to create novel entities that can be employed in vitro and in vivo in a variety of means, such as to immunize against pathogens, and for example, build tolerance to antigens.
  • the epitopes are inserted into the so-called heavy or light chain variable domain of a given immunoglobulin molecule.
  • known recombinant DNA technologies come to bear in the present invention, helping create novel immunoglobulin entities that retain functionality by localizing to particular cell types mechanistically via the so-called constant domains but otherwise functionally exploited to provide a novel localization of a particular antigenic determinant or epitope.
  • Recombinant DNA technology has reached the point currently of being capable, in principle, of providing the methodology sufficient to identify, isolate and characterize DNA sequences, configure them for insertion into operative expression vectors and transfect those vectors variously into recombinant hosts such that those hosts are harnessed in their ability to produce the polypeptide encoded by the DNA sequence.
  • many variations attend the methodology associated with recombinant DNA technology, and particular means are not without inventive faculty. Nonetheless, methods are generally known in the published literature enabling requisite mental equipment for the art skilled to practice recombinant DNA technology in the production of polypeptides from a given recombinant host system.
  • Ir ⁇ munoglobulins are the main effectors of humoral immunity, a property linked with their ability to bind antigens of various types.
  • immunoglobulins that contain antigenic determinants or epitopes against particular such antigens.
  • Immunoglobulin molecules are unique in their functionality of being capable of localizing to certain cell types, probably by means of mutual recognition of certain receptors that are located on the cell membrane. Immunoglobulins demonstrate a second general property whereby they act as endogenous modulators of the immune response. Igs and their idiotypic determinants have been used to immunize at the B- and/or T-cell level against a variety of exogenous antigens. In many cases, the im unity they evoke is comparable with that induced by the antigen itself.
  • V domain of antibodies was demonstrated. Oudin, et al . , Academy of Sciences D 257. 805 (1963) and Kunkel, et al. , Science 140. 1218 (1963) . Subsequently, further research pointed out the existence of discrete areas of variability within V regions and introduced the notion of hypervariable (HV) or complementarity-determining regions (CDR) . u, et al . , J . EXP. Med. 132. 211 (1970). Many studies since have indicated that the immunogenic property of Ig molecules is determined presumably primarily by amino acid sequence contained in the CDRs. Davie, et al. , Ann. Rev. Immunol. A, 147 (1986) .
  • HV hypervariable
  • CDR complementarity-determining regions
  • the basic immunoglobulin or antibody structural unit is well understood.
  • the molecule consists of heavy and light chains held together covalently through disulfide bonds.
  • the heavy chains are also covalently linked in a base portion via disulfide bonds and this portion is often referred to as the so-called constant region which is thought responsible for a given immunoglobulin molecule being mutually recognizable with certain sequences found at the surface of particular cells.
  • the N-terminal regions of the so-called heavy chains branch outwardly in a pictorial sense so as to give an overall Y-shaped structure.
  • the light chains covalently bind to the Y branches of the two heavy chains.
  • a domain of approximately 100 amino acids in length which is variable, and therefore, specific for particular antigenic epitopes incidental to that particular immunoglobulin molecule.
  • variable region contained in the N-terminus Y branches It was a goal of the present research to manipulate these variable regions by introduction or substitution of novel determinants or epitopes so as to create novel immunoglobulin molecules that would possibly retain the localization functionality and yet contain functional heterologous epitopes.
  • novel immunoglobulin molecules hereof could be employed for use within the organism at foreign sites, thereby imparting immunity characteristics in a novel site-directed manner.
  • the present research and invention are based upon the successful threshold experiment, producing model, novel immunoglobulin molecules found to be fully functional by virtue of their ability to localize on certain cell/receptor sites and elicit reactivity to the antigens specific for the introduced novel antigenic determinant or epitope.
  • the present invention is based upon the successful production of novel immunoglobulin molecules having introduced into the N-terminus variable region thereof a novel epitope not ordinarily found in the immunoglobulin molecule used as a starting molecule.
