WO1991016353A1 - Derives d'anticorps specifiques au thrombus - Google Patents

Derives d'anticorps specifiques au thrombus Download PDF

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Publication number
WO1991016353A1
WO1991016353A1 PCT/EP1991/000767 EP9100767W WO9116353A1 WO 1991016353 A1 WO1991016353 A1 WO 1991016353A1 EP 9100767 W EP9100767 W EP 9100767W WO 9116353 A1 WO9116353 A1 WO 9116353A1
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fibrin
cell
thrombus
chain
scupa
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PCT/EP1991/000767
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English (en)
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Yves Laroche
Paul Holvoet
Marc De Maeyer
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Corvas International N.V.
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Publication of WO1991016353A1 publication Critical patent/WO1991016353A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to: novel thrombus (preferably fibrin)-binding molecules ("SCAs”), derived from thrombus (preferably fibrin)-specific antibodies; novel thrombolytic agents (“SCAPAs”) derived from SCAs; and genes coding for such SCAs and SCAPAs.
  • SCAs novel thrombus-binding molecules
  • SCAPAs novel thrombolytic agents
  • This invention also relates to the uses of the SCAs and SCAPAs and to methods of producing them.
  • An injury to a blood vessel normally results in the activation of the complex hemostatic process (Colman et al., 1987) and the formation of a blood clot at the site of injury.
  • the blood clot is predominantly composed of blood platelets emmeshed in a network of fibrin, and its formation protects the organism from bleeding.
  • the clot is later remodelled and removed by degradation of fibrin by proteolytic enzymes such as plasmin.
  • thrombus which is a solid mass or plug formed in a living heart or in blood vessels which can cause severe complications (e.g., myocardial infarctions).
  • a thrombosis is due to either local obstruction of blood vessels or distant embolization.
  • the relative amounts of the formative elements of thrombi i.e., blood platelets, erythrocytes and fibrin
  • depend on the place in the vascular system where the thrombi are formed (Freiman, 1987) .
  • Fibrin is formed by polymerization of fibrinogen, a protein with a very complex organization.
  • Each fibrinogen molecule is composed of six complexly linked polypeptides (i.e., 2 A-alpha, 2 B-beta and 2 gamma polypeptides) .
  • the molecule can be divided into a number of domains, the most important of which are the two terminal D-domains and the central E-domain.
  • covalent crosslinks are formed between D and D and between D and E domains of adjacent fibrinogen molecules, resulting in a highly interconnected network of fibers with a sufficient mechanical stability to serve as a hemostatic plug (Hantgan et al., 1987).
  • Degradation of fibrin by plasmin results in a variety of degradation products, one of which is the so-called D-dimer which comprises two cross-linked D domains of adjacent fibrinogen molecules.
  • thrombosis and its complications has mainly been directed towards: prevention of local extension of a thrombus by administration of anticoagulants such as heparin; and/or acceleration of the rate of dissolution of the thrombus by administration of thrombolytic agents such as plasminogen activators which are substances that initiate formation of plasmin (a proteolytic enzyme that degrades fibrin) .
  • plasminogen activators which are substances that initiate formation of plasmin (a proteolytic enzyme that degrades fibrin) .
  • the most important plasminogen activators are tissue-type plasminogen activators ("tPA”) , urokinase-type plasminogen activators ("uPA”) and streptokinase and its derivatives (Bachmann, 1987) .
  • tPA is a protein of 530 amino acids which can be isolated from many tissues but which seems to be mainly produced by endothelial cells as a single-chain polypeptide.
  • the amino acid seguence of this polypeptide and the nucleotide seguence that encodes it have been described by Pennica et al. (1983).
  • tPA is cleaved by plasmin between Arg278 and Ile279 resulting in a molecule consisting of two polypeptide chains held together by one disulfide bridge. In the presence of fibrin both forms are egually active.
  • tPA can be divided in a finger domain containing low- affinity fibrin binding sites, a domain with homology to epidermal growth factor ("EPG") , two kringle domains with high-affinity fibrin binding sites, and the serine protease catalytic domain. Because of its binding capacity to fibrin, plasminogen activation by tPA is highly fibrin-specific.
  • EPG epidermal growth factor
  • urokinase precursor designated as prourokinase or single-chain uPA (“scuPA”)
  • scuPA single-chain uPA
  • tissues such as endothelium of the kidney as a continuous polypeptide of 411 amino acids and a molecular weight of about 54 kiloDalton (kD) (“scuPA-54k”) .
  • kD kiloDalton
  • ScuPA is converted to the active form, designated as urokinase or two-chain uPA ("tcuPA”) by a proteolytic cleavage (e.g., by plasmin) between Lysine-158 and Isoleucine-159 which results in a molecule consisting of two polypeptide chains held together by a single disulfide bond.
  • proteolytic cleavage e.g., by plasmin
  • Three domains can be identified in prourokinase: a EPG domain, one kringle domain and a serine protease catalytic domain. These domains show important homologies with the corresponding domains of tPA. However, neither the EPG domain, nor the kringle domain of scupa have affinity for fibrin.
  • a low molecular weight form of scuPA (“LM -scuPA”) is formed by an additional proteolytic cleavage between lysine 135 and Lysl36, releasing the first 135 amino acids (i.e., the EPG and the kringle domains).
  • the LMW-scuPA is converted to a low molecular weight two- chain urokinase of about 33 kD (“LMW-tcuPA”) by a proteolytic cleavage between Lysl58 and Ilel59.
  • EP 247674 describes a single-chain uPA of about 32 kD which apparently is derived from the scuPA by a proteolytic cleavage between Glul43 and Leul44 and which can be recovered in stable conditions and in fair yields from the culture fluid of human lung adenocarcinoma cells of the type CALU-33 (ATCC cell line HTB-55) .
  • This form was designated as "scuPA-32k” to differentiate it from the previously known scuPA-54k.
  • ScuPA-32k like scuPA-54k, can be proteolytically activated, e.g., by plasmin.
  • scuPA-54k does not bind directly to fibrin, it activates fibrin-associated plasminogen much more readily than plasminogen in the plasma (Gurewich et al., 1984). Although scuPA-32k lacks both the EPG and the kringle domain of scuPA-54k, it still displays fibrin selectivity. In contrast, the two active forms, described above, have lost their fibrin selectivity.
  • Plasminogen activators as thrombolytic agents has been hampered by the fact that they will also dissolve existing hemostatic plugs in an organism, thus leading to hemorrhaging. Plasminogen activators that lack fibrin affinity, such as streptokinase and urokinase, will also activate circulating plasminogen resulting in impairment of platelet function and degradation of circulating fibrinogen and clotting factors V and VIII (in addition to the fibrin in the thrombus) and leading to systemic effects which have generally been designated as the "lytic state" (Marder and Bell, 1987) .
  • plasminogen activators such as tPA or scuPA, which demonstrate fibrin-selective plasminogen activation, thus releasing plasmin only in the vicinity of a clot.
  • tPA and the active forms of uPA have very short circulatory half-lives (5 and 7 minutes for tPA and urokinase, respectively - Sherry, 1987) due to their interaction with receptors on cells of the liver.
  • their activatory half-life is reduced by means of a rapid and irreversible inactivation by plasminogen activator inhibitor I (Haber et al., 1989).
  • Both scuPA-54k and scuPA-32k are resistant to this inhibitor and have longer activatory half-lives. Nevertheless, treatment with any of these agents has had to be over extended periods and in combination with heparin to avoid the occurrence of reocclusion.
  • plasminogen activators In attempts to improve on the naturally occurring plasminogen activators, several approaches have been followed (see, e.g., Haber et al., 1989; Haber, 1990). Generally, plasminogen activators have been sought which display, in addition to high thrombus (e.g., fibrin)-selective activity, longer circulatory and activatory half-lives.
  • thrombus e.g., fibrin
  • Antibodies are proteins that are secreted by specialized cells (i.e., the B-lymphocytes) as a part of the immune response of an organism to introduction of foreign molecules (i.e., antigens). Any antibody is highly specific for one particular antigen. With the development of hybridoma technology, monoclonal antibodies, which are highly homogenous with respect to their antigen specificity, could be produced in virtually unlimited guantities (see, e.g., Harlow and Lane, 1988) .
  • antibodies The structure of antibodies is well known (see, e.g., Albers et al., 1989). They have a Y-shape and consist of two identical heavy (H) chains of about 440 amino acids and two identical light (L) chains of about 220 amino acids. The various polypeptide chains of a single antibody molecule are connected to each other by four disulfide bridges. In mammals, there are five different classes of antibodies, each of which is characterized by a different type of H-chain (the alpha, delta, epsilon, gamma and mu H-chains) . In addition, antibodies can contain two different L chains (the kappa and lambda L-chains) .
