Conjugates containing antibody, specific for ed-b-domain of fibronectin, and their application for detection and treatment of tumours

FIELD: medicine.

SUBSTANCE: versions of the bond intended for linkage with the external domain B (ED-B) of a fibronectin are offered. The bond includes an antigen-binding fragment of one-chained antibody L19 and a cysteinum-containing linker for hanging of a radioactive label. Versions of a pharmaceutical composition for diagnostics and treatment of angiogenic diseases on the basis of the specified bond are opened. Application of bond for linkage with radioactive bond is described. The method of reception of bond in eucariotic cells is opened, including in Pichia pastoris and a set for reception is radioactive labelled agents on the basis of bond.

EFFECT: high-avid bond accumulation in solid tumours.

23 cl, 4 dwg, 5 tbl, 15 ex

 

The technical field to which the invention relates.

The present invention relates to new methods of diagnosis and treatment of tumors using novel peptides for radionuclide.

A brief discussion of the background of invention

Tumors may not exceed a certain weight without the formation of new blood vessels (angiogenesis), and for many tumors revealed a correlation between the density of microvessels and invasiveness of tumors (Folkman, Nature Med., 1, 1995, p.27-31). In addition, angiogenesis associated with the majority of eye diseases that lead to vision impairment (Lee and others, Surv. Ophthalmol. 43, 1998, s-269; M. Friedlander and others, Proc. Natl. Acad. Sci. U.S.A. 93, 1996, s-9769). Molecules possessing the ability to perform selective directional migration markers of angiogenesis, can be valuable from a clinical perspective for diagnosis and therapy of tumors and other diseases, which are characterized by vascular proliferation, such as diabetic retinopathy and age related macular degeneration. In most of the growing solid tumors is the expression of markers of angiogenesis along with the increase in tumor vessels, and so they are readily available for specific binding substances that are injected (Pasqualini and other, Nature Biotechnol, 15, 1997, s-546; Neri and other Nature Biotechnol, 15, 1997, s-1275). Directed the termination of neovascularization can lead to a heart attack and collapse of the tumor (O'reilly and others, Nature Med., 2, 1997, s-692; Huang and others, Science, 275, 1997, s-550).

ED-B domain of fibronectin, consisting of 91 amino acids, the sequence of which is identical in mice, rats and humans, when embedding by alternative splicing in the molecule fibronectin-specific accumulated around associated with neovascularization structures (Castellani and others, Int. J.Cancer 59, 1994, s-618) and may represent a target for intervention at the molecular level. So, currently using fluorescence established that single-chain Fv fragments of antibodies to the ED-B (scFv) selectively accumulate around blood vessels of the tumor-bearing tumor mice and that the affinity for the antibody, apparently, determines the characteristics of the directional migration (Neri and others, Nature Biotechnol, 15, 1997, s-1275; WO 97/45544).

In addition, antibodies and antibody fragments that specifically associated with the ED-B domain of fibronectin, where the binding is characterized by the value of the dissociation constants, in subnanomolar range, and also labeled with radioactive atoms, their derivatives described in WO 99/58570. Already studied the biodistribution in the body of the bearing tumor mice of one of these high-affinity fragments of human antibodies, and it is marked using the125I fragment of the antibody, labeled L19 (Tarli, etc., Blood, I. 94, No.1, 1999, p.á192-198). Radioactively the sword is s conjugates, containing L19-antibodies, and their use for detection and treatment of angiogenesis described in WO 01/62800.

The previously described recombinant obtain functionally active single-chain Fv fragments of antibodies to the ED-B domain In-a isoform of fibronectin in Pichia pastoris (Marty and others, Protein Expression and Purification 21, 2001, c.156-164).

It also describes the incorporation of radioactive label99mTc in scFv-fragment antibodies by C-terminal containernode peptide (George and others, Proc. Natl. Acad. Sci. USA, t, 1995, s-8362 and Verhaar, etc., J. of Nuc. Med., v.37 (5), 1996, c.868-872).

However, from the point of view of application in the clinical setting is still a need to create fragments of antibodies that have improved pharmacokinetic characteristics and which can be easily marked with radioactive isotopes, such as technetium or rhenium, because these radionuclides are the most preferred as radioactively-labeled pharmaceutical agents.

The object of the invention

Thus, the object of the invention are antibody fragments that have improved pharmacokinetic properties, in particular the specificity against the target and/or stability in vivo, and which can be easily associated with radioactive isotopes, such as technetium or rhenium.

Summary of the invention

The present invention concerns the compounds containing peptide, which is

AA) sequence antigennegative centre antibodies to the external domain B (ED-B) of fibronectin, which contains hypervariable sites HCDR3 and/or LCDR3, are presented in table 1, or its variation, such as deletion, insertion and/or replacement, affecting up to 5 amino acids for HCDR3 region and up to 6 amino acids for LCDR3 region, which has the same function as the peptide having the sequence Seq. Id. No.1;

AB) sequence antigennegative centre antibodies to the external domain B (ED-B) of fibronectin, which contains hypervariable sites HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, are presented in table 1, or its variation, such as deletion, insertion and/or replacement, affecting up to 3 amino acids for HCDR1 region, including 8 amino acids for HCDR2 region, up to 5 amino acids for HCDR3 region, up to 6 amino acids for LCDR1 region, up to 4 amino acids for LCDR2 region and up to 6 amino acids for LCDR3 region; which has the same function as the peptide having the sequence Seq. Id. No.1;

AB) sequence, which is presented in Seq. Id. No.1 (LI 9) or a variation of Seq. Id. No.1, such as deletion, insertion and/or replacement, affecting up to 30 amino acids, and which has the same function as the peptide having the sequence Seq. Id. No.1; and

b is) the amino acid sequence XAA 1-Haa2-Haa3-Cys (Seq. Id. No.2), where the characters Xaa1, XAA2and XAA3each independently of one another represents any naturally occurring amino acid, or

BB) the amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No.3), where the characters Xaa1, Xaa2, XAA3and XAA4each independently of one another represents any naturally occurring amino acid, or

BV) amino acid sequence (His)n(Seq. Id. No.4), where n denotes an integer from 4 to 6,

moreover, With the end of the sequence described in (AA), AB) or AB), associated with the N-end of one of the sequences disclosed in Seq. Id. No.2, Seq. Id. No.3 or Seq. Id. No.4, through a peptide bond.

The compounds preferably are single-stranded fragments of antibodies, in particular scFv-fragments. In addition, the compounds preferably conjugated with radioactive isotopes, for example, with a radioactive isotope of technetium, such as94mTc99mTc, rhenium, such as186Re,188Re, or other isotopes, such as203Pb67Ga68Ga43Sc,44Sc,47Sc,110mIn111In97EN,62Cu64Cu67Cu68Cu86Y88Y90Y121Sn161Tb153Cm166But,105Rh,177Lu,72A and 18F.

The present invention relates also to pharmaceutical compositions containing as active ingredient the above-mentioned compound in combination with physiologically acceptable excipients, diluents and/or carriers.