  • oligopeptide epitopes in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. 1
  • peptides acquire an ordered conformation, and antigenized antibodies ( ⁇ Ab) can serve as useful mimics of antigens and ligands.
  • the present invention is thus directed to novel immunoglobulin molecules having at least one novel heterologous epitope contained within the N-terminus variable domain thereof, said novel immunoglobulin molecule having retained functionality with respect to its C-terminus constant domain of the heavy chain specific for a particular cell/receptor type, and having novel, specific epitope in vitro and in vivo reactivity.
  • the present invention is further directed to pharmaceutical compositions containing, as essential pharmaceutical principle, a novel immunoglobulin hereof, particularly those in the form of an administrable pharmaceutical vaccine.
  • the present invention is further directed to methods useful for building tolerance to certain antigens, including those associated with autoimmune diseases, or for down-regulating hypersensitivity to allergens, or for providing active or passive immunity against certain pathogenic antigens, by administering to an individual in perceived need of such, a novel immunoglobulin molecule as defined above.
  • the present invention is further directed to novel recombinant means and methods useful for preparing, identifying and using the novel immunoglobulin molecules hereof including DNA isolates encoding them, vectors operatively harboring such DNA, hosts transfected with such vectors, cultures containing such growing hosts and the methods useful for preparing all of the above recombinant aspects. More specifically, the present invention is directed to the introduction of the conformation of the Arg-Gly-Asp (RGD) tripeptide, a tripeptide involved in the interaction of a variety of adhesive proteins.
  • RGD Arg-Gly-Asp
  • a three-dimensional model of the engineered antibody loop reveals the structure and physicochemical characteristics likely required for the ligand activity. This study demonstrates that expression of the RGD motif in a jS-loop of an antibody imparts adhesive ligand properties to the antibody molecule.
  • the present invention is described herein with particular detail for the preparation of model, novel immunoglobulin entities. This description is provided, as it was conducted, using recombinant DNA technology. Further detail herein defines methods by which one can test a given immunoglobulin to assure that it exhibits requisite functionality common to its starting material immunoglobulin and specially as to its novel epitopic antigenic activity. Given this information with respect to the particular novel immunoglobulin molecules described herein, coupled with general procedures and techniques known in the art, the art skilled will well enough know how to configure recombinant expression vectors for the preparation of other novel immunoglobulin molecules falling within the general scope hereof for use as herein described.
  • RGD is a tripeptide motif present on a variety of adhesive proteins, including fibronectin 2 , vitronectin 3 , fibrinogen 4 and von Willebrand factor 5 .
  • RGD functions as the binding site for a number of integrins 6 and participates in biological processes such as adhesion of cells to the extracellular matrix 7 , platelet aggregation", and migration of tumor cells 9 10 .
  • Figure 1 is a diagram illustrating the construction of the pNylNANP expression vector.
  • Figure 2 is an SDS-PAGE of the ylNANP and T recombinant Ig.
  • Figure 3 shows the binding of 125 I-labelled monoclonal antibody Sp-3-B4 to engineered antibody ylNANP.
  • Figure 4 is a Western blot binding of 1 S I-labelled antibody Sp3-B4 to engineered antibody ylNANP and localization of the engineered (NANP) 3 epitope in the H chain.
  • Figure 5 shows results of cross-inhibition of 125 I-labelled antibody Sp3-B4 binding to synthetic peptide (NANP) 3 (panel A) or engineered antibody ylNANP (panel B) by ylNANP Ig or peptide (NANP) 3 .
  • Figure 6 illustrates the general configuration of the two molecules [referred to as ⁇ ,RGD and 7,(RGD) 3 ] and the site of insertion of the hydrophilic motifs in CDR3.
  • Figure 7 illustrates the adhesion of human non-small cell lung carcinoma cells (TV1) to fibronectin is inhibited by ⁇ ,(RGD) 3 .
