WO1999019361A1 - Bonding molecules against receptor-ligand-complexes - Google Patents

Abstract

The invention relates to bonding molecules against receptor-ligand-complexes which are obtained by immunization or immune selection with a receptor-ligand-complex, whereby the receptor and ligand are bound to one another by at least one covalent bonding, or one, two or three identical or different receptors and one, two or three identical or different ligands are bound to one another by at least one covalent bonding. In addition, the receptors are a protein or a protein which is modified by lipids, sugar, ribonucleic acid, phosphates, protoporhyrines, flavin adenine nucleotides or metals and the ligands are a protein, peptide, low-molecular synthetic, glycoside or a protein which is modified by lipids, sugar, ribonucleic acids, phosphates, protoporphyrine, flavin adenine nucleotides or metal.

Description

 description

B i nde o I ekü I e against Receptor-L i gand-Komp I exe

The use of B i ndemo I eku Ien against receptor-ligand complexes makes it possible to use structures that are selectively associated with a disease as a target for therapeutic agents.

The discovery of such structures has long been recognized by the pharmaceutical industry as a task. For example, many well-known phar aka are small molecule inhibitors of enzymes that are associated with certain pat home mechanics. Often the are inhibited

However, enzymes are neither selective for the disease nor overexpressed in the disease to be treated. This lack of selectivity often causes undesirable effects on normal tissues, which adversely affect the tolerance of the pharmaceutical (enzyme inhibitor).

Other drugs interact, for example, with receptor structures on the cell surface and inhibit the interaction of the receptor with its natural ligand (receptor antagonists). This prevents the transmission of a signal through the ligand via the receptor into the cell (blockage of the signal 11 transduction). Since the receptor / ligand interactions produced by the receptor antagonists in 11 and 11 just like the above-mentioned enzymes occur not only in diseased but also in healthy tissues, the use of receptor antagonists is also important mostly associated with adverse side effects.

It was therefore recognized that side effects would most likely not occur or would only occur to a very limited extent if the receptor antagonists I would inhibit it in the diseased tissue and measure the healthy tissue uneventfully.

Many attempts have been made to develop therapeutics which are intended to be selective for developing endothelia in vitro and in vivo. These suggestions can be found, for example, in some publications such as EP 0 696 456 A2, page 3, Senger et al, October 24 1994 USP 08 / 327,709, Rockwell et al, February 10 1994, WO 95/21868, Thorpe and Burrows, 1992, WO 93/17715.

In many cases, the potential therapeutic agents proposed there recognize antigens which are more strongly expressed on proliferating endothelium than on resting endothelium (Thorpe and Burrows, 1992 WO / 93/17715).

Antibodies known to date are specifically directed against a receptor on the cell surface or a ligand (for example EP 0 696 456; Mi llauer et al. (1993), Cell 72, 835-846) and thus influence a receptor or its ligands even when at rest (kovent i one II e receptor antagonists).

However, the binding molecules described in the present invention react with epitopes which are neither present on the receptor nor on the ligand aI I e i ne, but instead arise at the points at which a receptor interacts with its ligand. Binding molecules that are specifically directed against the receptor (s) ligand (s) complex I ex are selective for the receptor in the activated state and are therefore selective for the region of the tissue that migrates.

Suitable binding molecules include antibodies, antibody fragments (scFV, DABs, CRABs, diabodies, M inif I exant i bo-dies, M ini ant i od i es, tetravalent mono- or polyspecific antibodies), peptides, cyclic peptides, peptides i - metica and derived low-molecular ulare synthetics. The described (receptor (s) -L i gand (s) -comp I exe are target structures particularly suitable for therapeutic use for the reasons mentioned above.

The invention thus relates to binding demonstrations which are obtained by immunization or immunoselection with a receptor ligand copex, the receptor and ligand being linked to one another by at least one covalent bond, process for their manufacture as well as their use as pharmaceuticals or d i agnost iku. The invention further relates to nucleic acid chemistry which codes for the antibodies, vectors or expression vectors which contain these nucleic acids, and to animals, cells or cell lines by means of which the antibodies mentioned can be produced .

The invention relates to B i ndemo I ek ü I e such as Antibodies or antibody fragments which are characterized in that they bind specifically to the receptor ligand component I ex. A covalent bond between the receptor and ligand must be established for the production or immunization and immunization of the antibodies, whereas this is not necessary for the binding of the produced antibody to its epitope. An association of the receptor with its ligand through non-covalent interactions or stabilizing third molecules, e.g. This is sufficient on the cell membrane. It is also irrelevant for binding whether the receptor ligand compex is available in a soluble or menstrual form.

Further B i ndemo I ek ύ I e with specificity for a receptor complex can e.g. B. can also be obtained via the SELEX process (NeXstar Pharmaceuti ca I s Inc.). These B i ndemo I ek u I e are synthetic 0 I i gonuk I eot i de, so-called aptamers. A modification of these amptamers are the Spiegelmers (Nolte et al. (1996), Nature Biotechnology Igy 14, 1116-1119), which consist of nucleotides that do not occur naturally and therefore have a higher stability. The term receptor means the following structures:

Molecules on cell surfaces that can bind a ligand, or parts thereof that can bind a ligand. These are, for example, proteins, glycol ipide or proteins modified by lipids, sugars, ribonucleic acid, phosphates, protoporphyrins, F a i nnuk I eot i de or metals. Receptors are e.g. B. Tyros i nphosphok i nasen such as Fit 1, KDR, Tie, Tek. A summary of other suitable receptors is given in Table 1 and Appendix 1.