The present invention relates to the use of a peptide which is

AA) sequence antigennegative centre antibodies to the external domain B (ED-B) of fibronectin, which contains hypervariable sites HCDR3 and/or LCDR3, are presented in table 1, or its variation, such as deletion, insertion and/or replacement, affecting up to 5 amino acids for HCDR3 region and up to 6 amino acids for LCDR3 region, which has the same function as the peptide having the sequence Seq. Id. No.1;

AB) sequence antigennegative centre antibodies to the external domain B (ED-B) of fibronectin, which contains hypervariable sites HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, are presented in table 1, or its variation, such as deletion, insertion and/or replacement, affecting up to 3 amino acids for HCDR1 region, including 8 amino acids for HCDR2 region, up to 5 amino acids for HCDR3 region, up to 6 amino acids for LCDR1 region, up to 4 amino acids for LCDR2 region and up to 6 amino acids for LCDR3 region; which has the same function as the peptide having the sequence Seq. Id. No.1;

AB) sequence, which is presented in Seq. Id. No.1 (L1 9) or a variation of Seq. Id. No.1, such as deletion, insertion and/or replacement, affecting up to 30 amino acids, and which has the same function as the peptide having the sequence Seq. Id. No.1; and

BA) the amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No.2), where the characters Xaa1, XAA2and XAA3each independently of one another represents any naturally occurring amino acid, or

BB) the amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No.3), where the characters Xaa1, XAA2, XAA3and XAA4each independently of one another represents any naturally occurring amino acid, or

BV) amino acid sequence (His)n(Seq. Id. No.4), where n denotes an integer from 4 to 6, and With the end of the sequence described in (AA), AB) or AB), associated with the N-end of one of the sequences disclosed in Seq. Id. No.2, Seq. Id. No.3 or Seq. Id. No.4, through a peptide bond,

to associate with a radioactive isotope, such as a radioisotope of technetium or rhenium.

The antibody fragment L19 has the following sequence (Seq. Id. No.I):

(VH)
EVQLLESGGGLVQPGGSLRLSCAASGFTFS
SFSMSWVRQAPGKGLEWVSSISGSSGTTYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAED
TAVYYCAKPFPYFDYWGQGTLVTVSS
(linker)
GDGSSGGSGGASTG
(VL)
EIVLTQSPGTLSLSPGERATLSCRASQSVS
SSFLAWYQQKPGQAPRLLIYYASSRATGIP
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQ
OTGRIPPTFGOGTKVEIK

Division, the insertion and/or replacement, affecting up to 30 amino acids, represents a deletion, insertion and/ or replacement 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 25, 26, 27, 28, 29 or 30 amino acids of Seq. Id. No.1. However, in hypervariable sites (CDR) of the inventive peptide, such as peptide has the sequence presented in Seq. Id. No.1, variation, such as deletion, insertion and/or substitution of amino acids (AA), shall not exceed the maximum amount of variation shown in table 1 (HCDR denotes a CDR of the heavy chain; LCDR CDR denotes light chain).

Table 1
SitesThe length of the CDR (AK)SequenceMaximum (preferred) number of variations in the provisions of the sequence
HCDR15SFSMS3 (2, 1)
HCDR217SISGSSGTTY
YADSVKG
8 (7, 6, 5, 4, 3, 2, 1)
HCDR37PFPYFDY5 (4, 3, 2, 1)
LCDR112RASQSVSSSF
LA
6 (5, 4, 3, 2, 1)
LCDR2 7YASSRAT4 (3, 2, 1)
LCDR310CQQTGRIPPT6 (5, 4, 3, 2, 1)

CDR was determined according .A.Kabat with co-authors, "Sequences of Proteins of Immunological Interest", published by the U.S. Department of Health and Human Services, National Institutes for Health, Bethesda, MD, 50th ed., 1991.

Preferred are peptides that contain the sequence represented in Seq. Id. No.1 (LI 9), or a variation of Seq. Id. No.1, where fission, the insertion and/or replacement affects up to 20 amino acids.

The peptide, which is a variation of the CDR sequences listed in table 1, and, in particular, the variation Seq. Id. No.1, representing deletions, insertions, and/or replacement, and which has the same function as the peptide having the sequence Seq. Id. No.1, designated as peptide that binds to the ED-B domain of fibronectin, and this binding is characterized by the value of the dissociation constants (Kd), located in subnanomolar range (i.e. less than 10-9), as measured by BIAcore (see WO 99/58570, example 2 and table 2).

Preferred amino acid sequence XAA1-Haa2-Haa3-Cys (Seq. Id. No.2) represent the sequence Gly-Gly-Gly-Cys (Seq. Id. No.5) and Gly-Cys-Gly-Cys (Seq. Id. No.6). Most preferred is posledovatel the ability Gly-Gly-Gly-Cys (Seq. Id. No.5).

Preferred amino acid sequence XAA1-Haa2-Haa3-Cys (Seq. Id. No.3) represent the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No.7) and Gly-Cys-Gly-Cys-Ala (Seq. Id. No.8). Most preferred is the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No.7).

Compounds which contain amino acid sequence (His)n(Seq. Id. No.4), preferred are compounds in which n denotes an integer of 6.

Preferred radioactive isotopes of technetium or rhenium are isotopes94mTc99mTc186Re and188Re. The preferred radioactive isotope is99mTc.

Detailed description of the invention

Single-chain fragment antibody L19 (Seq. Id. No.1) previously marked using the125J for the study of bearsdley of this compound in the body bearing tumor mice (Tarli, etc., Blood, t, No.1, 1999, p.á192-198). These results allowed us to establish that it is possible to carry out selective directional migration to the blood vessels of tumors in vivo. While it has been unexpectedly found that the pharmacokinetic characteristics of single-chain antibody fragment L19 can be substantially improved by its conjugation with the peptide specified in BA), BB) or BV), and labeling with radioactive isotopes of technetium or rhenium. Isotope99mTc represents RA is ioactivity isotope, selected for carrying out the normal applied in a clinical setting single photon emission computed tomography (SPECT) because of its radiochemical characteristics (easy to obtain with99Mo/99mTc-generator, the ability to emit a single gamma-quanta with energy of 140 Kev, the ability to provide a strong flow of photons and the ability to degrade rapidly (half-life 6 hours), and also because of its relatively low cost. For therapeutic purposes the most preferred is tagging using chemically similar isotopes186Re and188Re (Hsieh V.T. and others, Nucl. Med. BioL, 26 (8), 1993, s-972 and s-976, Zamora P.O. etc., Anticancer Res., 17 (3B), 1997, s-1838).

Peptides proposed in the present invention are derived recombinant scFv-fragment antibody L19 (Seq. Id. No.1) to the extracellular ED-B domain of fibronectin and get them using genetic engineering according to the process represented in figure 1. Get the following peptides: L19 (Seq. Id. No.1)

L19His:

1EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSS
51ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF
101PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS LSPGERATLS
151CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR FSGSGSGTDF
201TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKAAAL EHHHHHH

(Seq. Id. No.9)

AP38:

1EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSS
51ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF
101PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS LSPGERATLS
151CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR FSGSGSGTDF
201TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC

(Seq. Id. No.10)

AP39:

1EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSS
51ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF
101PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS LSPGERATLS
151CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR FSGSGSGTDF
201TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC A

(Seq. Id. No.11)

L19-GlyCysGlyCys:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSS
51ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF
101PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS LSPGERATLS
151CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR FSGSGSGTDF
201TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC

(Seq. Id. No.12)

L19-GlyCysGlyCysAla:

1EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSS
51ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPF
101PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS LSPGERATLS
151CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR FSGSGSGTDF
201TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC A

(Seq. Id. No.13)

Obtaining peptides described in the examples below (see "Experimental section").