  • the following inhibitors were used: ⁇ RGD ⁇ (D), ⁇ GD ( O ) , 7,NANP ( ⁇ ), fibronectin ( ⁇ ) , GdRGDSP
  • Figure 8 shows the lysis of K-562 cells by NK cells of peripheral blood lymphocytes from healthy donors is inhibited by "*Ab 7 1 (RGD) 3 .
  • Figure 9 depicts molecular models of the V regions containing RGD or (RGD) 3 .
  • the RGD-containing molecule is shown in the left panels, and (RGD) 3 in the right panels.
  • the L chain is colored in pink, and the H chain is colored in green.
  • the loops are colored yellow except for: Arg, blue; Gly, white; and Aspartic acid, red.
  • Panels (a) and (d) depict the molecules as solid ribbons through their backbone atoms with the (RGD) 3 loop projecting much farther from the main body of the V region than RGD. Both loops are fairly rigid, as shown by high temperature molecular dynamics, and are roughly planar.
  • the side- chains of the loop residues point away from each other, accentuating the dipolar character of the loops.
  • Panels (J )-( ) and (e) - (f) show contours of the electrostatic fields of the two molecules.
  • Panels (b) and (e) are side views of the RGD and (RGD) 3 loops, respectively.
  • Panels (c) and (f) are top views. Positive contours are shown in blue, and negative contours are shown in red. Contour values are +/- 0.01, +/- 0.02, +/- 0.03, +/- 0.05, +/- 0.07, +/- 0.1, and +/- 0.15 kT/e.
  • RGD is largely negative
  • (RGD) 3 has alternating positive and negative lobes.
  • (RGD) 3 also is seen to be more positive at the N-terminal end of the loop, and negative at the C-terminal end. In the top views, RGD is still largely negative, presumably due to the proximity effects of the rest of the protein. On the other hand, (RGD) 3 is markedly dipolar in this view.
  • “Expression vector” includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operatively linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors may be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. "Operative,” or grammatical equivalents, means that the respective DNA sequences are operational, that is, work for their intended purposes. In sum, "expression vector” is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids” referred to as circular double stranded DNA loops which, in their vector form, are not bound to the chromosome.
  • plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • novel immunoglobulins of the present invention may otherwise permissively differ from the parent in respect of a difference in one or more amino acids from the parent entity, insofar as such differences do not lead to a destruction in kind of the basic activity or bio-functionality of the novel entity.
  • Recombinant host cells refers to cells which have been transfected with vectors defined above.
  • Extrinsic support medium is used to support the host cells and includes those known or devised media that can support the cells in a growth phase or maintain them in a viable state such that they can perform their recombinantly harnessed function. See, for example, ATCC Media Handbook. Ed. Cote et al . , American Type Culture Collection, Rockville, MD (1984) .
  • a growth supporting medium for mammalian cells for example, preferably contains a serum supplement such as fetal calf serum or other supplementing component commonly used to facilitate cell growth and division such as hydrolysates of animal meat or milk, tissue or organ extracts, macerated clots or their extracts, and so forth.
  • Other suitable medium components include, for example, transferrin, insulin and various metals.
  • the vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms.
  • Heterologous with reference herein to the novel epitope for a given immunoglobulin molecule refers to the presence of (at least one) such epitope in the N-terminus domain of an immunoglobulin that does not ordinarily bear that epitope(s) in its native state. Hence, that chain contains heterologous epitope sequence(s).
  • Such heterologous epitope sequences shall include the classic antigenic epitopes as well as receptor binding domains or binding regions that function as receptor sites, such as the human CD4 binding domain for HIV, hormonal receptor binding ligands, retinoid receptor binding ligands and ligands or receptors that mediate cell adhesion.
  • Chimeric refers to immunoglobulins hereof, bearing the heterologous epitope(s) , that otherwise may be composed of parts taken from immunoglobulins of more than one species. Hence, a chimeric starting immunoglobulin hereof may have a hybrid heavy chain made up of parts taken from corresponding human and non-human immunoglobulins.