Table 1

CD number other names main main cellular tiie mein ligand furtcüσπ diitribution

CD 129 IL-9R gro t T * eeil », 64kDa 1 111--99 promotlng maασphages, activity for T ceU mcga aryoblaßtβ tunumra

CD 135 Flt3 Flk2 receptor tyiosinc CD 34 ceUs, 130-150 kDa F 3 / F kinate carcinoma cells ligand

The term "ligand" means the following structures:

Solve molecules that bind to a receptor. These are, for example, proteins, glycol ipides, peptides, glycopeptides, 0 I i gosacchar i de, ni edermo I ek u I are synthetics, glycosides or by lipids, sugars, ribonucleic acids, phosphates, protoporphyrins, FI av i naden i nnuk I eot i de or Metal l modified proteins. Ligands are e.g. B. VEGF, VEGF-B, VEGF-C, Interleukin 12, PIGF, TEKL-1, TEKL-2.

The covalent bond between receptor and ligand is preferably formed by a peptide or a bi-functional crosslinker. In the case of protein receptors and carbohydrate II proteins, other covalent bonds can also be used. Amino acid sequences can be used as peptide from 1 to 30 amino acids are used, e.g. B. the sequence (GI y * Ser) 3.

The covalent bond between receptor and ligand is produced, for example, in such a way that purified and / or unpurified receptor and ligand moecules with a bifunctional crosslinking agent such as Bis-Su I f osucc inii dy I suberate, N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccini idyl-4-azidobenzoate, p-nitrophenyl-2-diazo-3,3,3-trifluoro-propionate (Pierce, Rockford, I ll inois , USA) and then the covalently linked receptor-ligand complex is separated from the individual components.

Suitable immunogens or antigens are also recombinant fusion proteins, containing the sequences of a protein receptor, peptide - as a compound (linker) of receptor and ligand - and a protein or peptide.

Suitable antigens for the production of the antibodies according to the invention are also one, two or three identical or different receptors which can be covalently linked to one, two or three identical or different ligands. The following receptor ligand compexes can result:

Ri-Lj-Ri; R, -L ₂ -Rai Ri-Lj-R ,;

R _j -LrR.-L _j -R ,;

Rι- ι-R ₂ Lι-Rι; Rι- ι-Ra-L R ₂ ; R ^ -Ra-L ^; R ^ LL _j -R ,; R ₂ -L, -L, -R ₂ ; Rι-L, -L, -R ,.

R1 and R? stand for two different receptors and L1 and Lz stand for two different ligands; "-" stands for a covalent bond between receptor and ligand. The ligand can also be a homodimer or heterodimer.

Two identical receptors are preferably coupled with one ligand. Example swe ise binds a vascular endothelial growth factor (VEGF) -L i gand to two kinase do ain insert re ceptor (KDR) receptors. VEGF-binding domains are preferably bound to a VEGF ligand by a KDR receptor or an fms-like tyrosine kinase receptor (FI t receptor) as a soluble component. Further possible and preferred receptor ligand compexes include those described in Shawver et al, Drug Discovery Today, Vo I 2; 50-63, 1993 receptors and ligands described in Figures 4 and 5.

In addition, the complex of membrane proteins Fas antigen and tumor necrosis factor receptor (Yonehara et al., J. Exp. Med. 169, 1747-1756, 1989), which only occurs on cells that develop, can be found ) can be used as immunogen.

The invention further relates to the following DNA sequences of a single-chain antibody fragment (scFv), which represents the variable domains of a light (Vi) and heavy (V _H ) antibody chain connected via a linker (VH-Linker-Vι). The linker has the sequence:

GGA AGT TGC TCT GGC CGT GGC GGA TCG Gly Ser Gly Ser Gly Arg Gly Gly Ser

and mutant derivatives thereof

The DNA sequences 1-10 according to the invention code for scFV fragments which are directed against the sFLT / VEGF-Komp I ex (see example 1).

SEQ I D NO.1

5'- TGG CCC AGG TGA AGC TGT AGC AGT CAG GGG GAG AGC TTG TGA ΛGC

CAG GGG CCT CAG TCA AGT TGT CCT GCΛ CAG CTT CTG GCT TCA ACΛ TTA

AΛG ACT CCT ATA TGC ACT GGG TGA AGC ΛGΛ GGC CTG AAC AGG GCC TGG

AGT GGA TTG GAA GGA TTG ΛTC CTG CGA ΛTG GTΛ ATA CTA AAT ATG ACC CGA AGT TCC AGG GCA AGG CCΛ CCΛ TAΛ CAG CAG ACA CAT CCT CCA CAC

ΛGC CTA CCT GCA GCT CAG CAG CCT GAC ATC TTΛ GGA CAC TGC CGT CTA

TTA CTG TGC TAG ATG CTA TGG TΛΛ CTA CGT GTA TTA CTA TGC TAT GGA

CTA CTG GGG GGC AAA GGA ACA CGG TCA CCG TTC TCC CCC AAT TGA AGC

CGT TCA GGG GAA GTT GCT CTG GCC GTG GCG GAT CGG ACA TCG AGC TCA CTC AGT CTC CAG GGA AGT TGC TCT GGC CGT GGC GGA TCG GΛC ΛTC