  • H-chain defines effector functions other than antigen specificity, such as interactions with antibody receptor molecules on different cells.
  • the difference between the two types of L-chains has not yet been identified.
  • the largest class of circulating antibodies (IgG) have a gamma H-chain.
  • the H- and L-chains consist of variable and constant domains.
  • the L-chain has a variable domain of about 110 amino acids ("V L ”) and a constant domain also of about 110 amino acids (“C L ”) .
  • the H-chain has one variable domain ("V H ") and three constant domains ("C H1 ", "C H2 " and "C H3 "), each of about 110 amino acids.
  • V H variable domain
  • C H1 ", "C H2 " and “C H3 " constant domains
  • CDRs complementarity determining regions
  • the amino acid sequences of the CDRs are highly variable among antibodies while the sequences of the parts of the variable domains next to and in between the CDRs (i.e., the so-called “framework regions") are much more conserved.
  • the antigen binding site interacts with a well-defined region of the antigen which is designated the "epitope".
  • variable domains of the heavy and light chains are encoded by different gene segments which are properly organized in the fully differentiated B- lymphocyte through recombination events.
  • the V L region thus consists of a large N-terminal part which is encoded by the so-called variable (“V") gene segment and a short C-terminal part which is encoded by the so-called joining ("J") gene segment.
  • V variable
  • J joining
  • the V H domain largely consists of a large N-terminal part, which is encoded by a V gene segment, and two smaller parts, encoded by the diversity (“D”) gene segment and J gene segment, respectively.
  • V L and V H are the only components necessary for antigen binding. It has been shown that proteins can be prepared by connecting the nucleotide sequences coding for the V L and V H regions with a linker seguence coding for a linker polypeptide ("L") , and expression of these hybrid DNA molecules can be obtained in E. coli (Bird et al., 1988; Huston et al., 1988; Chaudhary et al., 1989, 1990). The resulting V L -L-V H or V H -L-V L proteins retained their antigen- binding capacity and can be designated as single-chain antibodies.
  • L linker seguence coding for a linker polypeptide
  • Anti-fibrin antibody 59D8 directed against the amino-ter inal six-amino acid sequence of the fibrin beta chain, was chemically conjugated to urokinase and tPA (Bode et al., 1987), and anti-fibrin antibody MA-15C5, directed against human fibrin D-dimer, was conjugated to scupa (Collen et al., 1989; Dewerchin et al., 1990; Collen et al., 1990) .
  • Recombinant DNA technology has also been used to replace parts of the heavy chain of the 59D8 antibody with portions of tPA and uPA catalytic domains (EP 271227, EP 355068 and EP 347078). In general, the results of these attempts have been mixed, and an ideal thrombolytic agent has not yet been identified (Haber et al., 1989; Haber, 1990).
  • This invention provides an SCA comprising a single-chain antibody which can bind in a highly specific manner to at least one thrombus constituent, preferably fibrin. It is preferred that the SCA comprise all or preferably the effective antigenic- binding parts of a monoclonal antibody directed against the thrombus constituent, particularly fibrin, quite particularly fibrin cross-links, in a thrombus. It is particularly preferred that the SCA comprise all or preferably the effective antigenic-binding parts of the heavy and light variable domains (V H and V L , respectively) of the monoclonal antibody, linked through a first linker peptide ("L- f c”) so as to form a single chain.
  • V H and V L first linker peptide
  • This invention also provides a method of using the SCA for imaging of thrombi and for making novel thrombolytic agents comprising the SCA as a thrombus constituent-binding portion, preferably a fibrin- binding portion.
  • This invention further provides an SCAPA which is a plasminogen activator comprising an SCA as a thrombus-binding portion ("SCA-portion") connected to a plasminogen activating portion ("PA-portion”) .
  • SCA-portion a plasminogen activator
  • PA-portion plasminogen activating portion
  • the .PA-portion comprise at least the catalytic domains" of a plasminogen activator, preferably of tPA or uPA, particularly of scuPA.
  • the C-terminal end of the SCA-portion is preferably directly linked to the N-terminal end of the PA- portion, but both portions can also be linked through a second linker peptide ( n l ⁇ d " ) .
  • This invention still further provides a DNA molecule coding for the SCA ("sea gene”) or for the SCAPA (“scapa gene”) , a chimaeric DNA sequence ("chimaeric gene”) containing the sea or scapa gene and a vector containing the chimaeric gene.
  • the chimaeric gene comprises the following operably linked DNA fragments in the same transcriptional unit: 1) a promoter capable of directing expression of a sea or scapa gene in a procaryotic or eucaryotic host cell; 2) a sea or scapa gene; and 3) suitable 3• regulatory sequences.
  • the chimaeric gene can optionally contain, between DNA fragments 1) and 2) , a signal sequence that encodes a polypeptide ("signal peptide") directing the secretion of the SCA or SCAPA from the procaryotic or eucaryotic host.
  • This invention further provides a method for obtaining the SCA or the SCAPA by: introducing the chimaeric gene in a procaryotic or eucaryotic host cell so that it is actively expressed within the host cell; culturing the host cell; and then recovering the SCA or SCAPA from the culture.
  • Fig. 1 Amino acid sequence of the variable region of the kappa chain of the monoclonal antibody MA-15C5.
  • the numbering of the amino acids follows the generalized numbering described by Kabat et al. (1987) .
  • Single lines indicate the borders of the CDR and framework regions.
  • Fig. 2 Amino acid sequence of the variable region of the gamma-chain of the monoclonal antibody MA-15C5.
  • the numbering of the amino acids follows the generalized numbering described by Kabat et al. (1987) .
  • Single lines indicate the borders of the CDR and framework regions.
  • the double line indicates the end of the region encoded by the J gene segment.
  • Fig. 3 Nucleotide sequence of the cDNA coding for the variable and constant region of the kappa chain of the monoclonal antibody MA-15C5.
  • the amino acid sequence of the variable region is also given (see also Fig. 1) .
  • the seguence also comprises the signal sequence. Important restriction sites used during cloning procedures are indicated.
  • Fig. 4 Nucleotide sequence of the cDNA coding for the variable region of the gamma chain of the monoclonal antibody MA-15C5 with the amino acid sequence as given in Fig. 2.
  • the codons for the first four amino acids of the V L domain are missing.
  • the sequence also comprises part of the coding sequence of the C H1 domain of the MA-15C5 gamma chain. Important restriction sites used during cloning procedures are indicated.
  • the double line indicates the end of the region encoded by the J gene segment.
  • Fig. 5 Nucleotide sequence and deduced amino acid sequence of the human uPA cDNA. Important restriction sites used during cloning procedures are indicated. The cleavage site of LMW-tcuPA (single vertical line) and the N-terminus of scuPA-32k (double vertical line) are indicated.
  • Fig. 6 Amino acid sequences of preferred SCAs of this invention.
  • the numbers and amino acids in square brackets refer to the amino acid sequence of V L of MA-15C5 as given in Fig. 1.
  • the numbers between brackets refer to the amino acid sequence of the first linker peptide (L ⁇ ) .
  • the letters and amino acids between accolades refer to the amino acid sequence of V H of MA-15C5 as given in Fig. 2.
  • the sequences derived from the V L and V H anchor regions are underlined. Residues marked with an asterisk are residues that are mutated with respect to the original sequence.
  • Fig.7 Nucleotide sequence of the tac promoter and the PhoA signal sequence.
  • the encoded amino acid sequence of the PhoA signal peptide is also shown.
  • the promoter and signal peptide can for instance be used for the expression and secretion of foreign proteins in E ⁇ _ coli.
  • Fig. 8 - A Nucleotide sequence of the signal sequence of the kappa chain of MA-15C5 monoclonal antibody. The encoded amino acid sequence of the kappa-chain signal peptide is also shown.
  • - B Nucleotide sequence of a signal sequence coding for a consensus signal peptide of a human IgG gamma chain. The 11 C-terminal amino acids of this signal peptide are those of the natural signal peptide of the gamma chain of MA-15C5 monoclonal antibody.
  • the signal peptides of Figs. 8a and 8b can, for instance, be used for the expression and secretion of foreign proteins in eucaryotic cells.
  • Fig. 9 Nucleotide sequences and corresponding amino acid sequences of the first linker peptide of selected sea genes of the present invention.
  • the seguences of the actual first linker peptides are underlined.
  • the single-chain antibody of the SCA of this invention is derived from a monoclonal antibody that is specific for a constituent of thrombi, preferably fibrin, particularly fibrin cross-links.
  • the monoclonal antibody is a murine monoclonal antibody, such as MA-15C5, raised against human fibrin D-dimer.
  • MA-15C5 has been described by Holvoet et al. (1989) , and the construction of recombinant genes coding for the L- and H-chains of MA-15C5, from cDNA libraries of MA-15C5 hybridoma cells, has been described by Vandamme et al. (1990) and in European patent application (“EPA”) 90401090.7.