The antibody fragment L19 was originally obtained by expression in E. coli (see WO 99/58570). However, as installed, this system is unsatisfactory for large-scale production of scFv fragments of antibodies. Was studied another expression system, namely the system e is cpressey, based on the use of yeast, in particular the expression system based on the use of Pichia pastoris. When creating the present invention found that the yeast, for example, Pichia pastoris, as a rule, suitable for the expression of high biological activity of the antibody fragment, for example, a fragment OR, however, high level expression, providing up to 250 mg of fragment antibodies for 1 l culture, which is required for cheap get biopharmaceutical product can only be achieved when using a constitutive expression vector (for example, pGAP) and it cannot be achieved using induced by menthol vector (for example, pPIC9K). Another advantage of this constitutive expression system is its simplicity and robustness of the procedures fermentation compared with the use of inducible expression system based on the yeast. With the invention it has been unexpectedly found that the correct processing of the signal sequence of the fragment of the antibody, for example, a fragment OR occurred only when he used the expression cassette, in which the N-end of the fragment was directly fused to the site of cleavage KEH of the alpha signal sequence.

The peptides can be used for diagnostic and therapeutics what their goals, in particular for the diagnosis and therapy of invasive tumors and metastatic tumors. The preferred use for diagnostic purposes is used for SPECT (single photon emission computed tomography) and PET (positron emission tomography).

The above peptides are most suitable for labeling with the above radioactive isotopes, such as radioactive isotopes of technetium and rhenium, preferably radionuclides94mTc99mTc186Re and188Re. For labelling peptides peptides first restore using an appropriate reducing agent type, for example, tin chloride(II) or Tris(2-carboxyethyl)phosphine (TCEP). The resulting recovered peptides have free SH-groups, which can interact with the eluate99mTc-generator or eluate188Re-generator and chloride of tin(II) obtaining the compounds proposed in this invention (see details below in the experimental examples). Indirect labelling is carried out by pre-conjugation of chelating ligand and the subsequent formation of a complex with radioactive isotopes, for example, indium, yttrium, lanthanides, etc. Chelating ligand, as a rule, is derived from ethylenediaminetetraacetic acid (edtc), diethylenetriaminepentaacetic acid (DTPC), cyclohexa is -1,2-diaminetetraacetic acid (CDTC), the ethylene glycol-O,O'-bis(2-amino-ethyl)-N,N,N',N'-luxusni acid (HBED), Triethylenetetramine acid (TGC), 1,4,7,10-tetraazacyclododecane-N,N',N"-tetraoxane acid (DOTA), 1,4,7-triazacyclononane-N,N',N"-trioxanes acid (touristic organisation) and 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraoxane acid (TETC), and he is a chelating agent or for amino, or for Tilney groups of the peptide compounds. Chelating ligands are acceptable for the combination group, for example, active esters, maleimides, thiocarbamates or α-halogenated acetamide fragments. For conjugation of chelating ligands with amino groups, for example ε-NH2-groups of lysine residues, no prior recovery of peptide compounds. Labeled with radioactive isotope peptides can be used for radiodiagnosis and radiotherapy.

The resulting labeled with radioactive isotope peptides in experiments on animals were found unexpected benefits. For example, excretion through the kidneys to 70% or more, for example, 80,63% labeled peptide, for example, labeled with99mTc AR (Seq. Id. No.11) in Nude occurs within 24 h, whereas excretion through the kidneys L19 (Seq. Id. No.1), labeled with125I, in Nude mice was within 24 h only 67,79%. The ratio of the giving of the distribution in the tumor and blood labeled peptide, for example, labeled with99mTc AR was 5:1 or more, preferably 8:1 or more, such as about 10:1, after 5 h, whereas this ratio in the case L19 marked with125I was only about 3:1. It is unexpected when compared to other scFv fragments of antibodies labeled with99mTc, which is often characteristic of the less desirable characteristics of the distribution. For example, Verhaar, etc., J. of Nuc. Med., v.37 (5), 1996, s-872 have found that to be marked using the99mTc scFv-fragment antibody ratio distribution in the tumor and blood after 24 h was only 4:1, and after 24 h the kidneys accumulated 9%, which is a very high figure compared with the data obtained for peptides proposed in the present invention, for example, using labeled with99mTc AR accumulation in the kidneys was 1.3% (see example 13).

In addition, stability in vivo labeled peptides, proposed in the invention, for example, labeled with99mTc AR, significantly higher compared with the stability in vivo L19 marked with125I. In the claimed invention found that after 2 h after injection of the peptide, for example, labeled with99mTc AR, only 10% or less, for example, 3% of radioactivity in serum accounted for metabolite,while after 2 h after injection L19, marked with125I, 49% of the radioactivity in the serum accounted for metabolites, which can be a free iodine. Improved stability in vivo peptides, for example, labeled with99mTc AR, is also evident in its continued ability to contact the target, i.e. with ED-B. When creating the present invention found that after 2 h after injection of the peptide, for example, labeled with99mTc AR, 50% or more, for example, 74% of the radioactive label present in the serum, has the ability to connect with ED-B, while after 2 h after injection L19 marked with125I, only 27% of the radioactive label present in the serum, can contact ED-B. Connections proposed in the invention also have a high ability to accumulate in the tumor. For example, Tc-99m-AIR and In-111-MX-TPC-ε-HN(Lys)-AP39 found to have a high ability to accumulate into tumors component of 10.7% (Tc-99m) or 12.9 per cent (In-111) from the injection dose per gram (ID/g) after 1 h after injection (ri.). Thus, the absorption of the tumor is significantly higher compared to levels, which are known for other fragments of the antibodies labeled with In-111 or Tc-99m (see, for example, Kobayashi and others, J. of Nuc. Med., so 41 (4), 2000, s-762; Verhaar, etc., J. of Nuc. Med., v.37 (5), 1996, s-872).

The connection can be used for di is Gnostic and therapeutic purposes. They are usually administered to the patient parenterally, more preferably by intravenous injection. Human dose is preferably 0.1 to 1 mg per person when used in radiodiagnosis and 0.1-100 mg per person when used for radiotherapy.

Methods of obtaining and marking compounds proposed in the present invention, is illustrated in more detail in the following examples, which are given only for illustrative purposes and in no way limit the scope of the invention.

Experimental section

Example 1: preparation of derivatives of L19

As the original product used recombinant antibody (scFv L19, shorthand L19) to the extracellular domain B (ED-B) splicing variant of fibronectin. scFv L19 were isolated by selection from a range of synthetic human antibodies using phage expression (Neri and others, Nature Biotechnol. 15, 1997, s; Pini and others, J. Biol. Chem. 273, 1998, s). This fragment of recombinant antibodies is in the form of so-called single-chain antibody fragment (scFv) and consists of the VH - and VL-region associated linker sequence (see Seq. Id. No.1). This scFv L19 has a very high affinity against ED In (Kd:5,4×10-11M).

Using genetic engineering techniques has received various derivatives L19 (see figure 1). To modify L19 encoding scFv DNA amplif is provoked by PCR (polymerase chain reaction) using primers coding additional sequence, and cloned in the expression vector.

Derivatives L19:

L19: no additional limit modifications.

L19 His: C-terminal domain of His6(His-tag), is designed to chelate

chromatography on Ni and to associate with radioisotopes.

AR: C-terminal domain GlyGlyGlyCys, is designed to link (via Cys) substances that can be applied in therapy and diagnostics (e.g., radioisotopes).

AR: C-terminal domain GlyGlyGlyCysAla, is designed to link (via Cys) substances that can be applied in therapy and diagnostics (e.g., radioisotopes).

L19-GlyCysGlyCys: C-terminal domain GlyCysGlyCys, is designed to link (via Cys) substances that can be applied in therapy and diagnostics (e.g., radioisotopes).