GAG CTC ACT CAG TCT CCΛ GCΛ ΛTC ATG TCT GCΛ TCT CCΛ TGG GAG ΛΛG GTC ACC ΛTG ΛCC TGC AGT GCC TGC TCA ΛGT GTΛ ΛGT TΛC ATG CAC TGG TΛC CΛG CΛG ΛΛG TCΛ GGC ACC TC ATC AAΛ AGΛ CTG GCT TCT GGΛ TCC CΛG CTC GCT TCΛ GTG GCΛ GTG GΛT CTG GGA CCT CTT ΛCT CTC TCΛ CΛΛ TCΛ GCC GΛΛ TGG ΛGG CTG ΛΛG ΛTG CTG CCΛ CTT ATT ΛCT GCC AGC AAA GGA GTΛ GTT ACC CΛ TCG CTT TCT GGΛ ΛTΛ ΛΛΛ CGG GCG GCC GCA GΛΛ ΛCT GTT GΛΛ ΛGT TGT- 3 ^*

SE Q I D NO. 2

5'- ATG GCC CAG GTG AAA CTG CΛG GAG TCA GGA ACT ACC TTG TGA GTC CGT GGG CCG TGC TCT CΛT TTT CCT ACG GCT CAT CGG TCT TCA GΛC TGC

AGT AGG TCT GTG GAA CTG CCG TAA ACC ACA ΛCG CCT ACA GCA GGG TCT

GGΛ CTG GAA TTG ACC GAT TGA AGO CTG CAA CTG GTA TCC CTG GTT CTG

ΛTC CCA AGA TGC TGC GCT CCG ΛCΛ GTA CAA AAC CAA ACC CAT CCT CCA

ATA TCA ACG TAA CCG GΛC GGG TCA ACA GGA CCA ACG TCT CAG GΛC TCC TGC CGT CTA ATG CTG TTA CTG CAT GGΛ GΛT TGG TAC TTC CΛG TGT CTG

GΛT GCC AAC GGA CCΛ CGG TTA CCG TTT TCT TΛΛ TCT GGΛ GCG GTT

TCG GCG GGA GGT GGC TCT GGC GGT GGC GGA TCT GAC ΛTT GAG CTC

ACCCAGTCT CCΛ GCAATC ATGTCN GCA TCT CCA GGG GAG ΛAG GTC ACC

ATG ACC TGC AGT GNC AGC TCANGT GTA AGT TAC ATG CAC TGG TNC CAG

CΛG AAG TCA GGC ACT TCN CCC ΛΛΛ ΛTΛ TGG ΛTT TAT NAC ACΛ TCC ΛAN

CTG GCT TCT GGA GTC CCT GCT CGC TTC AGT GGC AGT GGΛ TCT GGG ΛCC

TCT TAC TCT CTC ACA ATC AGC NGC ATG GAG GCT GAΛ GAT GCT GCC ACT

TAT TAC TGC CAG CΛN NGG AGT AGT NAC CCN TAC ACG TTC GGN GGG GGG ACC AAGCTGGAGATG AAA CGG-3 '

or scFv with partially deleted V _H domaini SEQ ID NO.3

5'- GAA GCT GCA GGA TTT TGG GCC CΛA GGG ACA ACG TTC CCT TTT TCT TCΛ '

ΛGT GGA GGC GTT TCA GGC GGΛ GGT GGT TCT GGC GGT GGC GGΛ TCG

GΛC AAC TAG CTC ΛCT CAG TCT CCA GCA ATC ATG TCT GCA TCT CCA GGG

GAG AAG GTC ACC ATA ACC TGC ΛGT GCC ΛGC TCΛ ΛGT GTΛ ΛGT TΛC ΛTG

CAC TGG TTC CAG CAG ΛΛG CCA GGC ACT TCT CCC AAΛ CTC TGG ATT TAT

AGC ACA TCC AAC CTG GCT TCT GGA GTC CCT GCT CGC TTC AGT GGC AGT GGATCT GGG ACC TCT TAC TCT CTC ΛCΛ ΛTC ΛGC CGA ATG GAG GCT GΛΛ

GAT GCT GCC ACT TAT TΛC TGC CAG CAA AGG ΛGT ΛGT TΛC CCG CTC ΛCG

TTC GGT GCT GGC ACC AAG CTG GΛΛ ΛTC ΛΛΛ CGG GCG GCC GCA GAA ACT

GTTGΛAAGTTGTTTTTCA- 3 '

or scFv with partially deleted V _H domain:

SEQ ID NO.4

5 '- CGG TCG TGC CTT TCT ATG CGG CCC AGC CGG CCA TGG CCC AGG TGC AGC TGC AGG AGT CΛG GAC CAA GGG ACC ACG GTC ACC GTC TCC TCΛ GGT GGΛ GGG GGT TCA GGC GGA GGT GGC TCT GGC GGG GGCG GCT CΛC TCA GTC TCC AAC AAT CΛT GTC TGC ATC TCC ΛGG GGΛ GΛΛ GGT CAC CAT AΛC CTG CAG TGC CAG CTC AAG AGT AΛG TTA CΛT GCΛ CTG GTT CCA GCA GΛΛ GCC ΛGG CAC TTC TCC CATA ACT CTGACGT GGC TTC TGG AGT CCC TGC TCG CTT CAC TGG CAG TGG ATC TGG GAC CTC TTA CTC TCT CAC AAT CAG CCC GAA TGG ΛΛG CTG AAΛ ATG CTG CCA CTT ATT TAC TGC CAC. CTC GGG AΛT TAT TAT CCC CAA TGG TTC CCC TTT CGG AAG GGG GGΛ ΛCC AAC - 3 '