  • the present disclosure serves to enable reproduction of the specific immunoglobulins disclosed and others, and fragments thereof, such as the individual chains for in vitro assembly, using means within the skill of the art having benefit of the present disclosure. All of such means are included within the enablement and scope of the present invention.
  • the ligand function of the two ⁇ Ab were tested in an in vitro assay by measuring their ability to inhibit the attachment and spreading of tumor cells to a surface coated with fibronectin 10 12 .
  • Human lung carcinoma cell TV1 adhere to surface-bound fibronectin and spread thereafter to form monolayers 13 .
  • 7,(RGD) 3 inhibited the adhesion and spreading of these tumor cells in a dose-dependent manner (Figure 2, A) .
  • Inhibition by 7,RGD was apparent at the highest concentration (200 ⁇ g/ml) only.
  • Also used as inhibitors were two synthetic peptides from the sequence of fibronectin, RGDS and GdRGDSP. Of these, GdRGDSP caused inhibition only at a concentration of 1 mg/ml. RGDS did not inhibit.
  • 7,(RGD) 3 was a more efficient inhibitor than synthetic peptides known to block cell attachment to fibronectin 1014 15 .
  • Figure 2 (B) depicts the inhibitory effect of 7,(RGD) 3 on the adhesion/spreading of TV1 cells.
  • 7,(RGD) 3 but not 7,(RGD) bound tumor cells in a fluorescence-activated cell sorter (FACS) assay.
  • FACS fluorescence-activated cell sorter
  • 7,(RGD) 3 markedly inhibited lysis of K-562 target cells in a conventional 51 Cr-release assay. Inhibition by 7,RGD was somewhat weaker, a finding seemingly reflecting the lower accessibility of the adhesion motif.
  • GdRGDSP failed to inhibit. This rules out a nonspecific effect through the Fc receptor, and implies the involvement of the RGD motif and the requirement for a stabilized tertiary structure in this phenomenon.
  • Conformational models for the two engineered V regions were constructed and analyzed. Each model was refined through a combination of energy minimization and molecular dynamics. The lowest energy conformations were the ones that were analyzed further. As can be seen in Figure 4, both loops project outward from the main body of the protein, ensuring that they are fully accessible for binding to a putative receptor. A comparison of panels (a) and (jb) shows that the (RGD) 3 -containing loop extends much farther into solvent.
  • Figure 6 models for the structure of ⁇ Ab expressing RGD.
  • (a,jb) Diagram of pN7,RGD (a) and pN7,(RGD) 3 (jb) expression vectors.
  • the D region of the parental V H gene (KAYSHG; residues 93-98) was mutagenized in the known per se manner to introduce a single Kpnl/Asp718 site to yield the intermediate sequence KVPYSHG (residues 93-99) .
  • the amino acid 94A was deleted and substituted by the VP doublet encoded by the nucleotide sequence of the Asp718 cloning site.
  • oligonucleotides coding for one or three RGD copies were introduced between 94V and 95P of the mutagenized V H region.
  • the coding strand of CDR3 is shown with the RGD- coding sequence in parenthesis.
  • the expressed ⁇ Ab 7,RGD and 7,(RGD) 3 are mouse/human chimeras.
  • the H chain is the product of the fusion of a human 7,C region with the parental V H murine engineered to express one (c) RGD or three (d) repeats. As shown, the D region was modified by insertion of RGD or (RGD) 3 between 94V and 95P.
  • the inserted sequences are flanked by VP doublet [VP(RGD or RGD 3 )VP] .
  • the light (L) chain is the murine ⁇ j provided by the myeloma J558L host cell (H and L chains not to scale) .
  • V H RGD and V H (RGD) 3 coded by the 2.3 kb EcoRI fragments were cloned upstream from a human 7, constant (C) region gene contained in the 12.8 kb vector pN7,.