L19-GlyCysGlyCysAla: C-terminal domain GlyCysGlyCysAla, is designed to link (via Cys) substances that can be applied in therapy and diagnostics (e.g., radioisotopes).

Recombinant deriving L19

Described L19 derivatives were obtained in prokaryotic and eukaryotic expression systems.

a) Obtaining L19 in E.coli

DNA sequences encoding various derivative L19 (AR, AR, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His), cloned into prokaryotic expression vector (pDN5, Pini and others, J. Immunol. Methods 206, 1997, s, Pini and others, J. Bol. Chem. 273, 1998, s; pet, firms Novagen) using induced IPTG (isopropylthioxanthone) promoter and marker, causing resistance to ampicillin. To ensure secretion of the recombinant protein in periplasm this vector was used to obtain the expression cassette, in which the N-terminal scFv fused with the signal sequence Pel C. This ensured the creation of a stable producer strains after transformation of E. coli (TG1, BL21DE3 and NW) expression vector, followed by selection on the basis of resistance to ampicillin. To obtain scFv these strains were cultured in the presence of 1% glucose in the growth phase (37°C) for the suppression of the promoter. Expression of scFv in cultures induced by adding IPTG and by incubation at 30°C over a period of time up to 16 hours of Soluble and bind to the antigen scFv can be distinguished from the full extract of E. coli strains from fractions periplasm or that, as installed, is particularly effective from the standpoint of cleanup and exit from the culture supernatant. Obtaining carried out in shake flasks and fermenters up to 10 litres

b) deriving L19 in Pichia pastoris

The DNA sequence encoding L19His, AP38, AR, L19-GlyCysGlyCys and L19-GlyCysGlyCysAla, amplified by PCR and cloned in E. coli and in the expression vectors RSC and pGAP (firm Invitrogen) to obtain drogach Pichia pastoris. For the expression of heterologous genes RSC contains induced by methanol promoter (OH), and pGAP contains the constitutive promoter of the enzyme glycerylphosphorylcholine (GAPDH). In addition, these vectors contain a gene determining resistance in geneticin, and the gene causing resistance to zeocin for selection/amplification of a foreign gene and the signal sequence of the α-factor of yeast for expression and secretion of the recombinant product. Used for the expression of OR the expression cassette encodes fused protein (signal □-factor + derived L19), in which the signal sequence has been eliminated and only the site of cleavage KEH and were not eliminated all other cleavage sites required for processing occurring in natural conditions □-factor. Stable transfection clones Pichia pastoris (PP) was obtained by introduction by electroporation of linearized vectors in strains of Pichia pastoris (for example, RSC-A transformed strain GS115, a pGAP-AP39 transformed strain H) and subsequent selection using geneticin or zeocin. These clones can be used to obtain these derivatives L19 in the form of soluble secretory protein. The clones were cultured at 30°C in BMGY medium or in the main mineral environment. When using the clone is in, which are based pPIC, was added methanol to enhance the induction process phase expression. The recombinant product have the correct versions of the ends and high antigennegative activity. Possible solution (untreated biologically active product/l of culture supernatant) was dependent on cultivation conditions and process control: for example, pPIC9K-AP39/GSl15 (in shake flask output was 5 mg/l fermenter 10-15 mg/l); for pGAP-AP39/X33 (in shake flask output was 30-40 mg/l fermenter 100-250 mg/l).

Derivatives L19 was purified from the supernatant of cultures of Pichia pastoris or E. coli using affinity chromatography (loaded with protein And the column, the firm Pharmacia Streamline or loaded antigen ED In column) with subsequent gel filtration. Purified, containing A fraction, which was used for further processing, had homodimer structure (where the a subunit is covalently linked to the main part) and had high antigennegative activity.

Example 2A

Synthesis restored AR [restored L19-(Gly)3-Cys-OH]

To a solution containing 240 g (4,29 of nmole) S-S-dimer AR 156 μl SFR (phosphate buffered saline), 10% glycerol, was added 50 μl of a solution DEF (14,34 mg TCEP×HCl/5 ml aqueous Na2HPO4, 0,1M, pH 7.4). The reaction mixture was carefully shook the for 1 h at room temperature. SH-Monomeric AR was purified by gel filtration using a column NAP-5, Amersham, eluent: SFR). Analysis using LTO-SDS page of the selected product is allowed to set the number of S-S-dimer OR transformed into SH-Monomeric AR. Output: 79,4 µg/220 ál SFR (33,1%).

Example 26

Synthesis of Tc-99m-AP38 [Tc-99m-L19-(Gly)3-Cys-OH]

2.37 mg of disodium L-tartrate was placed in a vial, which was then added 79,4 µg restored OR in 220 ál SFR, and the solution was diluted in 100 ál of water Na2HPO4buffer (1M, pH of 10.5). Added 50 μl of the eluate Tc-99m generator (24 h) and 10 μl of a solution SnCI2(5 mg SnCl2/1 ml 0,1M HCl). The reaction mixture was shaken for 0.5 h at 37°C. Labeled with Tc-99m AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:39,7%
Radiochemical purity:92,5% (LTOs-SDS page)
Specific activity:17,7 MBq/nmol
Immunoreactivity:88,7%

Example 3A

Synthesis restored AR [restored L19-(Gly)3-Cys-Ala-OH]

To a solution containing 240 g (4,29 of nmole) S-S-dimer OR V ál SFR/10% glycerol, added 50 μl of a solution DEF (14,34 mg TCEP×HCl/5 ml aqueous Na2HPO4, 0,1M, pH 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH-Monomeric AR was purified by gel filtration using a column NAP-5, Amersham, eluent: SFR). Analysis using LTO-SDS page of the selected product is allowed to set the number of S-S-dimer OR transformed into SH-Monomeric AR. Output: 135,9 mcg/180 µl SFR (56,2%).

Example 3b

Synthesis of Tc-99m-AP39 [Tc-99m-L19-(Gly)3-Cys-Ala-OH]

4.2 mg of disodium L-tartrate was placed in a vial, which was then added to 135.9 µg restored OR in 180 µl SFR, and the solution was diluted in 100 ál of water Na2HPO4buffer (1M, pH of 10.5). Added 100 μl of the eluate Tc-99m generator (24 h) and 10 μl of a solution of SnCl2(5 mg SnCl2/1 ml 0,1M HCl). The reaction mixture was shaken for 0.5 h at 37°C. Labeled with Tc-99m AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:50,1%
Radiochemical purity:91,5% (LTOs-SDS page)
Specific activity:21,4 MBq/nmol
Immunoreactivity: 96,4%

Example 4

Synthesis of Re-188-AP38 [Re-188-L19-(Gly)3-Cys-IT]

2.37 mg of disodium L-tartrate was placed in a vial, which was then added 112 mg restored OR in 310 μl SFR, and the solution was diluted in 100 ál of water Na2HPO4buffer (1M, pH of 10.5). Added 100 μl of the eluate Re-188 generator and 50 μl of a solution of SnCl2(5 mg SnCl2/l ml 0.1 M HCl). The reaction mixture was shaken for 1.5 h at 37°C. Labeled with Re-188 AP38 was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:28,3%
Radiochemical purity:to 91.1% (LTOs-SDS page)
Specific activity:15,3 MBq/nmol
Immunoreactivity:89,9%

Example 5

Synthesis of Re-188-AP39[Re-188-L19-(Gly)3-Cys-Ala-OH]