or scFv with partially deleted V _H domain: SEQ ID NO. 5

5 * - CCC CCC CTC TTT GGT TGG GCC TTG GAATTGTTG TTC CTT TCT ATG TTG CCC AGC CGG CCΛ TGG CCC AGC TGC ΛGC TGC ΛGC ΛGT CΛG GAC GGC CCT GCΛTCA TGG GGC AGC CGG GGΛ TGC TGC GCCGCCC AGG CGG TCA GGT GGA GGC GGT TCA GGC GGΛ GGT GGC TCT GGC GGT GGC GGG TCT GAT ΛTT GΛG CTC ACC CΛG TCT CCA GCA CTC ATG GCT GCA TCT CCΛ GGG GAG AAG GTC ACC ΛTC ACC TGC ΛGTGTT AGC ΛGTG AGC CAC TGG TAC CAG CAG AAG TCA TAA ACC TCC CCC ΛΛA CCC TGG ATT TAT GGC ΛCA TCC AAΛ CCT GGC TTT CTG GGA GTT CCC TGTTTC GCTTCC AGT GGC CATTGGAAT CTG GGG- 3 '

or scFv with partially deleted V _H domain:

SEQ ID NO.6

5 * - TGG AGG GGG GTG GGG CCA AGG GGA CCA CGG TTC ACC GTT TTC CTC ΛΛA GGG GAG GCG GGT TCA GGC GGA GGT GGT TCT GTC GGT GGC GGG ATC AGA CAT TGΛ GCT CΛC CCA GTC TCC AGC AG CΛ GGA ΛΛA AGG TCA CCA TGΛ CCT GCA GGG CCΛ GCT CAA GTG TAΛ GTT CCΛ GTT ACT TGC ACT GGT ΛCC ΛGC ΛGA AGT CAG GTG CCT CCC CCA AAC TGT GGA TTT ATA GCΛ CΛT CCA ACT TGG CTT CTG GAG TCC CTG GTC G GTG GGT CTG GGΛ CCT CTT ΛCT CTC TCA CAΛ TCA GCΛ GTG TGG ΛGG CTG AAG ATG CTG CCA CTT ATT ACT GCC AGC AGT ACA GTG GTT ΛCC AAC TCA CGT TCG GCT CGG GGA CAA AGT TGG AAA TAA AAC GΛG 'CCG

SEQ ID NO.7

5 ^* .- ATGGCCCAGGTGAAGCTGCAGGAGTCTGGC GCTGAGTTGGTGΛΛA CCTGGGGCTTCAGTG ΛAG ΛCATCCTGC AAGGCTTCT GGC AAC TTC TA _C ΛTTGΛC CAT GTT ATT CAC TGGATA AAGGAG AAGCCT GAACAG C _GG CTG GAATGGATT GGATAT ΛTΛTCT CCC AAG GGC GGΛΛΛT GGTGATATTΛΛGTΛCΛΛT GAGAATTTC ΛAGGCTACACTGACT GCΛΛAC ΛAΛTCC TCC AAC ΛCT GCCTAC ATGCAGCTC ΛΛC AGC CAC ATCTGAAGATTCTGC ΛGT GTΛ TTTTTGTAGGGATTACTACGGTAGGGCTAGGGACTACTGGGGCCΛΛGG GAC CΛC GGT CAC CGT CTC CTT CAGGTGGAG GCGGTTTCAGGC GGAGGT GGCTCT GGGCGGTGG CGG ATC GGΛCΛT CGAGCT CAC TCΛGTCTCC ΛGC 5 TTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCΛC CΛT CTC CTGCΛGAGC CAGCNGAAGTGTTGATAATTATGGCATTAGTTTTAT GAACTGGTT CCA ΛCAGAAACC AGGACA GCC ACC CAAACTCCT CAT CTATGC TGC ATC CΛA CCAAGG ATCCGGGGT CCC TGC CΛGGTTTAGTGGCAGTGGGTCTGGGAC AGACTTCAGCCT CAACAT CCATCCTAT GGAGGAGGATGΛTΛC TGC ΛΛT ° GTATTT CTGTCAGCAAAGTAΛGGAGGTTCCTTGGAC GTT CGGTGG AGG GAC GΛGCTA

5 SEQ ID NO.8

5 '- ATGGCC CAGGTC AAGCTC AGC AGT CAT GAC CTG AACTAGTGAAGC CTGGGGCTTCGGTGAAGATGT CCT GCAAGG CTT CTGGATACACAT TCΛ ° CTGACT ATGTTATAT CTT GGGTGAAGC AGAGAACTGGAC AGG GCC TTG AGT GGATTG GAGAGATTTATC CTG GΛΛGTGTTAATACTT ACT ACΛGTG AGAAGT TCAAGGACAAGGTCACAC TGACTGCΛGACAAAT CCT CCAΛCA CAGCCT ACATGC AGCTCAGCA GCCTGACAT CTGCGGACT CTGCGGTCT ATTTCT GTG CGAGAGGAGTGTACT ACGGGT ACTTCGATGTCT GGG GCC 5 AAGGCACCACGGTCACCGTCT CCTCAGGTGGΛΛGCGGTT CAG GCGGAG GTGGCT CTG GCGGTGGCGGAT CGGACΛTCGAGCTCACTCAGT CTC CΛG CAATCATGT CTGCAT CTCTAGGGΛΛGAAGATCACCC TAACCT GCAGTG CCAGCTCGAGTGTAA GTTACATGC ACT GGT ACC AGC AGΛΛGT CΛGGCΛ 0 CTTCTC CCAAACTCTTGATTT ATAGCACAT CCΛACC TGGCTT CTG GAG TCC CTT CTC GCTTCAGTGGCAGTGGGT CTGGGΛCCTTTT ATT CTC TCΛ CAATCAGCA GCATGGAGGCTGAAGΛTGCTGCCACTTATTACT GTC AAC AGT GGAGTAGTT ACC CGCTCACGTTCGGTG CTGGGΛCΛΛ ΛGTTGG AAA TΛAAΛCGG-3 '5 SE QID NO. 9