  • recombinant genes can be used for the construction of an sea gene.
  • amino acid sequences of the variable domains of the kappa and gamma chains of MA-15C5 are shown in Fig. 1 and Fig. 2, respectively.
  • the CDR and framework regions are indicated in these Figures.
  • the corresponding nucleotide sequences are shown in Fig. 3 and Fig. 4, respectively.
  • the SCA can have the following general structure
  • V H and V L domains should be linked by an appropriate first linker peptide (L gb ) .
  • a suitable I_, b can be designed using the computerized procedures outlined in PCT patent publication WO
  • the L ⁇ can be designed by the so-called “spare parts” method as described by Claessens et al.
  • This method also involves the use, as a template, of an existing three-dimensional structure of an antibody molecule, with H- and L-chains similar to those of the fibrin-specific monoclonal antibody (e.g.,
  • MA-15C5 to be used for the construction of the SCA, to construct a 3D model of the fibrin-specific antibody or at least its framework regions.
  • 3D structures of proteins can be found in, for example, the Brookhaven
  • Identification of suitable fragments can be carried out by calculating the root mean square deviations ("rms") between the Cartesian coordinates of the alpha carbon atoms (or the main chain atoms) of the anchor regions and the overlapping regions of the protein fragments (Claessens et al., 1989) . Only those fragments, for which the rms falls below a certain threshhold determined by the user, are withheld for further study as an L ⁇ .
  • rms root mean square deviations
  • step 3 Selecting the most desirable fragments identified in step 2, which preferably conform to the following requirements:
  • the first linker peptide should not interfere with the ordered secondary structure or with the folding of the V L and V H domains. Secondary structure predictions can be performed according to the procedures described by Jibrat et al. (1987) .
  • the regions of the first linker peptide that are exposed to solvent should not contain patches of hydrophobic residues.
  • the first linker peptide should be sterically accomodated. Sterical accomodation of fragments can, for instance, be evaluated by calculating the non-bonded energy of the main chain atoms of the linker fragments with respect to the rest of the protein.
  • the amino acid sequences of the first linker peptide and/or the anchor regions can, if desired, be optimized by introduction of mutations (e.g., substitutions, deletions and/or additions) in order to reduce their non-bonded energy, to minimize their hydrophobicity and/or to improve their flexibility.
  • mutations e.g., substitutions, deletions and/or additions
  • regions of the first linker peptide, that immediately flank the anchor regions be mutated to residues that were originally present in the V L and V H domains.
  • V L and/or V H can be extended by an extension seguence in appropriate directions, and such a sequence can then serve as an anchor region.
  • the anchor regions be as near as possible to the appropriate C- and N- termini of the V L and V H or the V H and V L domains, respectively.
  • an anchor region at the C-terminus of the V H domain can also be located at the end of the V H region that is encoded by the J gene segment.
  • 3D structures of proteins e.g., SCAs
  • SCAs proteins
  • 3D structures of proteins can be obtained by methods such as crystallography (Wyckoff et al., 1985), nuclear magnetic resonance spectroscopy (W ⁇ thrich, 1986) , structure derivations based on available 3D structures from homologous proteins (see, e.g., Blundell et al., 1987) , or from structure predictions based on analysis of the primary structures (for a review, see Taylor, 1988) .
  • the 3D structures of proteins of this invention can be analyzed and modelled by the use of a dedicated computer software package such as the BRUGEL (R) molecular graphics software package (Delhaise et al., 1985 - Plant Genetic Systems N.V. , Ghent, Belgium).
  • BRUGEL R molecular graphics software package
  • the SCA is characterized by an amino acid sequence as shown in Fig. 6.
  • a first linker peptide which comes from a naturally-occurring protein and which seems to serve as a natural linker between major functional domains of the naturally-occurring protein, such as a hinge-like sequence of an immunoglobulin.
  • the corresponding SCA can be connected to at least the catalytic domain of a plasminogen activator (i.e., the PA-portion) .
  • the serine-protease catalytic domain of scuPA comprising amino acids 144 to 411 in Fig.
  • the SCA- and PA-portions can, in principle, be connected in two ways: the C-terminus of the PA-portion can be linked to N-terminus of the SCA or the C-terminus of the SCA can be linked to the N- terminus of the PA-portion.
  • a suitable second linker peptide (L ⁇ ) should be designed.
  • scuPA when the catalytic domain of scuPA is used for the production of the SCAPA of this invention, in which the C-terminal part of a SCA- portion is linked to the N-terminal part of a PA- portion, it is preferred that part or all of this region be used as a second linker peptide between the two portions.
  • preferred attachment sites on the scuPA are believed to be Alal32, Lysl36 and Leul44.
  • preferred attachment sites at the C-terminal part of the V ⁇ L ⁇ -V, of SCAs are believed to be Serll3 (i.e., the end of the V H part encoded by the J gene segment), Serl20 (i.e., the actual end of the fourth framework of the V H domain) , or any other amino acid between these two residues of the heavy chain.
  • a preferred C-terminal attachment site on the V H -L ab -V L of SCAs is believed to be located at Leul04 of the kappa-chain (numbering as in Fig. 1 and Fig. 2) .
  • part of the constant domain following the variable domains of the heavy and light chains of the fibrin-specific antibodies can also be used as the second linker peptide between the SCA- and PA-portions.
  • all or part of the A-domain of scuPA such as the EGF-like and/or the Kringle domains of scuPA, can be used as the second linker peptide.
  • proteolytic cleavage of the uPA catalytic domain e.g., by plasmin or thrombin
  • Phel57 of scuPA can, for instance, be mutated to Aspl57 to remove the Argl56- Phel57 thrombin cleavage site.
  • Lysl35 of scuPA can be mutated, for instance to Glnl35, to remove the Lysl35-Glnl36 plasmin cleavage site.
  • the SCA and the SCAPA of this invention can be produced by the expression, in host cells, of the sea and scapa genes, respectively, preferably the chimaeric gene of this invention.
  • the construction of these genes can be achieved in a conventional manner.
  • cDNAs coding for V L and V H can, for instance, be isolated from cDNA libraries from suitable hybridomas producing thrombus- specific, preferably fibrin-specific, antibodies (see, e.g., Vandamme et al, 1990).
  • DNA fragments coding for linkers L ⁇ and ⁇ can be directly synthesized.
  • the gene coding for prourokinase can be obtained as described by Holmes et al. (1985). Appropriate DNA fragments can be ligated to each other by conventional means so as to produce one contiguous DNA fragment coding for the SCA or SCAPA protein of this invention.
  • the sea and scapa genes can be expressed in suitable procaryotic or eucaryotic host cells by placing the genes under the control of a promoter capable of directing their expression in the host cells.
  • a promoter capable of directing their expression in the host cells Conventional promoters can be used.
  • Preferred promoters for use in E. coli are, for example, regulatable promoters such as: the P tac promoter (De Boer et al., 1983), the sequence of which is shown in Fig. 7, the P lac promoter (Fuller, 1982), the P trp promoter (Martial et al., 1979), the lambda P L promoter (Bernard et al., 1979) and the P R promoter (Zabeau and Stanley, 1982) .
  • Preferred promoters for use in mammalian cells have, for example, been described by Menck et al. (1987), Baker et al. (1988), Artelt et al. (1988) and Lee
  • a signal sequence can be placed in front of, and in reading phase with, the sea or scapa gene.
  • the signal sequence provides: a) a translation initiation site and b) the necessary functional sequence for exporting the SCA or SCAPA.
  • signal sequence is meant a DNA fragment coding for a polypeptide fragment ("signal peptide") which is normally associated with a protein, or subunit of a protein that is translocated out of the cytosol of the host cell —for example, to the periplasmic space in E.
  • the signal peptide is responsible for the translocation process during which the signal peptide is separated or proteolytically removed from the protein or subunit.
  • Signal sequences which can be used are those coding for the signal peptides listed by Watson (1984) or for signal peptides that conform to the general characteristics as outlined by Von Heyne (1988) .
  • coli is the one coding for the phoA signal peptide (Michaelis et al., 1983) which is shown in Fig. 7.
  • Preferred signal sequences, that can be used in eucaryotic cells are those coding for the signal peptides that are naturally associated with the heavy and light chains of antibodies.
  • the amino acid seguence and its encoding nucleotide sequence of the signal peptide of the kappa chain of the MA-15C5 monoclonal antibody is shown in Fig. 8A
  • the amino acid seguence and its encoding nucleotide sequence of the signal peptide of the gamma chain of MA-15C5 is shown in Fig. 8B.
  • Another preferred signal seguence is that coding for the signal peptide normally associated with the plasminogen activator, for instance the signal peptide associated with scuPA (Fig. 5 - residues Metl to Gly20 - see also Holmes et al., 1985).