2.37 mg of disodium L-tartrate was placed in a vial, which was then added 112 mg restored OR in 303 μl SFR, and the solution was diluted in 100 ál of water Na2HPO4buffer (1M, pH of 10.5). Added 100 μl of the eluate Re-188 generator and 50 μl of a solution of SnCl2(5 mg SnCl2/1 ml 0,1M HCl). The reaction mixture was shaken for 1.5 h at 37°C. Labeled with Re-188 AP39 ciali gel filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:33,5%
Radiochemical purity:92,3% (LTOs-SDS page)
Specific activity:of 18.5 MBq/nmol
Immunoreactivity:92,5%

Example 6A

Synthesis restored L19-Gly-Cys-Gly-Cys-OH

To a solution containing 240 g (4,29 of nmole) S-S-dimer L19-Gly-Cys-Gly-Cys-OH in 160 µl SFR/10% glycerol was added 75 μl of solution DEF (14,34 mg TCEP x HCl/5 ml aqueous solution of Na2HPO4, 0,1M, pH 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH-Monomeric L19-Gly-Cys-Gly-Cys-OH was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR). Analysis using LTO-SDS page of the selected product is allowed to set the number of S-S-dimer L19-Gly-Cys-Gly-Cys-OH, transformed into SH-Monomeric L19-Gly-Cys-Gly-Cys-OH. Output: 80,4 µg/210 μl SFR (33,5%).

Example 6b

Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-OH

2.37 mg of disodium L-tartrate was placed in a vial, which was then added 80,4 µg restored L19-Gly-Cys-Gly-Cys-OH in 210 μl SFR, and the solution was diluted in 100 ál of water Na2HPO2buffer (1M, pH of 10.5). Added 50 μl of the eluate Tc-99m generator (24 h) and 10µl solution of SnCl 2(5 mg SnCl2/1 ml 0.1 M HCl). The reaction mixture was shaken for 0.5 h at 37°C. Labeled with Tc-99m L19-Gly-Cys-Gly-Cys-OH was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:37,7%
Radiochemical purity:91,5% (LTOs-SDS page)
Specific activity:19,7 MBq/nmol
Immunoreactivity:89,7%

Example 7a

Synthesis restored L19-Gly-Cys-Gly-Cys-Ala-OH

To a solution containing 240 g (4,29 of nmole) S-S-dimer L19-Gly-Cys-Gly-Cys-Ala-OH and 155 μl SFR/10% glycerol was added 75 μl of solution DEF (14,34 mg TCEP×HCl/5 ml aqueous solution of Na2HPO40,1M, pH 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH-Monomeric L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR). Analysis using LTO-SDS page of the selected product is allowed to set the number of S-S-dimer L19-Gly-Cys-Gly-Cys-Ala-OH, transformed into SH-Monomeric L19-Gly-Cys-Gly-Cys-Ala-OH.

Output: 81,2 µg/215 μl SFR (33,8%).

Example 7b

Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH

2.37 mg of disodium L-tartrate placed the in the bubble, where was further added 81,2 µg restored L19-Gly-Cys-Gly-Cys-Ala-OH in 215 μl SFR and the solution was diluted in 100 ál of water Na2HPO4buffer (1M, pH of 10.5). Added 50 μl of the eluate Tc-99m generator (24 h) and 10 μl of a solution of SnCl2(5 mg SnCl2/1 ml 0,1M HCl). The reaction mixture was shaken for 0.5 h at 37°C. Labeled with Tc-99m L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:35,6%
Radiochemical purity:93,5% (LTOs-SDS page)
Specific activity:19,1 MBq/nmol
Immunoreactivity:88,7%

Example 8A

Synthesis restored OR for specific conjugation of chelators type etc, CDTC, TED, DTPC, TTGC, HBED, PILLBOX, touristic organisation and DO3A with SH-group of cysteine

50 μl of a solution DEF (14,34 mg TCEP×HCl/5 ml aqueous solution of Na2HPO4, 0,1M, pH 7.4) was added to a solution containing 400 micrograms (7.1 nmole) OR 450 ál SFR. The reaction mixture was gently shaken for 1 h at 37°C. the Recovered AR was purified by gel filtration using a column NAP-5, Amersham, eluent: sodium acetate b is fer, 0,1M, pH 5.0). Analysis using LTO-SDS page of the selected product has proved a complete transformation OR in the restored AR. Output: 140 µg/200 µl SFR (35%).

Example 8b

Synthesis MX-TPC-maleimide(1,4,7-triaza-2-(N-maleimidomethyl-para-amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl-6-formed)

512 mg (1 mmol) of {[3-(4-AMINOPHENYL)-2-(bicarboxylic)propyl][2-(bicarboxylic)propyl]amino}acetic acid (firm Macrocyclics Inc. Dallas, PCs TX, USA) and 707 mg (7 mmol) of triethylamine were dissolved in 3 ml of anhydrous DMF. Was added dropwise to 400 mg (1.5 mmole) of 2,5-dioxopiperidin-1 silt ester 3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propionic acid (company Aldrich) in 1 ml anhydrous DMF. The solution was stirred for 5 h at 50°C. was Slowly added 30 ml of diethyl ether. The reaction mixture was stirred for a further 30 minutes the Precipitate was collected by filtration. The crude product was purified by means OF GHUR (acetonitrile: water: triperoxonane acid/ 3:96,9:0,1→99,9:0:0,1). Yield: 61% (405 mg, and 0.61 mmole). MC-ESI: 664=M++1.

Example 8b

Synthesis of In-111-MX-TPC-maleimide-S(Cys)-AR-R

(R denotes the restored)

140 g (5 nmoles) AP39-R in 200 μl of sodium acetate buffer (0.1m, pH 5) were subjected to interaction with 50 ál of dissolved 1,4,7-triaza-2-(N-maleimidomethyl-para-amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl-6-methylheptane (0.25 mg TPC-maleimide 500 µl per the bacilli acetate buffer, 0,1M, pH 5) for 3 h at 37°C. the Reaction mixture was dialyzed (2×1 h) each time the counter 200 ml of sodium acetate buffer (0.1m, pH 6)using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA).

Added 80 μl of a solution [In-111]InCl3(HCl, 1N., 40 MBq, firm Amersham Inc.) and the reaction mixture is kept at 37°C for 30 minutes Labeled with In-111 TPC-maleimide-S(Cys)-AR-R was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:54%
Radiochemical purity:94% (LTOs-SDS page)
Specific activity:6,2 MBq/nmol
Immunoreactivity:86%

Example 9

Synthesis of In-11l-MX-TPC-ε-HN(Lys)-AP39

200 mcg (3.6 nmole unrestored AR 111 μl SFR was diluted in 300 μl of sodium borate buffer (0.1m, pH 8.5) and dialyzed (2×1 h) in a counter 200 ml of sodium borate buffer (0.1m, pH 8.5), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA). Added 50 μl of a solution of 1,4,7-triaza-2-(para-isothiocyanate)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl-6-methylheptane (MX DTPC) (033 mg MX-DTPC, dissolved in 500 μl of sodium borate buffer, 0.1m, pH 8.5) and the reaction mixture was stirred for 3 h at 37°C. the Reaction mixture was dialyzed (2×1 h and 1×17 h (overnight)) each time the counter 200 ml of sodium acetate buffer (0.1m, pH 6.5), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA).