5 - ATGGCC CAGGTGAAG CTGCGGAGTCTGGAGGAGGCTTGGTGC AAC CTGGGGGGTCΛCGGGGΛCTCTCTTGTGΛΛGGCTCΛGGGTTTACTTTTA 5 GTGGCTTCT GGATGAGCT GGGTTC GAC AGACAC CTGGGAAGACCC TGG ΛGTGGΛTTGGΛGACΛTTΛATTCTGATGGCAGTGCAATAA ACTACGCΛC CAT CCATAAAGG ATC GATTCACTATCTTCAGAG ΛCAΛTG ΛCΛΛGΛGCΛ CCCTGT ACCTGC AGATGAGCΛΛTGTGCGAT CTGAGGΛCΛCΛGCCACGT ATTTCTGTATGAGAT ATGATGGTTACTACTGGT ACTTCGΛTGTCT GGG ° GCCΛAGGGΛCCACGGTCACCGTCT CCT CAGGGGAAGCTGTTCAGGCGG ΛΛGTGGCTCTGG CGGTGGCGGATC GGACATTCACCT GΛC CCAGTC TCC ΛTCCTC CAT GTATGC ATC GCT GGG ΛGAGAGΛGTCΛC TAT CΛCTTG CAA GGC GAGTCAGGACATTAAAΛGCTATTT AAGCTG GTACCAGCAGΛΛ ΛCC ΛTG GAAATC TCCTAΛGAC CCT GAT CTATTATGC AΛC AAGCTT GGC TGG GGT CCC ATC ΛGΛ 5 AΛGΛTT CΛGTGGCAGTGGATCTGG GCAΛGATTATTC TCT AAC CAT CCT CAGCAG GGAGTCTGACGATACΛGC ΛΛC TTΛTTΛCTG TCTACAGCATGGTGΛGΛG CCC GCT CAC GTT GAC CGGTGCTGG CAAGCT GGΛGCT ACGG- GAA 3 '0

or scFv with partially deleted V _H domain:

5 SEQ ID 10

5 'ATGGCC CAGGTC CAGCTGCAGCAGTCAGGGGCC ΛΛGGGΛCCACGG

TCACCGTCTCCTCAGTTGGAGGCGGTTCAGGCGGAGGTGGCT CTG GCG

GTGGCGGAT CGGACATTGAGCTCACCCAGT CTC CAACAATCΛTGT CTA ° CATCTCCAGGGGAGAΛGGTCΛCCATGACCTGCΛGTGCCAGCTCAAGTG

TAΛATT ACATGT ACT GGT ACC AGC ΛGΛAGC CΛGAAT CCT CCC CCAGAC

TCCTGATTTATGACACAT CCΛΛCCTGGCTTCTGGΛGTCCCTGTTCGCT

TCΛGTGGCAGTGGGT CTGGGACCT CTT ACT CTCTCA CAATCAGCC GΛΛ

TGGΛGGCTGAAGATG CTGCCACTTΛTTACT GCCAGCAGTGGAGTAGTT 5

ΛCC CGCTCACGTTCGGTGCTGGGACCΛAGC TGG AAATAAΛΛC GG-3 ' The invention also relates to functionally equivalent variants of SEQ ID NO. 1 -10. The term "functionally equivalent variants" stands for modifications of the DNA sequences and their corresponding amino acid sequences, which stand for the light and / or heavy antibody chain and which bind to the receptor ligand component, but essentially not to the receptor or ligand alone, and their correspondingly formed antibodies, in comparison with the antibodies coded by SEQ ID NO.1-10, has a similar affinity for the receptor ligand component I ex. The affinity for sFLT-VEGF receptor-L-i gand Comp ex I (see. Example 1 p 18) determined by Scatchard Ana I ysis is from 10 ³ I / mol to 10- ¹⁵ / mol, preferably from 10 - ⁷ I / mol to 10- ^Z I / mol.

Another object of the invention is a molecular construct consisting of the amino acid chain which is encoded by SEQ ID NO.1-10 or its analogs and further amino acid chains linked to the sequence mentioned, these amino acid chains being different from the inventions Sequences and preferably parts of a human

Antibodies are, for example, parts of the constant areas of the heavy and light chains. Other molecular constructs are, for example, "single domain" fragments or "single chain" fragments.

The present invention also includes humanized monoclonal antibodies (humMAK) or functionally active parts thereof which contain the light antibody chains and heavy antibody chains according to the invention. Functionally active parts thereof are, for example, F (ab) or F (ab ') 2 fragments with one or more hanging regions. Functionally equivalent variants are also the complementary determination regions (CDRs) of the antibody sequences, and peptides or mimetics derived from the CDRs.