  • This is a PSV vector harboring a human 7, gene.
  • This vector encodes a human 7, gene downstream from the EcoRI site. It also carries a neo ycin resistance gene under the control of the SV40 promoter for the selection of stable transformant cells. See also Solazzo et a_l. Eur. J. Immunol. 19. 453 (1989) .
  • pN7,RGD and pN7,(RGD) 3 were electroporated in the murine J558L cell line (2 x 10 7 cells) using a field strength of 750 V/cm.
  • Transfected cells were cultured in Dulbecco's modified minimum Eagle's medium (DMEM) supplemented with 10% fetal calf serum
  • FCS FCS
  • G418 G418-resistant clones secreting high level of the ⁇ b were identified by screening supernatants using an enzyme- linked immunosorbent assay (ELISA) and horseradish peroxidase conjugated goat antibodies to human immunoglobulin (Ig) (Sigma) as probe.
  • ELISA enzyme- linked immunosorbent assay
  • Ig human immunoglobulin
  • ⁇ Ab from culture supernatants were concentrated by (NH 4 ) S0 4 precipitation and then purified on Protein A (Repligen) equilibrated with 3M NaCl, 1M glycine, pH 8.9. Elution was performed using 0.1 M, glycine HC1, pH 2.8, 0.5 M NaCl, and purified ⁇ Ab were neutralized with 1 M Tris-HCl, pH 8.0, followed by dialysis against 0.15 M phosphate buffered saline, pH 7.3 (PBS) .
  • B BamHI; RI, EcoRI; Neo, neomycin resistance; Amp, ampicillin resistance. See Figure 6.
  • Cells were cultured in RPMI 1640, supplemented as described in the legend to Figure 6. Cells were harvested, washed and resuspended in RPMI 1640 containing 2 mM CaCl 2 and MgCl 2 . Cells (2 x 10 6 /well) were incubated in a final volume of 100 ⁇ l for 18 hours at 37°C in 5% C0 2 atmosphere, with or without inhibitors. Non-adherent cells were removed by washing with PBS. Adherent cells were fixed with methanol and stained with 2% crystal violet (Sigma) in 2% ethanol. Cells were washed with PBS and the dye was solubilized by addition of 100 ⁇ l of 0.1 M citric acid in ethanol. Adherent cells were quantitated by reading the plates at 595 nm. Results are expressed as percent inhibition. Tests were done in triplicate.
  • Peripheral blood leukocytes were isolated from heparinized peripheral blood of healthy donors by Ficoll- Hypaque (Histopaque 1077, Sigma) density gradient, washed, resuspended in RPMI 1640 and incubated for 2 hours at 37°C to deplete the adherent mononuclear cells. Lymphocytes were harvested, washed and resuspended in culture medium (RPMI 1640 supplemented as described in the legend to Figure 1) . The NK activity was tested in vitro in a standard 4 hours 51 Cr-release assay against the human erythroleukemia K-562 cells 22 .
  • Target cells (10 6 ) were labeled with 150 ⁇ Ci of Na 51 Cr0 4 (Dupont de Nemours) for 45 minutes at 37°C. Cells were resuspended in culture medium and added to 96-well flat-bottom culture plates (Costar) (5 x 10 3 cells/well) at various effector to target cells ratios (50:1, 25:1, 12:1) in a final volume of 200 ⁇ l alone or in the presence of 100 ⁇ g/ml of 7, (RGD) 3 , 7,RGD, or 7,NANP as a negative control. After 4 hours incubation at 37°C in 5% CO, the plates were centrifuged and 100 ⁇ l of supernatant were harvested from each well.
  • Costar 96-well flat-bottom culture plates
  • the coordinates of RGD and (RGD) 3 were built using the program Homology [Biosym Technologies [Biosym Technologies, #172]]. In this regard, alignments of the sequences of the H and L chains of reference proteins with known coordinates were made using the scheme of Kabat and Wu 23 .