Added 80 μl of a solution [In-111]InCl3(HCl, 1 N., 40 MBq, firm Amersham Inc.) and the reaction mixture is kept at 37°C for 30 minutes Labeled with In-111 MX-TPC-ε-NH(Lys)-AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:70%
Radiochemical purity:85% (LTOs-SDS page)
Specific activity:of 7.6 MBq/nmol
Immunoreactivity:74%

Example 10

Synthesis of In-111-DOTA-P-benzyl-para-HCS-ε NM(Lys)-AR

200 mcg (3.6 nmole unrestored AR 114 μl SFR was diluted in 300 μl of sodium borate buffer (0.1m, pH 8.5) and dialyzed (2×1 h) in a counter 200 ml of sodium borate buffer (0.1m, pH 8.5), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA). Add recipients who do 50 μl of a solution of 1,4,7,10-tetraaza-2-(para-isothiocyanate)benzylchloride-1,4,7,10-tetraoxane acid (benzyl-para-SCN-DOTA, the firm Macrocyclics Inc., Dallas, PCs TX, USA) (1.5 mg benzyl-para-SCN-DOTA, dissolved in 5 ml of sodium borate buffer, 0.1m, pH 8.5) and the reaction mixture was stirred for 3 h at 37°C. the Reaction mixture was dialyzed (2×1 h and 1×17 h (overnight)) each time the counter 200 ml of sodium acetate buffer (0.1m, pH 6.5), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA).

Added 80 μl of a solution [In-111]InCl3(HCl, 1N., 40 MBq, firm Amersham Inc.) and the reaction mixture is kept at 37°C for 30 minutes Labeled with In-111 DOTA-P-benzyl-para-NCS-ε-HN(Lys)-AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:74%
Radiochemical purity:94% (LTOs-SDS page)
Specific activity:to 12.3 MBq/nmol
Immunoreactivity:73%

Example 11

Synthesis of Y-88-MX-TPC-ε NM(Lys)-AR

200 mcg (3.6 nmole unrestored OR in 115 μl SFR was diluted in 300 μl of sodium borate buffer (0.1m, pH 8.5) and dialyzed (2×1 h) in a counter 200 ml of sodium borate buffer (0.1m, pH 8.5), using the device type Slie-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA). Added 50 μl of a solution MX-DTPC (0.33 mg MX-DTPC dissolved in 500 μl of sodium borate buffer, 0.1m, pH 8.5) and the reaction mixture was stirred for 3 h at 37°C. the Reaction mixture was dialyzed (2×1 h and 1×17 h (overnight)) each time the counter 200 ml of sodium acetate buffer (0.1m, pH 6.0), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA).

Added 100 μl of a solution of [Y-88]YCl3(HCl, 1 N., 75 MBq, firm Oak Ridge National Lab.) and the reaction mixture is kept at 37°C for 30 minutes Labelled with Y-88 MX-TPC-ε NK(Lys)-AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:65%
Radiochemical purity:93% (LTOs-SDS page)
Specific activity:10,2 MBq/nmol
Immunoreactivity:72%

Example 12

Synthesis of Lu-177-DOTA-C-benzyl-para-NCS-ε-HN(Lys)-AP39

200 mcg (3.6 nmole unrestored OR in 110 µl SFR was diluted in 300 μl of sodium borate buffer (0.1m, pH 8.5) and dialyzed (2×1 h) in a counter 200 ml of sodium borate buffer (0.1m, pH 8.5)using the mustache is the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA). Added 50 μl of a solution of benzyl-para-SCN-DOTA (1.5 mg dissolved in 5 ml of sodium borate buffer, 0.1m, pH 8.5) and the reaction mixture was stirred for 3 h at 37°C. the Reaction mixture was dialyzed (2×1 h and 1×17 h (overnight)) each time the counter 200 ml of sodium acetate buffer (0.1m, pH 6.0), using the device type Slide-A-Lyzer 10,000 MWCO (Pierce firm Inc., Rockford, PCs Illinois, USA).

Added 200 μl of a solution of [Lu-177]LuCl3(HCl, 1N., 80 MBq, firm NRH-Petten, the Netherlands) and the reaction mixture is kept at 37°C for 30 minutes Labelled using Lu-177 DOTA-P-benzyl-para-NCH-ε NM(Lys)-AR was purified by gel-filtration using a column NAP-5, Amersham, eluent: SFR).

Radiochemical yield:74%
Radiochemical purity:95% (LTOs-SDS page)
Specific activity:19 MBq/nmol
Immunoreactivity:71%

Example 13

Distribution in organs and excretion of Tc-99m-AR expressed in Pichia pastoris, after a single intravenous (i.v.) injection of tumor bearing Nude mice

The substance of the proposed invention was administered intravenously dose about 74 kBq animals (body weight of about 25 g), bearing carcinoma line F9 (teratocarcinoma). The dose of radioactivity in various organs and radioactivity in faeces was assessed by using a γ-counter at various time points after administration of the substance. In addition, based on the concentration of the substance of the proposed invention, in the tumor and blood at different time points was calculated ratio in the tumor and blood.

Data on the biodistribution of Tc-99m-AR in the body Nude mice bearing F9 (teratocarcinoma) (mean values ± standard deviation, n=3)are presented in table 2:

Table 2
% dose/g of tissue% dose/g of tissue% dose/g of tissue
1 h ri.5 h ri.24 h ri.
Spleen1,97±0,0180,53±0,070,31±0,08
Liver1,91±0,0460,77±0,070,26±0,01
Kidney19,21±0,704,35±0,082 1,32±0,10
Easy3,43±1,011,41±0,0320,96±0,23
The stomach without content1,55±0,0431,35±0,220,48±0,10
The gut content1,42±0,101,26±0,340,29±0,05
Tumorof 10.72±3,215,13±1,453,48±1,28
Blood3,03±0,320,57±0,110,11±0,01

Data on the excretion of Tc-99m-AR from the body Nude mice bearing F9 (teratocarcinoma) (mean values ± standard deviation, n=3)are presented in table 3:

Table 3
% dose
24 h ri.
Urine80,63±3,33
Cal3,94±0,17

Data on the ratio of Tc-MT-OR the organization in the Oli/blood in Nude mice bearing F9 (teratocarcinoma) (mean values ± standard deviation, n=3)presented in figure 2

The results of this study indicate a very high ability of substance to offer in the invention, to accumulate in solid tumors in combination with a very high capacity for excretion.

Example 14

Distribution in the organs of In-111-MX-TPC-ε-HN(Lys)-AR after a single i.v.-injection of tumor bearing Nude mice

The substance of the proposed invention was administered intravenously at a dose of about 48 kBq animals (body weight of about 25 g)carrying carcinoma line F9 (teratocarcinoma). The dose of radioactivity in various organs and radioactivity in faeces was assessed by using a γ-counter at various time points after administration of the substance.

Data on the biodistribution of In-111-MX-TPC-ε-HN(Lys)-AP39 in Nude mice bearing F9 (teratocarcinoma) (mean values ±standard deviation, t=3)are presented in table 4:

Table 4
% dose/g of tissue
1 h ri.3 h ri.24 h ri.
Spleen1,94±0,49 1,28±0,131,18±0,24
Liver2,61±1,322,59±0,36of 2.26±0,75
Easy1,52±1,572,36±0,300,76±0,21
The stomach without content1,44±0,811,40±0,310,65±0,28
The gut contentof 5.05±5,261,07±0,340,67±0,11
Tumor12,90±4,817,44±1,344,33±0,84
Blood5,55±1,891,80±0,200,11±0,02

Data on the ratio of In-111-MX-TPC-ε NM(Lys)-AR in the tumor/blood in Nude mice bearing F9 (teratocarcinoma) (mean values ± standard deviation, n=3)are presented in table 5.