Furthermore, the molecular constructs according to the invention or the humMAK effector or reporter contain molecules, for example a chelate for complexation with metal ions (eg EDTA, DTPA), heterologous toxic enzymes (eg ricin), a second binding region of other specificity or with kata I yt i see properties and / or common or toxic or non-toxic enzyme derived from non-human primates. The constructs according to the invention or the humMAK according to the invention can also be used, for example, according to the method of Schwarz (Schwarz, A. & Steinstasse, A. (1987), A novel approach to Tc-99 labeled monoclonal antibodies. J. Nuc I. Med., 28, 721) with Tc-99m or according to generally known methods with Re or Y. The marking with Tc-99m is preferred for the diagnostic area.

The invention generally also includes any other marking with alpha, beta or gamma rays. The sequences according to the invention can be linked with any amino acid residues, depending on the use, either chemically or with the help of genetic engineering methods by chemically combining the sequence according to the invention or the heavy or light chains with the molecule to be linked according to generally known methods or be linked by genetic engineering. The sequences according to the invention themselves can also be produced chemically or by genetic engineering. In the event that the humMAK does not consist of an intact antibody molecule, but of a Fab or F (a ') 2 fragment or scFv, the genes for the heavy chains are lost when the Fc part of the antibody is removed - The passed 3 'regulation sequences (stop codon, 3' not to the area and Po I yA Add 11 i onss i gna I) by the C3 exon. the 3 'non-translucent region and the poly-A additio n s i gna I of a human MHC class I gene, preferably an HLA B27 gene, are replaced. The sequence according to the invention can in turn be produced either chemically or by genetic engineering using methods known to those skilled in the art. The DNA sequences which code for the light and heavy chains consist either of genomic sequences or of cDNA sequences or of a mixture of both. The DNA sequences mentioned preferably consist at least partially of genomic DNA.

The host cell which is used for the expression of the humMAK described in this invention is a prokaryotic or a eukaryotic cell, in particular Escherichia coli, a yeast such as Saccharomyces or CHO-ZeI - I en.

Further subjects of the present invention are therefore also cloning and expression vectors and transfected cells which contain the sequences according to the invention or their functional equivalents. In addition, the invention encompasses therapeutic and diagnostic formulations which contain the antibody sequences or sequences or mimetics derived therefrom and their production and the use of such compostments in the therapy and diagnosis of preferably inflammation, solid tumors, in particular breast cancer, gastric cancer, prostate cancer , Lung carcinoma, colon carcinoma, pancreatic carcinoma, Kapos isarcoma etc., as well as non-solid tumors such as leukemia etc.

The general methods with which the vectors can be constructed, the transformation technology and the culture technology are known to the person skilled in the art and are known, for example, from Maniatis (Sambrook, Fritsch, Maniatis; Molecular Cloning, A Laboratory Manual, Second Edition; Cold Spring Harbor Press, S 16.2-16.22, 16.30-16.40, 16.54-16.55, (1989)).

To produce the antibodies according to the invention, the procedure is, for example, that the covalently bound receptor ligand component I ex as such or in conjunction with an adjuvant for immunization or the complex for immune Selection of V regions of the naive or otherwise generated antibody gene banks.

A vertebrate is immunized with the receptor-ligand complex I ex or with the carier-bound receptor-ligand complex in a manner known from later literature, for example by subcutaneous or intaperial injection of the immunogen, if necessary with an adjuvant such as KFA (complete Freund's adjuvant) or IFA (incomplete Freund's adjuvant) or Qu i I A. If necessary, to increase the immune response one or more times " be boosted ". The selection of the species is not critical, for example mice, rats, rabbits, monkeys (Macaca f asc i cu I ar i s), sheep, goats or camels I e are suitable.

As an alternative to the method of obtaining polyclonal antibodies described above, monoclonal antibodies can of course also be produced. This is done, for example, by immunizing mice, as described above, and then fusion of the mice, for example with NS 1 myeloma cells and cloning of suitable cells. If necessary, the monoclonal antibodies obtained in this way can be multiplied, for example, by injecting the antibody-producing cells into nude mice. In principle, the production of such monoclonal antibodies is known to the person skilled in the art and is described in the literature.

As an alternative, mRNA can also be isolated from the malt cells in the unified animal, then produced using reverse transcriptase cDNA and, after amplification if by means of PCR (polymerase chain reaction), cloning takes place of antibody genes in a suitable host. By means of immunization using an immobilized receptor ligand component, host cells are selected which only bind to the receptor ligand component. The appropriate antibodies are then consumed in a suitable host cell. pe r ex primie rt.

Alternatively, a naive human, murine or primate V library can be prepared from mRNA from peripheral B cells in the phage library system and suitable antibodies can be isolated from it by immunoselection.

As an alternative to the above method, amp cli cation is preferably cloned into a phage library using PCR. The antibody fragments expressed on the phage surface are selected with the immobilized receptor ligand component I ex. Screening identifies phage clones that recognize the complex immobilized on a solid phase (positive clones). These positive clones are tested in a second screening for binding to the individual components of the complex. Clones that are positive in the 1st screening and negative in the 2nd screening are processed further. Genes of the clones selected in this way are subcloned into suitable vectors and expressed in a suitable host.

