DE4341471A1 - New small peptide(s) that inhibit tumour necrosis factor - Google Patents

Abstract

Peptides and their derivs. (I) that are complementary determining regions (CDR) are (a) of sequence. ITCTASQSVSNDVVW (1) or GGITCTASQSVSNDVVWGG (2) (b) allelic variations of (1) or (2) with at least one amino acid (AA) substitution, deletion or addn. or (c) post-translational modifications of (1), (2) or their allelic variations, provided that in (b) and (c) the peptide retains CDR activity. Also new are (I) of formula R1-Ao-A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-R2 (3) A0 and A16 = bond or GG; A1, A9, A13 and A14 = I , L or V; A2, A4, A6, A8 and A10 = T or S; A3 = C or S; A5 = A, G, or S; A7 = Q or N; A11= Q, N or H; A212 = D or E; A15 = W or Y; R1 = H, amino, protecting gp. or at least one additional AA; R2 = OH, carboxy protecting gp. or at least one additional AA; 1-3 of A1 to A15 may be replaced by bonds.

Description

The invention relates to peptides or peptide derivatives (DCRs = Complementarity determining regions) specific to the human tumor necrosis factor alpha (TNF α) or its receptors bind. Furthermore, the Invention also the use of these CDRs as TNF α-antagonists or Diagnostics for TNF α.

State of the art

The human tumor necrosis factor-alpha (TNF α) is a protein from the Group of cytokines. He becomes primarily in response to Gewebeschädigun gen, bacterial endotoxins, viruses and other cytokines from activated macro produced phages. He is a major regulator in the immune system and in inflammatory processes, by preventing the organism from tissue degradation and Protects infections. Furthermore, he promotes the reconstruction of damaged Tissue. TNFα has been described by E. CARSWELL et al. (1975) Proc Natl Acad Sci USA 72: 3666-3700 discovered in serum from mice. There shows the factor in certain tumors cytostatic and cytotoxic effects, the one Function as an endogenous tumoricidal factor.

The structure of human TNF α and the expression of TNF α coding DNA is described in the publications of PENNICA et al. (1984) Nature 312: 724-729; by MARMENOUT et al. (1985) Europ J Biochem 152: 515-522; by WANG et al. (1985) Science 228: 149-154; and by SHIRAI et al. (1985) Nature 313: 803-806.

In addition to the protective functions, TNF is α in a variety of patholo involved in genetic processes in which an elevated level of TNF .alpha causes life-threatening conditions. So, TNF α plays at following sick An essential role plays: septic shock, cachexia, cerebral Malaria, graft-versus-host reaction (graft versus host reaction), Arthri tis, premature birth, osteoporosis and multiple sclerosis. (For an overview, see informative: BEUTLER and CERAMI (1989) Ann Rev Immunol 7: 625-655; and BONAVISTA and GRANGER (eds) (1990) "Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy ", Kerger, Basel).  

Since TNFα is involved in a variety of pathogenic conditions, TNFα antagonists are of great therapeutic importance. TNF α acts on its target cells via two 55 kD and 75 kD membrane receptors (kD = kilodalton = molecular mass). (HOHMANN et al. (1989) J Biol Chem 264: 14927-14934)
Little is known about the mechanisms of signal transmission in the cell interior. The previous attempts to inhibit the effect of TNF α concentrate on blocking the interaction of TNF α and its receptors. This can be done either via binding of an inhibitor to TNF α or to the receptors. There are examples for both strategies. The inhibition of the toxic effects of TNFα is achieved by binding non-toxic TNFα mutants and monoclonal antibodies to the TNFα receptors. (J.-I. YAMAGlSHI et al. (1990) Prot Engin 3: 713-719 and T. ESPEVIK et al. (1990) J Exp Med 171: 415-426)
The TNF α mutants and the monoclonal antibodies are relatively large molecules and therefore only of limited use for therapeutic use because they are difficult to penetrate tissue and also have a high biological half-life. Antibodies continue to form disturbing immune complexes through their Fc portion. Also occur anti-idiotype antibodies. Cross-reactions with "self" structures can lead to autoimmune disease.

Smaller molecules such as antibody fragments or peptides are therefore therapeutic far better suited (R. SUTHERLAND et al. (1987) Cancer Res 47: 1627-1633 and M. WITLOW and D. FIPULA (1991) Methods 2: 97-105).