  • the coordinates for the backbone atoms of amino acid residues in the conserved regions were obtained from two reference structures. Those for the H chain came from the mouse Fab fragment J539 24 , and those for the L chain came from the anti-hemagglutinin 17/9 25 . In the conserved region, when the amino acid type of the reference protein and the model matched, the coordinates for the side-chains were copied directly.
  • the side-chain was built from a standard amino acid template library. Bond lengths and angles were kept at their standard values. For the chi angles, the conformations of five other reference structures were examined. A conformation was chosen that was compatible with the majority of the other proteins. Loop coordinates also came from reference immunoglobulin structures, using the protocol of Chothia and Lesk 26 .
  • the coordinates for the RGD and (RGD) 3 loops were generated using the method of Jones and Thirup 27 wherein an ⁇ -carbon distance matrix was constructed for peptide segments on either side of the loop. The matrix was compared to those made from proteins of high resolution found in the Brookhaven Protein Data Bank 21 *. Peptide segments (loops) were selected for which the root mean square (RMS) differences of the distance matrices were lowest and the number of residues in the loop were the same. Ten such loops were obtained, and one was chosen for each model that had the least steric overlap with the rest of the protein, as judged by eye. The final conformation for the loop in each model was determined through a combination of energy minimization and molecular dynamics using the program Discover 29 .
  • RMS root mean square

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Abstract

La présente invention concerne l'introduction de la conformation du tripeptide Arg-GLy-Asp (RGD), tripeptide qui est impliqué dans l'interaction d'une variété de protéines adhésives. Selon l'invention, un anticorps exprimant jusqu'à trois RGD de répétition dans la troisième région de complémentarité de la chaîne lourde bloque efficacement l'adhésion de cellules tumorales humaines à la fibronectine et inhibe la lyse de cellules érythroleucémiques humaines K-562 par des cellules tueuses naturelles (NK).
PCT/US1993/012356 1992-12-24 1993-12-17 Immunoglobulines produites par genie genetique WO1994014848A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022377A1 (fr) * 1995-01-19 1996-07-25 Armitage, Ian, M. Activation de lymphocytes t par des immunoglobulines antigeniques modifiees
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
EP1032420A1 (fr) * 1997-11-14 2000-09-06 Euro-Celtique S.A. Molecules d'immunoglobuline a partie variable de synthese et a specificite modifiee
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
IMMUNOMETHODS, Volume 1, issued 1992, BILLETTA et al., "Ligand Expression Using Antigenization of Antibody: Principle and Methods", pages 41-51. *
PROCEEDINGS OF THE NATION ACADEMY OF SCIENCES, Volume 88, issued June 1991, BILLETTA et al., "Immunogenicity of an Engineered Internal Image Antibody", pages 4713-4717. *
SCIENCE, Volume 238, issued 23 October 1987, RUOSLAHTI et al., "New Perspectives in Cell Adhesion: RGD and Integrins", pages 491-497. *
THE FASEB JOURNAL, Volume 6, No. 5, issued 28 February 1992, BILLETTA et al., "Immunogenicity of Hydrophylic Sequences in Antigenized Antibodies", No. 6226. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
WO1996022377A1 (fr) * 1995-01-19 1996-07-25 Armitage, Ian, M. Activation de lymphocytes t par des immunoglobulines antigeniques modifiees
US6294654B1 (en) 1995-01-19 2001-09-25 Inger Sandlie Modified immunoglobulin molecule incorporating an antigen in a non-CDR loop region
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
EP1032420A1 (fr) * 1997-11-14 2000-09-06 Euro-Celtique S.A. Molecules d'immunoglobuline a partie variable de synthese et a specificite modifiee
EP1032420A4 (fr) * 1997-11-14 2004-09-15 Euro Celtique Sa Molecules d'immunoglobuline a partie variable de synthese et a specificite modifiee

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