  • the use of a signal peptide is preferred for the production of the SCA or SCAPA, it is not necessary.
  • a cell can be transformed with just a sea or scapa gene encoding a SCA or SCAPA under the control of a suitable promoter, and the SCA or SCAPA, expressed by the transformed cell intracellularly, can be obtained by lysing the cell.
  • Preferred host cells to express the chimaeric gene of this invention are insect cells.
  • the chimaeric gene is placed under the control of the strong polyhedrine promoter, particularly of the Autoqrapha californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda using the procedures and vectors described, for example, by Summers and Smith (1987) and Luckow and Summers (1987, 1989) and in US patent 4745051.
  • Other baculovirus expression vectors such as those described in EP 345152 and EP 340359 and PCT publications WO 89/01038 and WO 89/01037, can also be used.
  • the SCA and SCAPA can also be prepared by construction of a chimaeric gene capable of being expressed in other host cells, such as E ⁇ coli, B.subtilis, yeasts and mammalian cells (e.g., CHO cells) , preferably mammalian cells.
  • a chimaeric gene capable of being expressed in other host cells, such as E ⁇ coli, B.subtilis, yeasts and mammalian cells (e.g., CHO cells) , preferably mammalian cells.
  • Appropriate promoters, regulatory sequences (including 3 ' regulatory sequences, as well 5* regulatory sequences and enhancer sequences) and, if necessary, signal sequences for such a chimaeric gene are well-known to those skilled in the art.
  • the chi.maeri.c gene of thi.s invention can be introduced into host cells, the host cells can be cultured, and the SCA or SCAPA can be purified from the host cell culture by conventional means.
  • Secreted SCA or SCAPA can be purified, for example, by affinity chromatography on immobilized epitope (e.g. D-dimer) and/or immunoadsorption to insolubilized antibody raised against the PA-portion.
  • the SCA of this invention can be constructed using the variable domain of thrombus-specific, preferably fibrin-specific, monoclonal antibodies of other than MA-15C5.
  • the corresponding sea gene can be constructed and expressed in analogous ways to those described above.
  • Monoclonal antibodies that can be used are, for instance, those described by Kudryk et al. (1984), Elms et al. (1983), Scheefers-Borchel et al. (1985) and Hui et al. (1986) and in Australian patent publication AU-B-25387/84.
  • the SCA or SCAPA of this invention When the SCA or SCAPA of this invention is used for multiple intravenous applications in patients, it may be preferred to minimize its immunogenicity. This can be achieved (see, e.g., LoBuglio et al., 1989) by replacing the nucleotide sequences coding for the murine framework regions by the corresponding sequences coding for framework regions derived from variable domains of human antibodies as described, for example, by Riechmann et al. (1988) and Verhoeyen et al. (1988) and in EP 328404.
  • the SCA of this invention can be used for imaging of thrombi.
  • the SCA can be labelled with an opacifying agent, such as an NMR or X-ray contrasting agent, or radioactively labelled in a conventional manner.
  • the SCAPA of this invention can be used as a thrombolytic agent to treat patients with myocardial infarction, peripheral arterial thrombosis, and stroke, as well as deep venous thrombosis and pulmonary embolism.
  • the SCAPA has a number of advantages over existing thrombolytics.
  • the thrombus-specific SCA- portion targets the SCAPA, and consequently its plasminogen activation activity, to the thrombus.
  • the use of an SCA derived from an antibody specific for fibrin, particularly fibrin cross-links (such as MA-15C5) is especially preferred. This ensures that the corresponding SCAPA will remain in contact with the thrombus for a longer period of time during the degradation of fibrin.
  • the half-life of the SCAPA is likely to be predominantly determined by its SCA-portion (Collen et al., 1989), it is also expected that the half-life of such a molecule will be greater than its PA-portion alone. It is believed that the half-life of the SCAPA of this invention also can be increased by producing it: 1) in a non-glycosylated form or in a super-glycosylated form or in a form in which some glycosylation is added to the SCAPA (i.e., to one or more regions of the SCAPA) and other glycosylation is removed from the SCAPA (i.e., from one or more other regions of the SCAPA); and/or 2) in a form which is resistant to plasminogen activator inhibitors; and/or 3) with all or at least a significant part of the A domain (at the N 1 end of the catalytic domain) of its PA-portion, particularly of scuPA, serving as the second linker peptide.
  • the SCAPA of this invention will display a lower immunogenicity and a better thrombus penetration due to its reduced molecular weight.
  • the final conformation of the SCAs and SCAPAs of this invention will depend upon the independent folding of their separate domains (V L , V H and plasminogen activator catalytic domain) and not upon the association of disulfide bridges with separate polypeptide chains. It is believed that this will simplify production of these molecules in host cells transformed with sea or scapa genes of this invention as described above.
  • transformed insect or mammalian host cells can properly process and secrete the SCAs and SCAPAs of this invention, so that they are properly folded for binding to a thrombus constituent and without significant loss of binding activity (as compared to the thrombus- specific antibodies, from which they are derived) .
  • the SCAs and SCAPAs of this invention can, if desired, be produced in a glycosylated or super- glycosylated form in insect or mammalian cells transformed with, respectively, sea or scapa genes or mutated sea or scapa genes in which glycosylation sites have been added.
  • the SCAs and SCAPAs can be conveniently expressed in transformed host cells in a non-glycosylated form by mutating the sea and scapa genes at sites which would otherwise encode amino acid sequences which could be glycosylated.
  • potential glycosylation sites could be eliminated, for example: in the portion of the nucleotide sequence of Figure 4 encoding the heavy chain domain (V H ) of the MA-15C5 antibody, by mutating its AAT nucleotides encoding Asn at positions 261-263 to the nucleotides GAT encoding Asp; and/or in the portion of the nucleotide seguence of Figure 5 encoding scuPA, by mutating its AAT nucleotides at positions 1063-1065 to the nucleotides GAT.
  • SCAPAs of this invention can be conveniently expressed in transformed host cells in a form more resistant to a plasminogen activator inhibitor (e.g., PAI-1) by mutating the plasminogen activator catalytic domain encoded by the scapa gene, so that it encodes, for example, a mutant tPA-encoding region as described by Madison et al. (1989, 1990) or a mutant SCUPA-encoding region in which, from nucleotide 691 to nucleotide 702 in Figure 5, the amino acids Arg Arg His Arg are changed to smaller uncharged amino acids such as Ala or to negatively charged amino acids such as Glu.
  • a plasminogen activator inhibitor e.g., PAI-1
  • PAI-1 plasminogen activator inhibitor
  • oligonucleotides were designed according to the general rules outlined by Kramer and Fritz (1988) and synthesized by the phosphoramidite method (Beaucage and Caruthers, 1981) on an Applied Biosystems 380A DNA synthesizer (Applied Bisosystems B.V. , Maarssen, Holland) .
  • Example l Design of first linker peptides for the V ⁇ .-L ⁇ -V ⁇ and ⁇ _--- ⁇ __- ⁇ . SCAs
  • the MA-15C5 antibody contains a kappa light chain and a gamma heavy chain.
  • the Brookhaven Database was searched for structures of immunoglobulines with similar heavy and light chains.
  • the protein with code pdb2hfl which is a Fab-lysozyme complex (Sheriff et al., 1987), fulfilled these requirements.
  • a model of the MA-15C5 V L and V H was obtained by substitution of all residues of the pdb2hfl structure that differed from the MA-15C5 V L and V H sequences with their corresponding residues in the MA-15C V L and V H .
  • the anchor region of V H was defined as the segment comprising residues 3 to 7 (i.e., QLKQS) which forms the end of a 9-sheet.
  • a suitable anchor region at the C-terminus of the MA-15C5 V L was found to be the segment comprising residues 102 to 106 (i.e., TKLEI) which also forms the end of a /3-sheet.
  • the gap between the attachment points of the two linkers is 30.8 Angstrom.
  • a first linker peptide (L ab ) of at least 8 amino acids should be sufficient to bridge the gap. Note that there are still two residues (i.e., KR) flanking the C-terminus of the V L anchor region, and two residues (i.e., QV) that flank the N- terminus of the V H anchor region.
  • the fitting of the fragment terminal regions with the V L and V H anchor regions was assessed by a least square fit of the atomic coordinates of: 1) the alpha carbon atoms and 2) all main chain atoms (MacLachlan, 1979) . This analysis resulted in a root mean square deviation (rms) which should be minimal.
  • the best fragment was found to be the segment comprising residues 22-42 from proteinase K (pdb2prk - Betzel et al., 1988).