In addition, effective low molecular weight TNF α antagonists are not known.

task

The object of the invention is the preparation and the use of CDRs or their Derivatives that do not belong to the group of TNF α mutants. In particular are small molecules of interest that are easy to prepare and have good compatibility for humans or animals. An effect as an antigen should be excluded as completely as possible.  

solution

The problem is solved by peptides or peptide derivatives (CDRs),

• a) which have the following sequence:
  Ile Thr Cys Thr Ala Ser Gln Ser Val Ser Asn Asp Val Val Trp
  (ITCTASQSVSNDVVW in one-letter code)
  (see Sequence Identifier No. 1 (Sequence Protocol No. 1))

or

• b) which as active CDRs allelic modifications of the previously under a) have mentioned amino acid sequence, wherein at least one Amino acid of the amino acid sequence is substituted, deleted or inserted, without significantly influencing the activity of the active CDRs,

or

• c) which as active CDRs posttranslational modifications of the Sequences under a) and b), wherein the modifications are not significantly affect the activity of active CDRs.

definitions

The abbreviations used in the text are determined by the rules that by the IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 11: 1726 (1972) and Biochem J. 219: 345 (1984)). The following common abbreviations are used: Ala = alanine; Arg = arginine; Asn = Cys = cysteine; Gln = glutamine; Gly = glycine; His = histidine; Ile = Iso leucine; Leu = leucine; Lys = lysine; Met = methionine; Phe = phenylalanine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Tyr = tyrosine and Val = Valine.

The protecting group of the radical R₁ may consist of:
Alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl groups having 1 to 10 carbon atoms, preferred are naphthoyl, Naphthylacetyl-, Naphthylpropionyl-, benzoyl or acyl group having 1 to 7 carbon atoms.

The protecting groups of the radical R₂ may consist of:
an alkoxy or aryloxy group having 1 to 10 carbon atoms or an amino group.

Further protecting groups - both for R₁ and R₂ - are in Houben-Weyl (1974) Georg Thieme Verlag, 4th edition described. The description of the protection groups in the cited reference is part of the disclosure.

The sequence of the peptide or peptide derivative according to the invention can be terminal and / or C-terminal end in place of a protecting group with wide ren frame amino acid sequences. This further frame Men amino acid sequences are for the binding of the invention Peptide or peptide derivatives are not essential, but they can be carriers of be other functions, such as chelates or cytostatic or include cytotoxic sequences. Such framework amino acid sequences zen occur in nature. It may be, for example, the between the variable region sequences arranged in the hypervariable regions an antibody. These sequences are called "Fram Work" (Frame sequences). As frame amino acid sequences are still not cleaved signal sequences of a secreted eukaryotic protein, wherein the protein is in a bacterium is expressed. Such signal sequences sometimes have no influence the function of the subsequent protein. It is also possible fiction, invention to couple corresponding peptides or peptide derivatives one behind the other, wherein Rah men amino acid sequences are arranged between the individual sequences.

To decide in a particular case, whether a particular inventive peptide or peptide derivative having at least one frame amino acid sequence and / or at least one protective group to the subject of the invention counts, is a comparison between

• (i) this peptide or peptide derivative with frame amino acid Se and / or with protective group and
• (ii) the same peptide without frame amino acid sequence and without protecting group

to hire. Both molecules should be essentially the same functio in the binding (see Example 2.1.) and the inhibition (see Example 2.2.). respectively.

The listed amino acids are L-amino acids.  

Allelic modifications

Most deletions, insertions, and substitutions do not seem to go through significant change in the characteristics of the peptides of the invention or To result in peptide derivatives. Since it is hard to get the exact effect of a Substitution, deletion or insertion must be indicated in advance Function of the modified CDR with the function of the CDR invention Sequence can be compared. In the examples are to be used for this purpose Methods indicated. This information is not exhaustive. As standard the CDR serves with the Seq Id No 1.

Amino acids can be substituted as shown in Table 1 without while significantly influencing the function of the CDRs. In each one Case is to be decided by the activity test, which influence the Verän to the function of the protein.

Usual substitution of amino acids in a peptide Original amino acid for example, made substitution Ala Gly, Ser bad Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala, Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu mead Leu, Tyr, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

The functions or the immunological identity are significantly changed, if substituents are chosen that are less conservative than those in Table 1 are shown amino acids. Let such significant changes to achieve substitutions with amino acids that are more in their own Structure and differ in the functional groups. basics Changes have the effect that the three-dimensional Structure is changed and / or that, for example, the leaflet structure or the helical structure is affected. Also interactions of charges and the hydrophobic chains are to be considered in the changes.

The mutations are compared by the homology (similarity) of two defined CDRs. The term homology includes similar ami nosäuren (for example Table 1) and gaps in the sequences of the Ami nosäuren (homology = similarity). The CDRs of the invention have Amino acid sequences having homology (see Seq. Id. No .: 1) have at least 13 amino acids, preferably 14 amino acid.