Table 5
1 h ri.3 h ri.24 h ri.
The ratio of tumor/bloodwas 2.76±2,004,16±0,7536,36±3,78

The results of this study indicate a very high ability of substance to offer in the invention, to accumulate in solid tumors in combination with very good biodistribution and the ratio of tumor/blood.

Example 15

Visualization using Tc-99m-AR. expressed in Pichia pastoris, after a single i.v.-injection of tumor bearing Nude mice

The substance of the proposed invention was administered intravenously at a dose of about a 9.25 MBq animals (body weight of about 25 g)carrying carcinoma line F9 (teratocarcinoma). At various time points after administration of the substance has been rendering using a gamma camera.

The results of planar scintigraphy bearing F9 (teratocarcinoma) Nude mice using Tc-99m-AR presented in figure 3 and 4. Figure 3 presents the Scintigram obtained after 5 h after injection of the substance, and figure 4 presents the Scintigram obtained 24 h after injection of the substance.

The results of this study indicate that the use of substances proposed in the invention opens up great possibilities for the visualization of dense SDA is Olga.

1. The connection to associate with the domain ED-B of fibronectin, which is a fragment of single-stranded radiolabelled antibodies and includes a peptide which contains antigennegative website for an external domain B (ED-B) of fibronectin selected from the group including:
AA) a sequence containing the hypervariable sites HCDR3 and/or LCDR3, are presented in table 1, from the sequence Seq. Id. No.1;
AB) a sequence containing the hypervariable sites HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, are presented in table 1, from the sequence Seq. Id. No.1;
AB) sequence, which is presented in Seq. Id. No.1 (L1 9);
and the primary amino acid sequence selected from the group including:
BA) the sequence represented in Seq. Id. No.2,
BB) the sequence represented in Seq. Id. No.3,
BV) sequence presented in Seq. Id. No.4;
With the end of the sequence described BAA), AB or AB), associated with the N-end of one of the sequences disclosed in Seq. Id. No.2, Seq. Id. No.3 or Seq. Id. No.4, through a peptide bond.

2. The compound according to claim 1, where the amino acid sequence of Seq. Id. No.2 is a sequence Gly-Gly-Gly-Cys (Seq. Id. No.5) or Gly-Cys-Gly-Cys (Seq. Id. No.6).

3. The compound according to claim 1, where the amino acid sequence of Seq. Id. No.3 is a sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No.7) or Gly-Cys-Gly-Cys-Ala (Seq. Id. No.8).

4. The compound according to claim 1, where the value of n in the amino acid sequence of Seq. Id. No.4 is 6.

5. The compound according to claim 1, optionally conjugated with a radioactive isotope and is intended for the diagnosis and treatment of angiogenic diseases.

6. The compound according to claim 5, conjugated with a radioactive isotope selected from the radioactive isotope of technetium, such as94mTC99mTc, rhenium, such as186Re,188Re, or other isotopes, such as203Pb67Ga68Ga43Sc,44Sc,47Sc,110mIn111In97EN,62Cu64Cu67Cu68Cu86Y88Y90Y121Sn161Tb153Sm166Ho,105Rh,177Lu,72As and18F.

7. The connection according to claim 6, where the radioactive isotope is a99mTC or188Re.

8. The compound according to claim 1, where the peptide is in a restored form.

9. The pharmaceutical is Skye composition, designed for the diagnosis of an angiogenic disease, comprising as active ingredient a therapeutically effective amount of a compound according to one of claims 1 to 8 in combination with physiologically acceptable excipients, carriers and/or diluents.

10. The pharmaceutical composition intended for the treatment of angiogenic disease, comprising as active ingredient a therapeutically effective amount of a compound according to one of claims 1 to 8 in combination with physiologically acceptable excipients, carriers and/or diluents.

11. The composition according to PP and 10, characterized in that up to 70% or more which is excreted in mice after 24 h through the kidneys.

12. Composition according to any one of p-11, characterized in that after 5 h after injection mice the ratio of its distribution in the tumor/blood is 5:1 or more.

13. The composition according to claim 9, intended for use in diagnosis.

14. The composition of claim 10, intended for use in therapy.

15. The use of compounds according to claims 1-4 for binding with a radioactive isotope.

16. The application indicated in paragraph 15, where the radioactive isotope is selected from a radioactive isotope of technetium, such as94mTC99mTc, rhenium, such as186Re,188Re, or other isotopes, such as203Pb67Ga68Ga43 Sc,44Sc,47Sc,110mIn111In97EN,62Cu64Cu67Cu68Cu86Y88Y90Y121Sn161Tb153Sm166Ho,105Rh,177Lu,72As and18F.

17. The application of article 16, where the radioactive isotope is a99mTC or188Re.

18. The method of obtaining the compounds according to one of claims 1 to 4, characterized in that the peptide Express in eukaryotic cells.

19. The method according to p, wherein the eukaryotic cells are yeast cells.

20. The method according to p, wherein the eukaryotic cells are cells of Pichia pastoris.

21. The method according to p, characterized in that the peptide is expressed constitutively.

22. The method according to p, characterized in that the N-end of the peptide bind directly to the site of cleavage KEH of the α-signal sequence.

23. Set to obtain labeled with radioactive isotopes pharmaceutical agent comprising the compound according to one of claims 1 to 8 and auxiliary substances.
Priority items:

03.01.2002 according to claims 1-5, 7-12, 15, 18-22;

25.02.2002 according to claims 1-5, 7-12, 15, 18-22;

02.01.2003 on PP, 13-14, 16-17, 23.



 

Same patents:

FIELD: chemistry, biochemistry.

SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.

EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.

22 cl, 13 dwg, 8 tbl

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns biotechnology. It is described bispecific antibody which binds also the factor of blood coagulation IX or the activated factor of blood coagulation IX, and the factor of blood coagulation X, and functionally replaces the factor of blood coagulation VIII or the activated factor of blood coagulation VIII which strengthens enzymatic reaction. The pharmaceutical composition containing the described antibody is revealed. The present invention can be used as an alternative agent for functional replacement of cofactor which strengthens enzymatic reaction.

EFFECT: creation of bispecific antibody which can replace functional proteins, strengthens enzymatic reaction.

14 cl, 18 dwg, 37 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

23 cl, 12 ex

FIELD: bioengineering.

SUBSTANCE: versions of the molecule binding CD45RO and CD45RB, and the anti-CD45RO and anti-CD45RB antibody are invented. In one of versions, the said molecule contains at least one antigen-binding site and includes the subsequently located hypervariable sites CDR1, CDR2 and CDR3. The molecule represents the humanised or monoclonal antibody. CDR1 has the amino acid sequence NYIIH, CDR2 has the amino acid sequence YFNPYNHGTKYNEKFKG and CDR3 has the amino acid sequence SGPYAWFDT. The molecule can additionally contain the subsequently located hypervariable sites CDR1', CDR2' and CDR3'. CDR1' has the amino acid sequence RASQNIGTSIQ, CDR2' has the amino acid sequence SSSESIS and CDR3' has the amino acid sequence QQSNTWPFT. In another version, the molecule contains both heavy and light chains where the amino acid sequences contain the corresponding CDR. The versions of the corresponding coding polynucleotide are disclosed; expression vector and based on it expression system. The host cell is disclosed basing on the expression system. The application of the molecule in treatment of autoimmune diseases, graft rejection, psoriasis, intestine inflammatory disease and allergy is described. The pharmaceutical composition for the said application is disclosed.