The invention further relates to antibody (Ak) enzyme fusion proteins, for example Ak-RNase (Saxena, SK et al., J Biol. Chem. 267: 21982-6, 1992), Ak-DNase I (Kreuder V et al. (1984), Eur. J. Biochem. 139, 389-400), Ak-Onconase

(Wu, Y. et al. J Biol Chem. 268: 10686-93, 1993), Ak-Perforin (Tschopp, J. & Nabholz, M. Annu. Rev. Immunol. 8: 279-302, 1990) or Ak-TIA (Tian et al 1991, Ce II 67, 629-39), where the enzymes are attached to the 3 'end of the antibody. Other fusion proteins consist of Ak and one

Activator molecule of coagulation, e.g. B. tPA (tissue plas inogen activator) or from Ak and a molecule of the coagulation cascade, for. B. tissue factor (TF) or truncated tissue factor (WO 96/016533, 1994). Another enzyme with a cytotoxic function is recombinant mistletoe lectin (M. Langer et al., 1997. EJC Vol. 33, Suppl. 5, 49). Cell lysis can be achieved by coupling antibodies to e.g. B. Lymphotσx in (Aggarval et al. (1984) J. Biol. Chem. 259, 686-691) or to a leukalexin (e.g. that described by Liu et al. (Liu et al. (1987) Ce l I 51, 393-403)). The antibodies can also be coupled to toxins, e.g. B. the recombinant Pseudomonas exotoxin A (ETA '; Barth et al. 1997. EJC Vol.33. Supp I.5, S22, 39).

These fusion proteins can also be produced by subcloning the scFv in pDN22 (pUC119-Der i vat) via Sf i I and Not l -Rest r i t i onsitte ntte I I en (D. Neri, personal communication).

This vector allows the expression of scFv with C-termina-

I a cysteine. The cysteine can be used for chemical

Modification of antibody fragments with t h io I spec i f i see substances, e.g. B. for coupling to a second protein via a D i su I fi bridge, or with a couplable Com p I ex d i l dner.

Furthermore, the antibodies can be subcloned on the antibody gene in pDN 268 (Neri et al. (1996), Nature Biotechno-

I oqy 14, 4, 485-490) and expression with ³² P are radioactively labeled. The vector pDN268 contains the recognition sequence for the human casein kinase II at the C-terminal as a phosphorization sequence. A variant is the production of scFv with a radioactively markable peptide id. ³² P is used for diagnosis and therapy.

The introduction of several cysteines into the peptides sequence allows radioactive labeling with ^99m Tc. An example of the linker sequence is the hinge region from the mouse I gG 1 molecule, or homologous human Ig hinge regions, which can be engineered as ant i para II e I peptide sequences.

Antibodies can also be produced, for example in single chain diabody format. Here, an antibody fragment with the specificity described above (hereinafter referred to as fragment A) linked to a second antibody fragment (fragment B). The second fragment is specifically directed against a factor from the coagulation cascade (e.g. C1q) or a surface molecule of T cells (CD8, CD24) or TF or TPA or TNF or another effector molecule. The first and second fragments are consecutively called scFv fragments (VH domain with the VL domain of the other fragment, e.g. V _H B with V _L A and V _H A with VB, with a 5 AS long left) a chain is cloned and connected via a 15 AS long left. Expression as a chain results in higher stability and higher yields.

example 1

Production of antibodies specific for an sFLT-VEGF-Komp I ex

VEGF (vascular endothelial growth factor) is a growth factor (ligand) which is secreted by tumor cells, activated macrophages, activated granulocytes and transformed synoviocytes and which is highly selective at two receptors (human. On the endothelium cytoplasmic membrane) : KDR / Flt-1, mouse: Flk) binds. The binding of VEGF as a homodimer to KDR (VEGF receptor) leads to receptor dimerization on the cytoplasmic membrane.

For the isolation and purification of functional receptors, the extracellular (VEGF-binding) domains of Fit are expressed as soluble receptors (sFIt = soluble fit) and isolated as functionally active molecules (Kendall, RL & Thomas, KA 1993. Proc. Nat I. Acad. Sci. USA 90, 10705-10709; Kendall, RL: et al. 1994, Biochem. Biophys. Res. Commun. 201, 326-330).

84 μg sFIt are incubated with 1.94 μg VEGF in PBS pH 7.4 for two hours at room temperature. Then the crosslinker BS ³ (B i s-Su I f osucc inimi dy I suberat) was added in a final concentration of 1 M and incubated for 15 min at room temperature. The reaction is stopped with 1 M Tris-HCl, pH 7.4 (final concentration 5mM). Free crosslinker and uncomplexed VEGF are removed from the solution by U 11 r af i 11 r ation (filter pore size: 30kD) and the solution is concentrated to a volume suitable for immunization.