Posttranslational modifications

The aforementioned post-translational modifications are understood Changes that can occur after translation. These include the Glycosylation, the formation of disulfide bridges, the chemical modifiers tions of the amino acids, such as the sulfation, which together described with the hirudin. (J.W. FENTON (1989) "Thrombin Inter Actions with Hirudin ", Seminars in Thrombosis and Hemostasis 15: 265-268) Furthermore, the amino acids can be changed as it is in the inter National Publication WO 91/10684.

advantages

The peptides or peptide derivatives of the invention have compared to her conventional TNF α binding molecules and TNF α antagonists some Advantages. The peptides or peptide derivatives according to the invention are small Mo molecules that have little to no antigenic activity. It eliminates the education of Fc complexes, as otherwise observed in antibodies. Likewise the complement system is not activated. Because big immune complexes are not arise, remain the complexes of the target molecules and the  CDRs according to the invention soluble, provided that the target molecules previously dissolved in Form have existed.

The peptides or peptide derivatives are readily linked to other molecules, such as diagnostics or cytotoxic molecules can be coupled. So are chelators with central Metal atom (for example gadolinium) as diagnostics and cis-diammine dichloroplatin known as cytotoxic substances. Further uses he also give the publications: EP publication 0 502 595; DE-Offenlegungsschriften DE-OS 40 25 788 and DE-OS 41 07 570; continue R. SUTHERLAND (1987) Cancer Res 47: 1627-1633; and M. WHITLOW and D. FILPULA (1991), Methods 2: 97-105; and Römpps Chemie-Lexikon / Otto Albrecht Neumüller, volume 2, pages 855 to 856, 8th edition, 1981, Stuttgart, (ISBN 3-440-04512-9). Due to the molecular size of the peptides or Peptide derivatives is a targeted reaction with a functional group of the Peptides or peptide derivatives according to the invention possible.

Furthermore, the peptides or peptide derivatives according to the invention are easy adjustable. Such short peptides or peptide derivatives can be synthesized by a technique which are known to those skilled in the art of peptide synthesis is. A summary of many of these techniques can be found in J.M. STEWART and J.D. YOUNG, San Francisco, 1969; and J. MEIERHOFER, Hormonal Proteins and Peptides, Vol. 2p46, Academic Press (New York), 1973 for the solid-phase method and E. SCHRODER and K. LUBKE, The Pep tides, Vol. 1, Academic Press (New York) 1965 for the liquid phase method be read. The steps of the synthesis are described in EP-OS 0 097 031 described. The general procedural steps from the European Publi cation can be applied analogously to the synthesis of the invention described here transmit appropriate peptides or peptide derivatives. Further literature on the Solid phase synthesis are: Solid Phase Synthesis, E. ATHERTON and R.C. SHEPPARD (1989) IRL Press, ISBN 1-85221-133-4 and Amino Acid and Pep tide Synthesis, J.ONES, Oxdford Science Publication (1992) ISBN 0-19-855668-3.

The peptides or peptide derivatives of the invention can be as in the example 1.4.

Further advantages over antibodies or their fragments exist in the better shelf life. The molecules are more stable and do not need any other proteins to be stabilized.  

Furthermore, the peptides or peptide derivatives have better tissues common as antibodies or fragments thereof. This makes them suitable especially as a therapeutic.

Further embodiments relating to the amino acid sequences

The invention further comprises peptides or peptide derivatives (CDRs),

• d) which have the following sequence:
  Gly Gly Ile Thr Cys Thr Ala Ser Gin Ser Val Ser Asn Asp Val Val Val Gly Gly Gly
  (GGITCTASQSVSNDVVWGG in one-letter code)
  (see Sequence Identifier No 2 (Sequence Protocol No. 2))

or

• e) which as active CDRs allelic modifications of the previously under d) have mentioned amino acid sequence, wherein at least one Amino acid of the amino acid sequence is substituted, deleted or inserted, without significantly influencing the activity of the active CDRs,

or

• f) which as active CDRs posttranslational modifications of one of Sequences under d) and e), wherein the modifications are not significantly affect the activity of active CDRs.