EFFECT: enables immunosuppressant induction; inhibiting T-cell response and primary lymphocyte response in mixed lymphocyte culture (MLC); prolongs survival period in mice with severe combined immunodeficiency SCID.

20 cl, 5 dwg, 2 tbl, 8 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.

EFFECT: valuable medicinal properties of plasmid DNA.

4 cl, 7 dwg, 6 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to a method for preparing protein conjugates. Method involves adding immunoglobulin or physiologically active polypeptide to a single end of nonpeptide polymer having two reactive terminal groups in the presence of reducing agent. From a prepared reaction mixture the complex containing nonpeptide polymer bound with immunoglobulin or physiologically active polypeptide is isolated. Then free reactive terminal group of nonpeptide polymer of the complex is added covalently to immunoglobulin or physiologically active polypeptide in the presence of reducing agent to obtain a protein conjugate. Protein conjugate containing physiologically active polypeptide bound, nonpeptide polymer and immunoglobulin that are added one to another covalently in indicated order is isolated. Invention provides preparing a protein conjugate useful for manufacturing a polypeptide drug owing to its enhanced stability in vivo, prolonged blood half-time index and decreased immunogenic properties.

EFFECT: valuable medicinal properties of conjugate, improved preparing method.

21 cl, 16 dwg, 14 tbl, 12 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.

EFFECT: valuable biological and medicinal properties of antibodies.

58 cl, 24 dwg, 6 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: obtained human antibody or its antigen-binding fragment specifically binds tumor necrosis factor hTNFα. The like antibodies show high affinity relative to hTNFα in vitro and in vivo. Antibodies according to the invention are taken as a full-length antibody or its antigen-binding fragment. The antibodies or their fragments are usable for detecting hTNFα and for inhibiting hTNFα activity in human beings suffering from a disorder in the case of which hTNFα activity is harmful.

EFFECT: wide range of applications of high affinity recombinant antibodies to hTNFα or their fragments of low dissociation kinetics.

15 cl, 11 dwg, 17 tbl

FIELD: immunology, antibodies.

SUBSTANCE: invention elates to human monoclonal antibodies to MN and antibody fragments to MN that are targeted to repeat sequence GEEDLP within proteoglycan domain. Binding with a desired epitope is confirmed by competitive immunoenzyme analysis method ELISA wherein ELISA signal is attenuated in combined incubation with peptide comprising this repeat sequence (PGEEDLPGEEDLP). Binding inhibition can be confirmed by the Biacore study also wherein binding required antibodies with immobilized MN or proteoglycan peptides can be inhibited by peptide repeat sequence. In addition to binding with human peptide repeat sequence anti-MN can inhibit adhesion of CGL-1 cells to plastic plates covered by MN. Human antibodies anti-MN can be used in treatment of cancer diseases or for diagnosis of cancer diseases wherein the level of MN is increased.

EFFECT: valuable medicinal properties of antibodies.

11 cl, 8 dwg, 2 tbl, 13 ex

FIELD: oncology and biotechnology.

SUBSTANCE: invention concerns conjugates used for treatment of malignant tumor. Conjugate includes staphylococcal or streptococcal wild-type superantigen or modified superantigen and antibody constituent. Bacterial superantigen is modified to reduce serum reactivity with preserved its antigenic activity. Amino acid sequence of superantigen incorporates A-E regions determining binding to TCR and MHC molecules class II. Invention is directed to preparing antitumor drug and also to preparing pharmaceutical composition.

EFFECT: use of the conjugate according to invention activate immune system and, therefore, resistance of mammalian against malignant tumor.

67 cl, 11 dwg, 1 tbl, 11 ex

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

FIELD: medicine, oncology, biochemistry.

SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.

EFFECT: valuable medicinal properties of protein complexes.

13 cl, 40 dwg, 18 ex

The invention relates to the field of immunobiotechnology and may find application in medicine

FIELD: biology; biotechnology.

SUBSTANCE: invention concerns biotechnology. It claims linking molecule represented by monoclonal antibody or its fragment capable of binding human NogoA polypeptide, human NogoA 623-640. Polynucleotide encoding the claimed molecule is presented. Expression vector including indicated polynucleotide is described. Host cell including indicated polynucleotide or vector is described. Pharmaceutical composition for treatment of diseases related to nerve reconstruction and containing indicated molecule is described.

EFFECT: obtainment of antibodies capable of binding NogoA 623-640.

9 cl, 1 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns medicine, particularly immunotherapy. Invention claims bispecific antibody and its application in tumour treatment. New antibody can link to EGF receptor and includes two antigene-binding sites linking to different epitopes of indicated receptor, thus ensuring higher receptor engagement by antibodies at the same dosage. It allows for higher antibody density on receptor and significant inhibition of tumour growth and/or enhancement of solid tumour or metastasis apoptosis.

EFFECT: higher receptor engagement by antibodies at the same dosage.

16 cl, 2 ex

FIELD: medicine; biotechnologies.

SUBSTANCE: anticoagulative antibody is more affinity fixed to FVIIa/TF complex, than to free TF. Discovered is pharmaceutical composition for thrombosis prevention and treatment, containing the described antibody. Offered is method of thrombosis prevention, consisting in introduction of therapeutically effective amount of the described antibody. Furthermore, offered is method of decrease and treatment of deep venous thrombosis (DVT), disseminated intravascular clotting (DIC), acute coronary artery thrombosis or cancer with coagulopathy signs in the patient, consisting that therapeutically effective amount of the described antibody is introduced to the patient. Discovered is method of inflammatory response regulation in the patient, consisting that therapeutically effective amount of the described antibody is introduced to the patient. Polynucleotide sequence encoding the described antibody is presented.

EFFECT: higher efficiency.

24 cl, 10 dwg, 9 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: claimed invention relates to field of biotechnology and immunology. Claimed is monoclonal antibody specific to human oculospanin, possessing cytotoxic activity with respect to human cancer cells. In one of versions antibody is obtained from mouse hybridome O3B8-2C9-4F3, FERM BP-08627. Described is set for cancer detection based on, at least, said antibody and second, specific to it, antibody. Described is pharmaceutical composition for treatment of cancer, whis cells express oculospanin, and application of antibody for manufacturing medication for treatment of cancer, whose cells express oculospanin. Use of invention ensures highly-specific antibodies to human oculospanin, which can find application for diagnostics of tumors, expressing human oculospanin.

EFFECT: ensuring highly-specific antibodies to human oculospanin, which can find application for diagnostics of tumors, expressing human oculospanin.

17 cl, 5 dwg, 10 ex

FIELD: medicine; bioengineering.

SUBSTANCE: are offered variants of antibodies, specifically recognizing two regions of peptide β-A4, characterised by amino-acid residual list. The first region of recognised peptide contains amino-acid sequence AEFRHDSGY or fragment thereof, while the second region contains amino-acid sequence VHHQKLVFFAEDVG or fragment thereof. Described are nucleic acid molecules coding molecules of antibodies, offered in invention, vectors and hosts containing specified nucleic acid molecules. Discovered are methods of antibodies production and optimisation, pharmaceutical compositions based on specified antibodies and method of production thereof, as well as antibodies-based set and various applications of antibodies. Invention application provides high-avidity antibodies of peptide β-A4 that can be applied for diagnostics of various peptide β-A4 mediated diseases.

EFFECT: efficient application of compositions.

29 cl, 15 dwg, 10 tbl, 16 ex

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