After immunization of Balb / c mice with 3 × 25 μg of the VEGF-sF I t-Komp I exes in Qu i IA, mRNA is isolated from the cells of the immunized animals (E. Hornes S. L. Korsnes, 1990, Genet. Anan. Tech. App I.7: 145-150). Subsequently, cDNA is synthesized with the reverse transcriptase (Sambrook, Fritsch, Maniatis 1990; Molecular Cloning, A Laboratory Manual, Second Edition; Cold Spring Harbor Press). Amp I ification by means of PCR using suitable pnier, followed by cloning using the vector pCANTAB 5E (Cambridge Ant i body Technology; Pharmacia Biotech Europe GmbH; Freiburg; Recombinat Phage Ant i body System) and the helper phage M13K07 (Vieira, J. & Messing, J., Methods in Enzymol. 153, 3, 1987) in Escheπchia col i (E. co I i) leads to a phage gene bank which contains the V region genes from the cells of the above immunized mice in the scFv format as fusion genes with the gene MI (Hü II prote i ne) of the phage. Phage that only react with the sFLT-VEGF-Komp I ex is selected by means of immune effect using sFLT-VEGF-Komp I ex immobilized on the solid phase. For this purpose, the phages and / or soluble antibody fragments are tested in an ELISA assay for binding to the immobilized VEGF-sFLT-Komp I ex. Binding antibodies are tested for binding against the receptor (sFLT) or ligand (VEGF) alone. (Phage) antibodies that are negative in the second test are processed further. In the Western blot, these antibodies show no binding to VEGF.sFLT or sKDR alone. The so i mmunse I ect i erten specific

Phages are propagated in E.col i by co-infection with the helper phage. The one on the surface of the phage clones expressed scFv are produced by infection of E.col i HB2151 as soluble antibodies.

Claims

claims

1. Binding molecules against receptor ligand component, characterized in that they are bound by immunization or immunoselection with a receptor ligand component, where receptor and ligand are bound together by at least one covalent bond. who are connected.

2. Binding molecules according to claim 1, characterized in that they are obtained by immunization or by immunoselection with a receptor ligand component I ex, one, two or three identical or different receptors and one, two or three identical or different ligands are connected to one another by at least one covalent bond.

3. Binding molecules according to claim 1 or 2, characterized in that the receptors are a protein or a protein modified by lipids, sugars, ribonucleic acid, phosphates, protoporphyrins, F i a i naden i nnuk I eot i de or metals.

4. Binding molecules according to claim 1 or 2, characterized in that the ligands are a protein, peptide, low molecular weight synthetic, glycoside, or a by lipids, sugars, ribonucleic acids, phosphates, protoporphyrin, FI ai naden i nnuk I eot i de or metal are modified protein.

5. Binding molecules according to claim 1 or 2, characterized in that at least one of the receptors is an unmodified or modified part of a naturally occurring receptor.

6. binding mole üle according to claim 1 or 2, characterized in that at least one of the ligands is an unmodified or modified part of a naturally occurring ligand or a low molecular weight synthetics.

7. binding molecules according to claim 1 or 2, characterized in that they bind only to the receptor-ligand complex, but not to the receptor and / or the ligand alone.

8. binding molecules according to one or more of claims 1 to 7, characterized in that at least one of the receptors a K i nase-do aini nsert-conta ini ng receptor (KDR), fms-l ike tyrosine kinase receptor (Fit) soluble K i nase-doma ini nsert-conta ini ng receptor (sKDR) or a soluble fms-like tyrosine kinase receptor (sFIt).

9. binding molecules according to at least one of claims 1 to 7, characterized in that at least one of the

Ligands is a vaseuiar endothelial growth factor (VEGF).

10. Binding molecules according to at least one of claims 1 to 7, characterized in that the covalent bond is formed by B i s-Su I fosucc i n i m i dy I suberat.

11. Binding molecules according to at least one of claims 1 to 7, characterized in that they are obtained by recombinant methods.

12. Binding molecules according to at least one of claims 1 to 7, characterized in that at least one is a chic, humanized, bi- or o I i gospez i f i ce binding o i ek u I.

13. Binding molecules according to at least one of claims 1 to 7, characterized in that they with the natural The receptor / ligand complex reacted to cells or in solution.

14. Binding molecules according to at least one of claims 1 to 13, characterized in that they have one

Animal, a cell or a cell can be produced.

15. A method for producing an antibody according to one or more of claims 1 to 14 by

a) immunizing an immunocompetent vertebrate with a receptor ligand compex and then isolating the antibody from the serum,

or

b) immunizing an immunocompetent vertebrate with a receptor ligand compex, isolating the immune cells from the vertebrate and immortalization by fusion with myeloma cells and isolating the appropriate specific hybrid domains,

or

c) by isolating the mRNA from malt cells from animals immunized according to teaching b), then connecting cDNA synthesis, amplifying the cDNA by means of PCR, cloning the amplified sequences into a suitable host and immunoselecting the Cells that only bind to the receptor-ligand complex with the help of an immobilized receptor-ligand complex, and then expressing the antibodies in a suitable host cell.

16. A method for producing a binding mole oil according to one or more of claims 1-15 using a phage library, which is derived from the mRNA of spleen cells the animals immunized with the receptor ligand comp I ex.

17. Binding molecule according to one or more of claims 1-16, which is wholly or partly by a polynucleotide with the sequence ID no. 1,2,7,8 or 9 is encoded for an antibody fragment from V regions of the heavy or light antibody chains (scFv).

18. Binding molecules according to one or more of claims 1-16, which is wholly or partly by a polynucleotide with the sequence ID No. 3,4,5,6 or 10 for an antibody fragment from V regions of the heavy or light antibody chains with partially deleted VH domain (scFv) is coded.

19. The binding molecule according to claim 19 or 20, which is partially or completely encoded by a polyunucleotide which contains between 80 and 99.9% sequence homology to the polynucleotides no. 1-10.

20. A method for producing a binding molecule according to claim 17-19 using a cloning or expression vector or a host cell which one of the sequences No. 1 - 10 or functional equivalents thereof.