The invention further comprises peptides or peptide derivatives (CDRs) with the Formula:

where positions Xaa are for the following amino acids or bonds stand:

Xaa₀ for a bond or for Gly-Gly;
Xaa₁ for Ile, Leu or Val;
Xaa₂ for Thr or Ser
Xaa₃ for Cys or Ser;
Xaa₄ for Thr or Ser
Xaa₅ for Ala, Gly or Ser;
Xaa₆ for Ser or Thr
Xaa₇ for Gln or Asn;
Xaa₈ for Ser or Thr;
Xaa₉ for Val, Ile or Leu;
Xaa₁₀ for Ser or Thr;
Xaa₁₁ for Asn, Gln or His;
Xaa₁₂ for Asp or Glu;
Xaa₁₃ for Val, Ile or Leu;
Xaa₁₄ for Val, Ile or Leu;
Xaa₁₅ for Trp or Tyr;
Xaa₁₆ for a bond or for Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid
and
R₂ is -OH or a carboxyl protecting group or at least one more amino acid
wherein one or two or three amino acids of the sequence Xaa₁ to Xaa₁₅ in place of the aforementioned amino acid have a bond.

It is preferred that serine only at not more than five positions of Sequences present.

Preference is given to CDRs according to the invention having the formula:

where positions Xaa are for the following amino acids or bonds stand:

Xaa₀ for a bond or for Gly-Gly;
Xaa₁ for Ile;
Xaa₂ for Thr;
Xaa₃ for Cys;
Xaa₄ for Thr
Xaa₅ for Ala;
Xaa₆ for Ser;
Xaa₇ for Gln
Xaa₈ for Ser;
Xaa₉ for Val;
Xaa₁₀ for Ser;
Xaa₁₁ for Asn;
Xaa₁₂ for Asp;
Xaa₁₃ for Val;
Xaa₁₄ for Val;
Xaa₁₅ for Trp;
Xaa₁₆ for a bond or Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid
and
R₂ is -OH or a carboxyl protecting group or at least one more amino acid
wherein one or two or three amino acids of the sequence Xaa₁ to Xaa₁₅ in place of the aforementioned amino acid have a bond.

More preferred are CDRs of the invention having the formula:

where positions Xaa are for the following amino acids or bonds stand:

Xaa₀ for a bond or for Gly-Gly;
Xaa₁ for Ile;
Xaa₂ for Thr;
Xaa₃ for Cys;
Xaa₄ for Thr
Xaa₅ for Ala;
Xaa₆ for Ser;
Xaa₇ for Gln
Xaa₈ for Ser
Xaa₉ for Val
Xaa₁₀ for Ser;
Xaa₁₁ for Asn
Xaa₁₂ for Asp;
Xaa₁₃ for Val
Xaa₁₄ for Val
Xaa₁₅ for Trp;
Xaa₁₆ for a bond or for Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid
and
R₂ is -OH or a carboxyl protecting group or at least one more amino acid.

From the group of the abovementioned peptide derivatives, the SEQ ID NO. 1 said compound the most preferred.

Further embodiments with respect to the radicals

In addition to the modification of the amino acid sequence of Pep according to the invention Tide or peptide derivatives is also a variation of the radicals R₁ and R₂ possible. The residues, however, require the binding ability of the CDRs and inhibition the effect of TNF α not influence. Nevertheless, you can get through Protecting parameters such as stability, half-life, bioavailability and Significantly affect penetration of matrix or membranes.

Preference is given to CDRs according to the invention in which R₁ is -H or an amino-protecting group,
and
R₂ is -OH or a carboxyl protecting group.

More preferred are CDRs of the invention in which R₁ is -H or an acyl group,
and
R₂ is -OH or an amino group.

Most preferred are inventive CDRs in which R₁ is -H
and
R₂ stand for -OH.

Production process of the peptides according to the invention

The invention further comprises the preparation of the CDRs according to the invention, wherein an N-α protected ω-amino-α-amino acid with a dialdehyde in  Presence of a reducing agent reacted and then the protection groups of the side chains and optionally the protective groups of the N-termi nus and / or the C-terminus are split off.

The invention also encompasses a process in which the CDRs according to the invention are prepared by condensing the amino acids in a homogeneous phase or by the solid-phase method,
wherein the carboxyl end of an amino acid to be coupled, whose amino groups and optionally functional groups of the side chain carry a protective group, reacts with the free amino end of the amino acid to be coupled or of the peptide fragment to be coupled in the presence of a condensation reagent,
and
in the case of a non-terminal amino acid, then the α-amino protecting group of the coupled amino acid is cleaved off and further amino acids are coupled to the peptide chain to be synthesized after the two steps described above
or
in the case of a terminal amino acid, if appropriate, the α-amino protective group of the coupled amino acid is subsequently split off
and
after coupling of the last amino acid in the case of the solid-phase method, the CDR is cleaved from the solid phase.