  • the following alignment could be made (the anchor regions or the L ⁇ sequence between them are underlined) : ...TKLEIKR QVQLKQS... (V L gap V H )
  • the first linker peptide was observed to be located at the side opposite to the antigen-binding site and should not interfere with binding. It was also seen that mutation of the Ilel06 and ArglOS residues into Gly or Ser residues also resulted in suitable SCAs ( Figure 6, constructions 2, 3, 2A and 3A) . Flexibility of the first linker peptide could be increased by replacement of the Q residue in the linker with R followed by O to 4 glycine residues (Fig. 6, constructions 4 and 4A) or by replacing the AGQ block of residues in the linker by one or more GGGS blocks of residues.
  • the first linker peptide should satisfy the stringent requirement of not interfering with the antigen binding sites.
  • the N-terminus is located at the start of a 3-strand situated avthe edge of one of two antiparallel /9-sheets packed on top of each other. Interference with the antigen-binding site can be prevented by designing an extension of the V L chain so that the 0-sheet at the N-terminus is entered by a ⁇ - turn preceded by a 3-strand.
  • An additional advantage of such an extension is that the gap between the C- terminus of the V H and the N-terminus of the (extended) V L is smaller so that a shorter first linker peptide is required.
  • the resulting configuration can be schematically represented as follows (the anchor region and extension are underlined) : SVTVSS DIKM... (V H gap V L )
  • SGSGSGTSY (pdb2hfl fragment) Residues 68 to 70 of pdb2hfl are preceded by a ⁇ - strand segment (residues 62 to 67) .
  • a ⁇ - strand segment (residues 62 to 67) .
  • the actual first linker peptide was then designed between the C-terminus of the V H domain (using the residues 108-111, i.e., SVTV, as an anchor region) and the N-terminus of the extended V L domain using the first four residues of the V L extension (i.e., SGSG) as an anchor.
  • a search of the 3D structures of proteins for suitable fragments resulted in the identification of a fragment from pdb2sod (superoxide dismutase ("SOD") — Tainer et al., 1982) with 11 residues (SOD residues 038-048 - EGDHGFHVHQF) between the anchor regions.
  • SOD superoxide dismutase
  • the configuration of the fit can be represented as follows (the anchor regions and first linker peptide are underlined) : SVTVSS SGSGSGDIKM— (V H gap extension-V L )
  • the total first linker peptide (EGDHGFHVHQFSGSGSG) between the original V H and V L domains is thus composed of this 11 residue pdb2sod fragment plus the six residue pdb2hfl extension sequence which was introduced at the N-terminus of the V L domain.
  • the structure was then subjected to 100 steps of a "Steepest Descent" (Fletcher and Reeves, 1964) energy minimalization procedure fixing all atoms except those of the first linker peptide.
  • the first two amino acids of the pdb2sod linker fragment (EG) were then mutated to serine residues to revert to the original V H C- terminus. Furthermore, the linker's hydrophobicity was reduced by mutating:
  • Example 2 Construction of sea genes and baculovirus expression vectors containing these genes
  • the Pstl-Hindlll fragment of Fig. 4 contains most of the V H domain and part of the N-terminal part of the C H domain of the MA-15C5 gamma chain. Only the first four amino acids of the V H domain are not present (Gln-Val-Gin-Leu) . This fragment was cloned into the PstI and Hindlll sites of pUC19 (Yanisch-Perron et al., 1985) .
  • Plasmid pMc5-8-uts can be obtained by cloning a universal translation stop sequence ("uts") with the following sequence :
  • pMc5-gamma6-S can be used directly for site directed mutagenesis.
  • a stop codon and a EcoRI site was introduced immediately after Serll3 by introduction of the sequence TGAATTC, yielding pMc5-G60-S.
  • the EcoRI sites, and the sequences between them were then deleted by digestion of pMc5-G60-S with EcoRI (filled in with Klenow) and religation.
  • the resulting plasmid was designated as pMc5-G60 ⁇ E-S.
  • the kappa chain was obtained on plasmid pCMBDHFR13-15C5KMu (Vandamme et al., 1990).
  • the EcoRI- Bglll fragment shown in Fig. 3, was cloned in the EcoRI and BamHI sites of pMc5-8, yielding plasmid pMc5-Kb.
  • This fragment comprises the signal peptide, the V L domain and the C L domain of the Ma-15C5 kappa chain.
  • pVL1393 (now available from British Biotechnology Ltd., Oxford, UK) can be obtained from pVL941, described by Luckow and Summers (1989) , by deletion of a 630 bp EcoRI-Xmalll fragment and by extension of the polylinker by insertion of the following sequence in the BamHI site of the pVL941 polylinker : GATCCCGGGTACCTTCTAGAATTCCGGAGCGGCCGCTGCAGATCT
  • GGCCCATGGAAGATCTTAAGGCCTCGCCGGCGACGTCTAGACTAG (Summers, personal communication) .
  • the construction of the sea gene coding for the SCA was done as follows. pMc5-G60 ⁇ E-S was digested with AccI (filled in with Klenow) and Xbal and the fragment, containing the V H encoding seguence, was cloned in the Styl (filled in with Klenow) and Xbal sites of pMc5-kb, yielding plasmid pMc5-KG60 ⁇ E-S. In one mutagenesis experiment, appropriate transition seguences between the V L and V H coding regions were then provided.
  • the sea genes in pMc5-K12A, pMc5-K14A, pMc5-K15A5 were then introduced in pVL1393-K by replacement of the BamHI-Xbal fragment (coding for the C-terminal end of the MA-15C5 kappa chain) of pVL1393-K with the BamHI- Xbal fragments of pMc5-K12A, pMc5-K14A, pMc5-K15A5, yielding plasmids pVL-K12A, pVL-K14 , pVL-K15A5. These plasmids can be used directly for transfection of insect cells.
  • Example 3 Construction of scapa genes and baculovirus expression vectors containing these genes
  • the 1475 bp Hindlll fragment of the scupa cDNA (Fig. 5) was cloned in the Hindlll site of pUC18, yielding plasmid pULscu-PA (Nelles et al., 1987).
  • the Ncol (filled in with Klenow)-Hindlll fragment of this plasmid was further subcloned in the BamHI (Klenow) and Hindlll sites of pMC5-8, yielding plasmid pMc5-scupa- Nco.
  • the C at position 1356 (Fig. 5) was mutated to a T (resulting in a destruction of a BamHI site)
  • the G at position 966 (Fig. 5) was mutated to an A (resulting in a destruction of a Fspl site)
  • the AAA codon at position 562 (Fig. 5) , coding for Lys- 135 in scuPA, was mutated to a CAA codon (Gin)
  • the TTT codon at positions 628-630 (Fig. 5) , coding for Phel57 of scuPA, was mutated to a GAT codon (Asp) .
  • the resulting plasmid was designated as pMc5-scupa-77-I.
  • the A at position 648 (Fig. 5) was mutated to a G (resulting in a destruction of an EcoRI site)
  • the G at position 1092 (Fig. 5) was mutated to an A (resulting in the destruction of a PvuII site)
  • the AGGs at positions 691-696 (Fig. 5) were each mutated to a CGT
  • the G at position 702 (Fig. 5) was mutated to a C (resulting in the creation of a SacII site)
  • the C at position 624 (Fig. 5) was mutated to a T (resulting in the creation of an StuI site) .
  • the resulting plasmid was designated as mPc5- scupa-77-II.
  • the Fspl-Xbal fragments of the pMc5-scupa-77-I and -II plasmids (each comprising the coding sequence of the scuPA catalytic domain) were cloned into the Hindlll (filled in with Klenow) and Xbal sites of pMc5-K12A, pMc5-K14A, pMc5-K15A5 (from Example 2) , after which the sequences between the C-terminal Serll3 (Fig. 2) codon of the Ma-15C5 V H and the N-terminal Alal32 codon of the scuPA were deleted.
  • the resulting plasmids were designated as pMc5-K12A-PA-I , pMc5-K14A- PA-I, pMc5-K15A5-PA-I , pMc5-K12A-PA-II, pMc5-K14A-PA-II and pMc5-K15A5-PA-II, respectively.
  • the scapa genes in the pMc5-K12A-PA-I and -II, pMc5-K14A-PA-I and -II, and pMc5-K15A5-PA-I and -II plasmids were then introduced in the transfection vector pVL1393-K (from Example 2) by replacement of a BamHI-Xbal fragment (coding for the C-terminal part of the MA-15C5 kappa chain) of pVL1393-K with the BamHI- Xbal fragments of pMc5-K12A-PA-I and -II, pMc5-K14A- PA-I and -II, and pMc5-K15A5-PA-I and -II, yielding plasmids pVL-K12A-PA-I, pVL-K14A-PA-I , pVL-K15A5-PA-I ,
  • Example 4 Expression of sea and scapa genes in insect cells
  • the sea and scapa genes of Examples 2 and 3 (in plasmids pVL-K12A, pVL-K14A, pVL-K15A5, pVL-K12A-PA-I and -II, pVL-K14A-PA-I and -II and pVL-K15A5-PA-I and II) are introduced and expressed in Spodoptera frugiperda (SF9) cells (ATCC no. CRL 1711) using the procedures and Autographa californica nuclear polyhedrosis viruses (AcNPV) described by Summers and Smith (1987).
  • SF9 Spodoptera frugiperda
  • Example 5 Purification of SCAs and SCAPAs from insect cell cultures of Example 4
  • the secreted SCAs expressed in Example 4 are purified by means of affinity chromatography on immobilized fibrin fragment D-dimer.
  • urokinase For purification of secreted SCAPAs expressed in Example 4, this step is followed by immunoadsorption on an insolubilized monoclonal antibody against urokinase, MA-4D1E8, as described by Nelles et al. (1987).
  • the fractions containing urokinase-related antigen are pooled and dialyzed against 0.3 M NaCl, 0.2 M arginine, 0.02 M Tris.HCl buffer pH 7.4, containing 0.01% Tween 80 and 10 KlU/ml aprotinin.
  • tcuPA is removed from samples equilibrated with dialysis buffer containing 0.2 M arginine by chromatography on benzamidine-sepharose. Fractions devoid of amidolytic activity are pooled. Aprotinin is removed by extensive washing on a Centrocon 30 microconcentrator (from Amicon, Danvers, MA, USA) .
  • Example 6 Purification of SCAPAs from insect cultures of Example 4
  • Example 4 The secreted SCAPAs expressed in Example 4 are also purified in a different way from that of Example 5. Each SCAPA is purified by ion exchange chromatography on SP-Sephadex (from LKB, Bromma, Sweden) , followed by gel filtration on Sephadex-GlOO superfine (from LKB) .
  • 1.5 1 of conditioned medium, with a pH adjusted to 5.5, is applied at 4'C and a flow rate of 20 ml/h on a 0.9 x 2 cm SP-Sephadex column equilibrated with 0.05 M NaH 2 P04, pH 5.5, containing 0.05 M NaCl, 0.01% Tween 80 and 10 KlU/ml aprotinin. Elution is performed with a 60 ml gradient from 0.05 M to 0.60 M NaCl in 0.05M NaH 2 P0 4 , pH 5.5. The fractions containing each SCAPA, as determined with an ELISA specific for uPA-related antigen, are pooled, and the pH is increased to 7.4 with 1 M NaOH.
  • the pooled fractions (representing 7 ml with a concentration of 0.28 mg of SCAPA per ml) are concentrated on a Centricon 30 microconcentrator (Amicon) to a final volume of 0.5 ml.
  • the concentrated sample is then applied at 4*C and at a flow rate of 4 ml/hr on a 1.0 x 110 cm Sephadex-GlOO superfine column equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, containing 0.3 M NaCl, 0.01% Tween 80 and 10 KlU/ml aprotinin.
  • the fractions containing the SCAPA are pooled.
  • Satisfactory antigen-binding activity of the SCAs and SCAPAs of Examples 5 and 6 is found in ELISA using immobilized fibrin fragment D-dimer and rabbit-anti- mouse antibodies specific for MA-15C5 and goat antibodies specific for total rabbit IgG fraction conjugated to alkaline phosphatase (Voller et al., 1976) . Satisfactory urokinase-related antigen activity is also found in ELISA according to Darras et al. (1986) .
  • the SCAs and SCAPAs are also characterized by SDS-PAGE under reducing and non-reducing conditions, and the amino termini of the proteins are determined to verify correct processing. Satisfactory equilibrium association constants of the SCAs for immobilized and dissolved purified fragment D-dimer are determined according to Hogg et al. (1987) . SCA is labeled by 125 I to show that there is satisfactory in vitro plasma clot binding capacity (Lijnen et al., 1986) and to determine in vivo half-life.
  • Example 8 Synthesis and Expression of a PVL-K12A' as in Examples 1 and 2
  • a suitable anchor region at the carboxyterminus of the V L domain of MA-15C5 was found to be the segment comprising residues Thr 10 -Lys 103 -Leu 104 -Glu 105 -Ile 106 , a segment that is at the end of a /3-sheet.
  • a suitable anchor region at the aminoterminus of the V H domain of MA-15C5 was found to be the segment comprising residues Gln 3 -Leu 4 -Lys 5 -Gln 6 -Ser 7 , a segment that also is at the end of a /9-sheet.
  • the attachment sites at the ends of the anchor regions define a gap in which the linker has to be fitted.
  • the 30.8 A spatial distance between these attachment sites determines a minimum number of amino acids that are required to bridge the gap. This minimum number was found to be 8.
  • the Brookhaven Protein Database was then searched for all peptide sequences consisting of 16 to 22 amino acids, so that the length of the peptide linker could be varied from 8 to 14 amino acids. To overlap the V L carboxyterminal anchor region, 5 more amino acids were required. To overlap the V H aminoterminal anchor region, 3 more amino acids were required. Thus, the number of amino acids had to vary between 16 and 22. This search yielded more than 10,000 peptide candidates. Secondary structure predictions were then performed according to Jibrat et al. (1987) to select those peptide segments that did not interfere with the ordered secondary structure or with the folding of the V L or the V H domain of MA-15C5. In this way, the number of linker peptide candidates was reduced to 82.
  • the Thr-Tyr-Tyr-Tyr- Asp fragment overlapped the Thr 102 -Lys 103 -Leu 104 -Glu 105 - Ile 106 V L carboxyterminal anchor region
  • the Ile- Asp-Thr-Gly fragment overlapped the Gln 3 -Leu-Lys 5 - GLn 6 -Ser 7 V H aminoterminal anchor region.
  • the fragments overlapping the anchor regions were mutated to the original anchor region amino acids.
  • the structure was then subjected to 100 steps of a steepest descent energy minimalization procedure (Fletcher and Reeves, 1964) , fixing all atoms except those of the linker peptide.
  • the cDNA encoding the synthetic peptide linker L12 was then inserted between the cDNA encoding the V L domain and the cDNA encoding the V H domain of MA-15C5, resulting in the construction of the synthetic cDNA pMC5-K12A* as described below.
  • the 419 bp Smal-Hindlll fragment from pUC19-G 6 (Vandamme et al., 1990) was ligated in the EcoRI- Hindlll treated pMa/c vector, in which the EcoRI recessing end was filled in with Klenow enzyme, yielding pMA/c-G6.
  • a "TGAATTC” seguence was inserted in pMa/c-G 6 by site-directed mutagenesis between nucleotides 350 and 351 (on the pUC19-G 6 fragment sequence) , introducing a TGA STOP codon at the presumed end of the J region of V H and an additional EcoRI site.
  • pMa/c-G ⁇ The resulting plasmid, pMa/c-G ⁇ , was digested with EcoRI, treated with Klenow enzyme and religated, yielding pMa/c-G 0 , in which the EcoRI restriction sites, together with the intervening sequences, are removed.
  • the 406 bp Accl-Xbal restriction fragment from pMA/c-G 0 of which the AccI recessing end was made blunt with Klenow enzyme, was transferred to Styl (filled in)-Xbal treated pMa/c-Kb to yield pMa/c-KG 0 .
  • the 226 bp fragment comprising the carboxyterminal part of the kappa constant region (C L ) and the kappa 3' untranslated sequence was deleted.
  • a single si.te-directed mutagenesi.s with the 72-mer oligodeoxynucleotide dCAAAGTTGGAAATCAAGCGTGCTGGTCAAGG- CTCTTCTGTTCAAGTTCAGCTGAAGCAGTCAGGACCTGGCC was performed on pMa/c-KG 0 to: i) delete the 328 bp DNA sequence separating the Arg 108 of the kappa chain from the codon for Lys 5 of the gamma chain; ii) reintroduce cDNA sequence coding for amino acids 1 to 4 missing at the NH 2 -terminus of V H ; and iii) insert the peptide linker L12 between the carboxyterminal end of V L (Arg 108 ) and the aminoterminal end of V H (Gin 1
  • Sf9 cells were grown at 27*C in Grace's insect cell culture medium supplemented with 10% (vol/vol) fetal calf serum, 3.3% (vol/vol) yeastolate, and 3.3% (vol/vol) lactalbumin hydrolysate (TMNF medium) essentially as described by Summers and Smith (1987) .
  • the Sf9 cells (2 x 10 6 cells in a 25 cm 2 flask) were transfected with 1 ⁇ g AcNPV DNA and 10 ⁇ g pVL-K12A' by the Ca-phosphate co-precipitation method (Gorman et al., 1985), and the resulting culture supernatant was harvested 5-7 days later for cloning of recombinant baculovirus and for measurement of human fibrin fragment D-dimer binding protein in solid-phase enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • the recombinant plagues resuspended in 1 ml of TMNF medium and 50 ⁇ l aliquots were used to infect fresh monolayers of Sf9 cells (2 x 10 6 cells in a 25 cm 2 culture flask) overlaid with 4 ml TMNF medium.