Use as a diagnostic

The CDRs according to the invention can, because of their binding ability as Diagnostic agent to be used. Analogous uses of binding mole in the publications EP Publication 0 502 595; DE-Disclosure 40 25 788 and 41 07 570 described.

Use as a therapeutic with coupled pharmaceutical reagent

Likewise, the CDRs according to the invention have pharmacological effects, if they are associated with a pharmacologically active further part of the molecule  they are. (R. SUTHERLAND (1987) Cancer Res 47: 1627-1633; and M. WHITLOW and D. FILPULA (1991), Methods 2: 97-105; and Rompps Chemicals Lexicon / Otto Albrecht Neumüller, Volume 2, pages 855 to 856, 8th edition, 1981, Stuttgart, (ISBN 3-440-04512-9))

Use as a medicine

The CDRs according to the invention have pharmacological properties and are therefore useful as pharmaceutical agents. The invention um also takes a drug containing one of the CDRs of the invention or a Mixture thereof contains. Furthermore, the invention belongs to a pharmaceutical Composition comprising one of the CDRs of the invention or a mixture Contains CDRs according to the invention, in the presence of pharmaceutically acceptable and acceptable excipients and carriers. Also includes the inventions a pharmaceutical composition which is one of the pharmaceutical active, inventive CDRs or mixtures thereof and a pharma ceutically acceptable salt or pharmaceutically acceptable excipients and carriers contains substances.

In particular, the CDRs according to the invention according to the sequence prototype No. 1 inhibits TNFα activity.

The CDRs according to the invention show an inhibition (IC₅₀ value) of the cyto toxicity induced by TNFα at concentrations of 20 to 80 μmol / l. Higher concentrations are applicable without disturbing the test system. Thus, the CDRs of the invention are in concentrations of 10 to 1000 μmol / l can be used.

The experimental results of this in vitro test show that the erfindungsge CDRs are used as drugs or for medical treatment can be. These test results can be derived from the in vitro test system to a system in vivo problem-free transfer, since the Zyto toxicity test is an established experimental set-up for detection the TNF α activity is used. (SECKINGER et al. (1990) J Biol Chem 264: 11966-11973).

The CDRs according to the invention can therefore be used for the treatment and prevention of septic shock, cachexia, cerebral malaria, graft-versus-host  Reaction (graft versus host reaction), arthritis, premature birth, Osteoporosis, autoimmune diseases and multiple sclerosis. The CDRs of the invention may be useful as a TNFα inhibitor in mammals, particularly in humans, used for the treatment of the aforementioned diseases become.

The invention further provides

• (i) the use of one of the CDRs of the invention or a mixture thereof for the manufacture of a medicament for the treatment of septic Shock, cachexia, cerebral malaria, graft-versus-host reaction (Graft versus host reaction), arthritis, premature birth, osteoporosis, Autoimmune diseases and multiple sclerosis;
• (ii) a method of treating septic shock, cachexia, cerebral Malaria, graft-versus-host reaction (graft versus host reaction), Arthritis, premature birth, osteoporosis, autoimmune diseases and multiple Sclerosis, which method ge administration of a CDR amount according to the invention, wherein the amount suppresses the disease, and wherein the amount of CDR is given to a patient having a requires such drug;
• (iii) a pharmaceutical composition for the treatment of septic Shock, cachexia, cerebral malaria, graft-versus-host reaction (Graft versus host reaction), arthritis, premature birth, osteoporosis, Autoimmune diseases and multiple sclerosis, what treatment one the inventive CDRs or mixtures thereof and at least a pharmaceutical excipient and / or carrier.

Different doses are suitable for the therapeutic effect. you depend, for example, on the CDRs used, on the host, on the type of Administration and the type and severity of the treatment stand off.

In general, however, satisfactory results are to be expected in animals daily doses range from 100 μg to 100 000 μg per kg Include body weight. In larger mammals, such as the People, a recommended daily dose ranges from 100 to 100,000 μg per kg of body weight. Preferably, a dose of 300 to 30,000 micrograms per kg body weight, more preferably a dose of 1000 to 10,000 μg per kg Body weight, most preferably a dose of 2000 to 5000 micrograms per kg  Body weight. For example, this dose will conveniently be in part doses administered up to four times a day. Satisfactory results are too when the CDRs of the invention are administered subcutaneously or intravenously become.

The present invention provides pharmaceutical compositions comprising one of the CDRs of the invention or their mixture and at least one pharmaceutically acceptable carrier or additive. Such compositions can be prepared by known methods. Reference may be made to Remington's Pharmaceutical Science, 15 ^th ed. Mack Publishing Company, East Pennsylvania (1980).