  • the resulting culture supernatants were harvested 48 h later for assessment of human fibrin fragment D-dimer binding in ELISA.
  • the recombinant virus, AcNpVLK 1 G 0 was then purified by 4 rounds of plague purification. For each round, the concentration of fibrin fragment D-dimer binding protein was assessed in ELISA.
  • scFV-K 12 G 0 For the large scale production of the SCA encoded by pMC5-K12A' and pVL-K12A', called “scFV-K 12 G 0 ", 40 x 10 6 Sf9 cells in 175 cm 2 culture flasks were infected with 200 x 10 6 plaque forming units of recombinant virus AcNpVLK 12 G 0 . After incubation for 48 h at 27 ⁇ C, the conditioned medium, containing up to 15 ⁇ g scFv- K 12 G 0 per ml, but. on average approximately 4.5 ⁇ g/ml, was removed and centrifuged at l,000xg for removal of cell debris.
  • scFv-K 12 G 0 was purified by ion exchange chromatography on SP-Sephadex followed by gel filtration on Sephadex-GlOO superfine.
  • 1.5 1 of conditioned medium with a pH adjusted to 4.5 was applied on a 0.9 x 2 cm SP-Sephadex column equilibrated with 0.05 M NaH 2 P0 4 , pH 4.5, containing 0.05 M NaCl, 0.01% Tween 80 and 10 KlU/ml aprotinin. Elution was performed with a 60 ml gradient from 0.05 M to 1.0 M NaCl in 0.05 M NaH 2 P0 4 , pH 5.5.
  • the fractions containing scFv-K 12 G 0 were pooled and the pH was increased to 7.4 with 1M NaOH.
  • the pooled fractions (representing 7 ml with a concentration of 0.7 mg scFv-K 12 G 0 per ml) were concentrated on a Centricon 10 microconcentrator (Amicon) to a final volume of 0.05 ml.
  • the concentrated sample was applied on a 1.0 x 110 cm Sephadex-GlOO superfine column equilibrated with 0.02 M Tris-HCl buffer, pH 7.4, containing 0.3 M NaCl, 0.01% Tween 80 and 10 KlU/ml aprotinin.
  • the fractions containing scFv-K 12 G 0 were pooled and found to migrate to a single 25,500 Mr band on reduced SDS gel electrophoresis.
  • NH 2 -terminal amino acid analysis of the scFv-K 2 G 0 revealed that the MA-15C5 kappa signal peptide was cleaved off by the insect cells just in front of mature kappa Asp 1 residue.
  • the scFv-K 12 G 0 was also found to bind to immobilized D-dimer with an affinity constant of 4 x 10° M "1 , as compared to 2.0 x 10 10 m "1 for intact MA-15C5. This finding indicates that, in scFv-K 12 G 0 , the MA-15C5 V L and V H domains can reassociate efficiently, resulting in the reconstitution of an intact, functionally active, antigen binding site.
  • scFv-K 12 G 0 When injected as a bolus (2.8 ⁇ g/kg) , scFv-K 12 G 0 was cleared from the plasma of rabbits with a half-life of 10 minutes and a clearance rate of 5.1 ml/min "1 , as compared to 90 minutes and 210 ml/min "1 for intact MA-15C5. These results indicate that scFv-K 12 G 0 can be useful for targeting radioisotopes or plasminogen activators to blood clots in vivo.
  • Example 9 Synthesis and Expression of a PVL-K12A-PA- II' as in Example 3
  • a transfer vector pVL-K12A-PA-II' encoding the SCAPA called "K 12 G 0 S 32 ", for expression in Sf9 insect cells was constructed starting from the plasmids pMA/c-K 12 G 0 and pVL-K12A' of Example 8, pULscu-PA (Nelles et al., 1987) and the pMA/c mutagenesis vector.
  • Arg 156 -Phe 157 thrombin cleavage site in urokinase was removed by mutating Phe 157 to Asp (nucleotides 628-630) with the 31-mer oligonucleotide dCCCAATAATCTTATCGCGAGGCCTCAGAG- TC.
  • a StuI restriction site was simultaneously created by changing the CCC Pro 155 codon to CCT.
  • the 33-mer oligonucleotide dGACAGAGCCCCCGCGGTGACGACGGTAGATGGC was used to modify 3 Arg codons: Argl78 and Arg 179 AGG rare codons (nucleotides 691-696) were replaced by CGT codons, while for screening purposes, the Argl ⁇ l CGG codon was changed to CGC, generating a SacII restriction site (nucleotides 699-704) .
  • the BamHI restriction site in urokinase (nucleotides 1352-1357) was deleted by changing the ATC lie 399 codon to ATT with the 18-mer oligonucleotide dGTGACTGCGAATCCAGGG.
  • This mutation was performed to facilitate further manipulation of the chimeric cDNA, using the BamHI restriction site present in the variable kappa light-chain coding seguence.
  • One of the two Fspl restriction sites (nucleotides 963-968) was removed by changing the GCG Ala 369 codon to GCA with the 20-mer oligonucleotide dCGGGATGGCTGTGCACACCT.
  • the Fspl enzyme cleaves the remaining site precisely in front of amino acid Ala 132 , which was used as the NH 2 - terminal amino acid of the truncated scuPA.
  • one of the two EcoRI restriction sites was deleted by changing the GAA Glu 163 codon to GAG with the 30-mer oligonucleotide dGATGGTGGTGAACTCTCCCCCAATAATCTT, and the PvuII restriction site (nucleotides 1090-1095) was removed by changing the CAG Gin 311 codon to CAA with the 19-mer oligonucleotide dGTCATTTTCAGTTGCTCCG.
  • the 613 bp BamHI-Hindlll fragment from pMa/c-K 12 G 0 which encodes the carboxyterminal sequence of scFv-K 12 G 0 and of which the Hindlll end was filled in with Klenow enzyme, was ligated in BamHI-FspI treated pMa/c-scu- PA' .
  • the resulting plasmid pMa/c-12VS contained the seguence encoding the carboxyterminal region of scFv- K 12 G 0 in front, but out of frame, of the aminoterminal sequence of the truncated catalytic domain of scuPA.
  • Deletion oligonucleotide-directed mutagenesis was performed on pMa/c-12VS to delete the 22 nucleotides that still separated the carboxyterminal amino acid (Ser 232 ) of scFv-K 12 G 0 and the first amino acid (Ala 132 ) of the truncated catalytic domain of scuPA, yielding pMa/c-12G 0 S 3 .
  • the 51-mer oligonucleotide dAGAGGAGGGCTTTTGTCCATCTGCTGAGGAGACGGTGACTGAGGTTCCTTG used was complementary to the 9 carboxyterminal amino acids of the scFv-K 12 G 0 molecule and to the 8 aminoterminal amino acids of the low molecular weight form (truncated catalytic domain) of scuPA.
  • Sf9 cells were grown at 27*C in Grace's insect cell culture medium supplemented with 10% (vol/vol) fetal calf serum, 3.3% (vol/vol) yeastolate, and 3.3% (vol/vol) lactalbumin hydrolysate (TMNF medium) , essentially as described by Summers and Smith (1987) .
  • the Sf9 cells (2 x 10 6 cells in a 25 cm 2 flask) were transfected with 1 ⁇ g AcNPV DNA and 10 ⁇ g pVL-K12A-PA- II 1 by the Ca-phosphate co-precipitation method (Gorman et al, 1985) , and the resulting culture supernatant was harvested 5-7 days later for cloning of recombinant baculovirus and for assessment of human fibrin fragment D-dimer binding protein in solid-phase enzyme-linked immunoassay (ELISA) .
  • ELISA enzyme-linked immunoassay
  • the recombinant plaques were resuspended in 1 ml of TMNF medium, and 50 ul aliquots were used to infect fresh monolayers of Sf9 cells (2 x 10° cells in a 25 cm 2 culture flask) overlaid with 4 ml TMNF medium.
  • the resulting culture supernatants were harvested 48 h later for assessment of human fibrin fragment D-dimer binding protein in ELISA.
  • the recombinant virus (AcNpVLK 12 G 0 S 32 ) was then purified by 4 rounds of plaque purification. For each round, the expression of fragment D-dimer binding protein and of uPA-related antigen was assessed in ELISA. The purity of the isolated recombinant virus was confirmed in filter-hybridization experiments (Kafatos et al. , 1979) .
  • K 12 G 0 S 32 was purified as described in Example 6 by ion exchange chromatography on SP-Sephadex followed by gel filtration on Sephadex-GlOO superfine.