Examples 1. CHEMICALS; PROTEINS AND CELL LINES 1.1. Chemicals for peptide synthesis

The Fmoc-protected amino acids are from Novabiochem (Switzerland) and Bachem (Bubendorf, Switzerland) available. Their production is state of the art Technology. In the synthesis of peptides on polystyrene resin, the amines Nosäuren in situ with PyBOP (Benzotriatol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate) and NMM (N-methyl-morpholine) activated.

1.2. Tumor Necrosis Factor α (Tumor Necrosis Factor α) (TNF α)

The preparation of TNF α is described in K. ASHMAN et al. (1989) Prot Engin 5: 387-391 described. The human ¹²⁵I-labeled TNFα is from the company Amersham (Brunswick) with a specific activity of 800 Ci / mmol obtainable.

The extracellular domains of soluble human 55 kD and 75 kD TNF Receptors sp55 and sp75 are also in the publication of ASHMAN described. The label with ¹²⁵I is in the publication L. HUSAON and F.C. HAY (1980) Practical Immunology, second ed. Oxford, page 96 ben.

1.3. Cell lines

The murine fibroblast cell line L929 is described in detail in the publication of SECKINGER et al. (1990) J Biol Chem 264: 11966-11973.

1.4. Production of CDRs

The synthesis of soluble peptide mixtures and peptides or peptide derivatives CDRs) is linked to a multiple peptide synthesizer (MPS) AMS 422 of ABIMED (Langenfeld) executable. (H. GAUSEPOHL et al., (1992) Peptides Research 5/6: 315-320).

The peptide synthesis, in which the CDR is fixed via an anchor group and only is split off at the end of the synthesis is carried out on polystyrene resin (Batch size 50 μmol). The solvent used is DMF. It will always performed double couplings. The synthesis cycle of the machine consists of the cleavage of the Fmoc protecting group with 20% piperidine  (twice 15 minutes) followed by a six-fold washing procedure with DMF before the amino acids are coupled (two times 15 minutes). In front The next Fmoc cleavage is again washed six times with DMF. In the reaction, the automaton activates 200 .mu.mol of amino acid in 400 .mu.l of DMF by adding 200 .mu.mol of PyBOP in 220 .mu.l of DMF and 400 .mu.mol NMM in 100 μl of DMF.

The N-terminus of the CDRs is replaced by the addition of DMF / acetic anhydride / DIPA in the Volume ratio 7: 2: 1 acetylated (stir for 30 minutes).

For splitting off the CDRs from the resin and splitting off the Seitenschutzgrup 2 to 3 ml of 90% TFA, 5% of triisobutylsiolane, 5% of water and 5% of phenol used. After a five-hour incubation, the CDRs are ice-creamed with approx. 35 ml precipitated cold ether. The precipitate is centrifuged off, the pellet (Sediment) with 15 to 20 ml ice-cold ether resuspended and washed. The centrifugation and washing is repeated five times before the CDRs in the smallest possible volume of 5% acetic acid are dissolved. Subsequently, the CDRs are freeze-dried. The CDRs are after the Cleavage C-terminal as amide before.

DIPA diisopropylethylamine
DMF dimethylformamide
Fmoc o-fluorenylmethoxycarbonyl
PyBOP benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate
NMM N-methyl morpholine
TFA trifluoroacetic acid

2. Functional detection of binding and inhibition 2.1. Competitive binding assay TNF α / TNF receptors

For the competition test, 10 ng / reaction vessel (well) of the soluble, recombinant part of the 55 kD or 75 kD receptor (in 50 μl of 0.1 mol / l NaHCO, pH 9.6) are bound to a microtiter plate (Dynateck) ( 4 ° C overnight). After washing once (3 minutes with 200 μl PBS / 0.1% Tween 80), the remaining binding sites are blocked by incubation for 3 hours at room temperature with incubation buffer (200 ml 6% Gelifundol®S from Biotest (Dreieich, Cat No. 1056061). , 0.1 Tween 80 in PBS). After a single wash, 0.5 ng ¹²⁵I-TNF α and the respective competitor (CDRs) are added (after 10 minutes Vorinkubation) in 50 ul incubation buffer and incubated for 2 hours at room temperature. The reaction vessels are washed three times and the bound activity is measured in the γ counter (count rate 73.1%). For evaluation, the cpm values measured during the control buffer (without competitor) (cpm = counts per minute) are set to 100%.
PBS = Phosphate Buffered Saline
Tween = polyoxyethylene sorbitan monooleate

2.2. Inhibition test of cytotoxicity of TNF α

To show that the TNF α-binding is also functionally effective measured whether the cytotoxic effect of TNFα on TNFα-sensitive L929 Cells is inhibited by the CDRs of the invention. (SECKlNGER et al. (1990) J Biol Chem 264: 11966-11973) The CDR according to Seq Id. 1 has an IC₅₀ value of 6 μmol / l.