  • the specific activity of the resulting K 12 G 0 S 32 towards a chromogenic substrate for urokinase was ⁇ 1,000 IU/mg before and 100,000 IU/mg uPA equivalent after conversion to its two-chain derivative with plasmin.
  • the specific activity of both the single-chain and two-chain form on fibrin plates was 100,000 IU/mg uPA equivalent.
  • low M r scuPA of this invention can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form, as compared to that of their uPA-32k counterparts.
  • this invention is not limited to the transformation of a specific host microorganism or the use, for this purpose, of a chimaeric gene containing any specific promoter, signal sequence, sea or scapa gene and/or 3' transcription regulation sequence of this invention, or the use of any specific SCA or SCAPA, expressed by such a transformed host, for the specific purposes mentioned above.
  • equivalents of the foregoing Examples will be readily apparent to those skilled in the art in view of the disclosure herein of the invention.
  • the DNA sequences of the described sea and scapa genes can be easily modified by: 1) replacing some codons with others that code either for the same amino acids or for other amino acids; and/or 2) deleting or adding some codons; provided that such modifications do not substantially alter the biological properties of the encoded SCAs or SCAPAs.
  • this invention is not limited to an SCA or an SCAPA derived from a monoclonal antibody directed to fibrin or fibrin D-links, such as MA-15C5 antibody.
  • This invention encompasses SCAs and SCAPAS derived from monoclonal antibodies directed to other thrombus constituents such as: a) antibodies to blood platelets, for example antibodies to resting and activated platelet surface receptors, e.g., antibodies to platelet membrane glycoprotein Ilb/IIIa (Bode et al., 1990) or antibodies (e.g. , MA-libs-1) specific for ligand-occupied receptor conformers (Frelinger et al., 1990) ; or b) antibodies to alpha 2-antiplasmin.
  • This invention also encompasses SCAs and SCAPAs derived from other monoclonal antibodies directed to fibrin such as the 59D8 antibodies (Bode et al., 1987).

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Abstract

Cette invention se rapporte à des anticorps à chaîne unique spécifiques à la fibrine, des agents thrombolytiques dérivés de tels anticorps et des fragments d'ADN codant pour de tels polypeptides. Les anticorps à chaîne unique peuvent être utilisés pour l'imagerie, alors que les agents thrombolytiques peuvent être utilisés pour la lyse in vivo de thrombi.
PCT/EP1991/000767 1990-04-23 1991-04-21 Derives d'anticorps specifiques au thrombus WO1991016353A1 (fr)

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EP0713496A1 (fr) * 1993-05-03 1996-05-29 President And Fellows Of Harvard College Inhibiteurs de la thrombine cibles sur la fibrine
WO1997015601A1 (fr) * 1995-10-20 1997-05-01 Novartis Ag Procede de preparation d'un fragment d'anticorps fv a simple chaine
WO1997024137A1 (fr) * 1995-12-28 1997-07-10 Tanox Biosystems, Inc. PROTEINE HYBRIDE A INTERFERON-α ET FRAGMENT Fc D'IMMUNOGLOBULINE A LIAISON ASSUREE PAR PEPTIDE NON IMMUNOGENE
AU690528B2 (en) * 1992-12-04 1998-04-30 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
US6303369B1 (en) * 1993-09-30 2001-10-16 Carl Spana Protein expression system
EP1151002A1 (fr) * 1999-01-29 2001-11-07 Imclone Systems, Inc. Anticorps specifiques au kdr et leurs utilisations
EP1203026A1 (fr) * 1999-07-29 2002-05-08 Dyax Corporation Fractions se liant la fibrine
US6624295B1 (en) 1998-08-28 2003-09-23 Genentech, Inc. Human anti-factor IX/IXa antibodies
US6984373B2 (en) 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
WO2006054059A1 (fr) * 2004-11-19 2006-05-26 Ucb Pharma S.A. Neutralisation de molecules d'anticorps presentant une specificite pour l'il-17 humaine
JP2009502139A (ja) * 2005-07-22 2009-01-29 ワイズセラピューティックス株式会社 抗ｃｄ２６抗体およびその使用方法
US8143025B2 (en) 2004-11-18 2012-03-27 Imclone Llc Antibodies against vascular endothelial growth factor receptor-1
US8580265B2 (en) 2011-01-14 2013-11-12 Ucb Pharma S.A. Antibody molecules which bind IL-17A and IL-17F
WO2015116753A1 (fr) * 2014-01-29 2015-08-06 Dana-Farber Cancer Institute, Inc. Anticorps contre le domaine extracellulaire de muc1-c (muc1-c/ecd)
US10617773B2 (en) 2016-08-05 2020-04-14 Dana-Farber Cancer Institute, Inc. Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD)

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WO1994009034A1 (fr) * 1992-10-12 1994-04-28 Agen Limited Anticoagulant ciblant les caillots, ses procedes de fabrication et d'utilisation

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AU690528B2 (en) * 1992-12-04 1998-04-30 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
EP0713496A1 (fr) * 1993-05-03 1996-05-29 President And Fellows Of Harvard College Inhibiteurs de la thrombine cibles sur la fibrine
EP0713496A4 (fr) * 1993-05-03 1999-03-24 Harvard College Inhibiteurs de la thrombine cibles sur la fibrine
US6303369B1 (en) * 1993-09-30 2001-10-16 Carl Spana Protein expression system
WO1997015601A1 (fr) * 1995-10-20 1997-05-01 Novartis Ag Procede de preparation d'un fragment d'anticorps fv a simple chaine
WO1997024137A1 (fr) * 1995-12-28 1997-07-10 Tanox Biosystems, Inc. PROTEINE HYBRIDE A INTERFERON-α ET FRAGMENT Fc D'IMMUNOGLOBULINE A LIAISON ASSUREE PAR PEPTIDE NON IMMUNOGENE
AU701579B2 (en) * 1995-12-28 1999-02-04 Tanox Biosystems, Inc. Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide
US5908626A (en) * 1995-12-28 1999-06-01 Tanox, Inc. Hybrid with interferon-β and an immunoglobulin Fc joined by a peptide linker
US7354585B2 (en) 1998-08-28 2008-04-08 Genentech, Inc. Methods of treating coagulapathic or thrombotic disorders
US6624295B1 (en) 1998-08-28 2003-09-23 Genentech, Inc. Human anti-factor IX/IXa antibodies
EP1151002A1 (fr) * 1999-01-29 2001-11-07 Imclone Systems, Inc. Anticorps specifiques au kdr et leurs utilisations
EP1151002A4 (fr) * 1999-01-29 2002-05-02 Imclone Systems Inc Anticorps specifiques au kdr et leurs utilisations
EP1203026A4 (fr) * 1999-07-29 2005-03-16 Dyax Corp Fractions se liant la fibrine
EP1203026A1 (fr) * 1999-07-29 2002-05-08 Dyax Corporation Fractions se liant la fibrine
US6984373B2 (en) 2000-12-23 2006-01-10 Dyax Corp. Fibrin binding moieties useful as imaging agents
US8143025B2 (en) 2004-11-18 2012-03-27 Imclone Llc Antibodies against vascular endothelial growth factor receptor-1
WO2006054059A1 (fr) * 2004-11-19 2006-05-26 Ucb Pharma S.A. Neutralisation de molecules d'anticorps presentant une specificite pour l'il-17 humaine
JP2009502139A (ja) * 2005-07-22 2009-01-29 ワイズセラピューティックス株式会社 抗ｃｄ２６抗体およびその使用方法
US9034600B2 (en) 2011-01-14 2015-05-19 Ucb Biopharma Sprl DNA encoding antibody molecules which bind IL-17A and IL-17F
US8580265B2 (en) 2011-01-14 2013-11-12 Ucb Pharma S.A. Antibody molecules which bind IL-17A and IL-17F
US9988446B2 (en) 2011-01-14 2018-06-05 Ucb Biopharma Sprl Methods of treatment using antibodies which bind IL-17A and IL-17F
US11919950B2 (en) 2011-01-14 2024-03-05 UCB Biopharma SRL Expression vector encoding antibody molecule which binds IL-17A and IL-17F
WO2015116753A1 (fr) * 2014-01-29 2015-08-06 Dana-Farber Cancer Institute, Inc. Anticorps contre le domaine extracellulaire de muc1-c (muc1-c/ecd)
US10059775B2 (en) 2014-01-29 2018-08-28 Dana-Farber Cancer Institute, Inc. Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD)
US11136410B2 (en) 2014-01-29 2021-10-05 Dana-Farber Cancer Institute, Inc. Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD)
US10617773B2 (en) 2016-08-05 2020-04-14 Dana-Farber Cancer Institute, Inc. Antibodies against the MUC1-C/extracellular domain (MUC1-C/ECD)

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