An exchange of one of the amino acids may be without significant influence on the activity of the CDRs of the invention. If only one of the amino acids is replaced by, for example, alanine, the inhibition of TNF-induced cytolysis remains approximately the same. Nevertheless, fluctuations by a factor of 10 are possible. If only one of the 15 amino acids is substituted by alanine, IC₅₀ values in the range of 7 to 40 μmol / l result. The concrete data are listed in Table 2.

Substitution of one amino acid of the sequence according to Seq. Id. 1 with alanine Exchanged amino acid IC₅₀ values Ala₁ instead of Ile₁ 7 Ala₂ instead of Thr₂ 9 Ala₃ instead of Cys₃ 9 Ala₄ instead Thr₄ 12 [Ala₅ instead of Ala₅ 6] Ala₆ instead of Ser₆ 6 Ala₇ instead of Gln₇ 8th Ala₈ instead of Ser₈ 9 Ala₉ instead of Val₉ 22 Ala₁₀ instead of Ser₁₀ 8th Ala₁₁ instead of Asn₁₁ 12 Ala₁₂ instead of Asp₁₂ 40 Ala₁₃ saturated Val₁₃ 14 Ala₁₄ instead of Val₁₄ 18 Ala₁₅ instead of Trp₁₅ 18

sequence Listing

SEQ ID NO: 1 Type of sequence: Synthetic amino acid sequence Sequence length: 15 amino acids Type of molecule: Synthetic molecule Properties: Binding to TNF α and antagonistic action Ancestry: CDRL1 of a light chain of an antibody without terminal glycines

SEQ ID NO: 2 Type of sequence: Synthetic amino acid sequence Sequence length: 19 amino acids Type of molecule: Synthetic molecule Properties: Binding to TNF α and antagonistic action Ancestry: CDRL1 plus 4 Gly of a light chain of an antibody

Claims (19)

1. peptides or peptide derivatives (CDRs),

• a) which have the following sequence:
  Ile Thr Cys Thr Ala Ser Gln Ser Val Ser Asn Asp Val Val Trp
  (ITCTASQSVSNDVVW in one-letter code)
  (see Sequence Identifier No 1 (Sequence Protocol No. 1))

or

• b) which as active CDRs have allelic modifications of the amino acid sequence mentioned above under a), wherein at least one amino acid of the amino acid sequence is substituted, deleted or inserted without significantly influencing the activity of the active CDRs,

or

• c) which as active CDRs post-translational modifications of any of the sequences under a) and b), wherein the modifications do not significantly affect the activity of active CDRs.

2. peptides or peptide derivatives (CDRs),

• d) which have the following sequence:
  Gly Gly Ile Thr Cys Thr Ala Ser Gln Ser Val Ser Asn Asp Val Val Trp Gly Gly (GGITCTASQSVSNDVVWGG in one-letter code)
  (see Sequence Identifier No 2 (Sequence Protocol No. 2))

or

• e) which have as active CDRs allelic modifications of the amino acid sequence mentioned above under d), wherein at least one amino acid of the amino acid sequence is substituted, deleted or inserted without significantly influencing the activity of the active CDRs,

or

• f) which as active CDRs have post-translational modifications of one of the sequences under d) and e), wherein the modifications do not significantly affect the activity of the active CDRs.

3. Peptides or peptide derivatives (CDRs) having the formula: wherein the positions Xaa are the following amino acids or bonds: Xaa₀ for a bond or for -Gly-Gly-;
Xaa₁ for Ile, Leu or Val;
Xaa₂ for Thr or Ser;
Xaa₃ for Cys or Ser;
Xaa₄ for Thr or Ser;
Xaa₅ for Ala, Gly or Ser;
Xaa₆ for Ser or Thr;
Xaa₇ for Gln or Asn;
Xaa₈ for Ser or Thr;
Xaa₉ for Val, Ile or Leu,
Xaa₁₀ for Ser or Thr
Xaa₁₁ for Asn, Gln or His;
Xaa₁₂ for Asp or Glu;
Xaa₁₃ for Val, Ile or Leu;
Xaa₁₄ for Val, Ile or Leu;
Xaa₁₅ for Trp or Tyr;
Xaa₁₆ for a bond or for Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid and
R₂ is -OH or a carboxyl protecting group or at least one other amino acid, and one or two or three amino acids of the sequence Xaa₁ to Xaa₁₅ have a bond in place of the aforementioned amino acid. 4. CDRs according to claim 3 having the formula: wherein the positions Xaa are the following amino acids or bonds: Xaa₀ for a bond or for -Gly-Gly-;
Xaa₁ for Ile;
Xaa₂ for Thr;
Xaa₃ for Cys;
Xaa₄ for Thr;
Xaa₅ for Ala
Xaa₆ for Ser;
Xaa₇ for Gln;
Xaa₈ for Ser;
Xaa₉ for Val;
Xaa₁₀ for Ser;
Xaa₁₁ for Asn;
Xaa₁₂ for Asp;
Xaa₁₃ for Val
Xaa₁₄ for Val;
Xaa₁₅ for Trp;
Xaa₁₆ for a bond or Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid and
R₂ is -OH or a carboxyl protecting group or at least one other amino acid, and one or two or three amino acids of the sequence Xaa₁ to Xaa₁₅ have a bond in place of the aforementioned amino acid. 5. CDRs according to claim 4 having the formula: wherein the positions Xaa are the following amino acids or bonds: Xaa₀ for a bond or for -Gly-Gly-;
Xaa₁ for Ile;
Xaa₂ for Thr
Xaa₃ for Cys;
Xaa₄ for Thr
Xaa₅ for Ala;
Xaa₆ for Ser
Xaa₇ for Gln
Xaa₈ for Ser
Xaa₉ for Val;
Xaa₁₀ for Ser;
Xaa₁₁ for Asn
Xaa₁₂ for Asp;
Xaa₁₃ for Val;
Xaa₁₄ for Val;
Xaa₁₅ for Trp;
Xaa₁₆ for a bond or for Gly-Gly;
R₁ is -H or an amino-protecting group or at least one further amino acid and
R₂ is -OH or a carboxyl protecting group or at least one more amino acid. 6. CDRs according to any one of claims 3 to 5, wherein R₁ is -H or an amino-protecting group,
and
R₂ is -OH or a carboxyl protecting group. 7. CDRs according to claim 6, wherein
R₁ is -H or an acyl group,
and
R₂ is -OH or an amino group. 8. CDR according to claim 7, wherein
R₁ for -H
and
R₂ stand for -OH. 9. A process for the preparation of peptides or peptide derivatives according to one of the preceding claims, wherein an N-α protected ω-amino-α-ami Nosäure reacted with a dialdehyde in the presence of a reducing agent and then the side chain protecting groups and optionally the Protective group of the N-terminus and / or the C-terminus are cleaved. 10. The method of claim 9, wherein the amino acids are condensed in a homogeneous phase or by the solid phase method, wherein the carboxyl end of an amino acid to be coupled, the Aminogrup groups and optionally functional groups of the side chain carry a protective group with the free amino end of the amino acid to be coupled or the peptide fragment to be coupled in the presence of a Kondensationsrea reacts, and
in the case of a non-terminal amino acid, then the α-amino protecting group of the coupled amino acid is cleaved off and further amino acids are coupled to the peptide chain to be synthesized after the two steps described above
or
in the case of a terminal amino acid, if appropriate, the α-amino protective group of the coupled amino acid is subsequently split off
and
after coupling of the last amino acid in the case of the solid phase method, the peptide or peptide derivative is cleaved from the solid phase. 11. peptides or peptide derivatives according to any one of claims 1 to 8 or prepared according to any one of claims 9 and 10 as a diagnostic agent. 12. peptides or peptide derivatives according to any one of claims 1 to 8 or prepared according to any one of claims 9 and 10 as a medicament, wherein the Peptides or peptide derivatives with a pharmacologically active further Part of the molecule are connected. 13. peptides or peptide derivatives according to any one of claims 1 to 8 or prepared according to any one of claims 9 and 10 as a medicament. 14. Pharmaceutical composition containing (i) one of the peptides or peptide derivatives according to any one of claims 1 to 8 or (ii) one of the Pep tide or peptide derivatives prepared according to any one of claims 9 and 10 or (iii) a mixture thereof, in the presence of pharmaceutically acceptable and acceptable excipients and carriers. 15. Use of one of the peptides or peptide derivatives after a of claims 1 to 8 or produced according to one of claims 9 and 10 for the manufacture of a medicament for the treatment of septic shock, Cachexia, cerebral malaria, graft-versus-host reaction, arthritis, premature birth, Osteoporosis, autoimmune diseases and multiple sclerosis.