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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/54—F(ab')2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• the invention relates to apoptosis inhibitors. Furthermore, the
• Apoptosis is the name for programmed cell death. This can be found e.g. in organ development and metamorphosis, tissue atrophy and tumor regression. Apoptosis is associated with condensation of the cytoplasm, loss of plasma membrane villi, segmentation of the nucleus and extensive degradation of chromosomal DNA (see Oehm, A. et al., The Journal of Biological Chemistry, volume 267, No. 15 (1992), pages 10709-10715).
• a cell surface protein designated APO-1 is often found in cells that are positive for apoptosis. This glycoprotein is assigned to the tumor necrosis factor / nerve growth factor receptor family. By crosslinking APO-1 via an anti-APO-1 antibody, apoptosis is induced in the cells mentioned (cf. Oehm, A. et al., Supra). The same seems to be brought about by binding a soluble or membrane-bound protein labeled APO-1 ligand to APO-1 (cf. Suda, T. and Nagata, S., J. Exp. Med., The Rocke ⁇ feiler University Press, Vol. 179 (1994), pages 873-879).
• the present invention is therefore based on the object of providing a means by which apoptosis can be inhibited.
• an agent which is characterized in that it contains one to all components of: (a) an APO-1 inhibitory compound,
• APO-1 inhibitory compound encompasses any compound suitable for inhibiting APO-1. This is preferably a blocking, non-cytotoxic anti-APO-1 antibody or an anti-APO-1 antibody without an Fc part, for example an F (ab), F (ab) 2 or F v fragment of one anti-APO-1 antibody. Such a fragment is produced in a customary manner, the person skilled in the art, for example, from the anti-APO-1 antibody described in Oehm et al., Supra or from that in Dhein, J. et al., The Journal of Immunology, Volume 149, No. 10, (1992), pages 3166-3173 anti-APO-1-F (ab) 2 fragment described. The latter can also be used directly.
• An APO-1 ligand analog can be mentioned as a further preferred “APO-1 inhibitory compound”. This still binds to APO-1, but no longer induces the intracellular APO-1 signaling pathway.
• Such an analog is prepared in a customary manner, the person skilled in the art e.g. from the APO-1 ligand described in Suda, T. and Nagata, S., supra.
• APO-1 ligand inhibiting or intercepting compound encompasses any compound suitable for inhibiting or intercepting the APO-1 ligand. This is preferably one of the following compounds: an anti-APO-1 ligand antibody, an APO-1, an extracellular APO-1 domain, a compound having at least one extracellular APO-1 domain and a carrier, a peptide having an APO-1 ligand binding site, and a compound having at least one peptide having an APO-1 ligand binding site and a carrier.
• Such a connection is established in the usual way.
• One skilled in the art will, e.g., produce an anti-APO-1 ligand antibody e.g. start from the APO-1 ligand described in Suda, T. and Nagata, S., supra. He will take care that the Fc part of the antibody is not considered foreign in an individual. He is aware of processes that make this possible.
• the person skilled in the art with regard to the production of APO-1 or an extracellular APO-1 domain e.g. start from the APO-1 described in EP-92 107 060.3 or its extracellular domain.
• it is used to prepare a peptide having an APO-1 ligand binding site e.g. use the combination of the above APO-1 ligand and the above APO-1 or its extracellular domain.
• a connection can be made with at least one peptide having an APO-1 ligand binding site and a carrier.
• a compound that inhibits the intracellular APO-1 signal includes any pathway suitable for inhibiting the intracellular APO-1 signal
• ICE interleukin-1ß converting enzyme
• DCI 3,4-dichloroisocoumarin
• YVAD-CHO an ICE-specific tetrapeptide
• CrmA a protein of vaccinia virus or derivatives thereof.
• ICE anti-sense To use nucleic acid or a derivative thereof as an ICE inhibitor.
• an inhibitor of ICE structure-related proteases particularly Nedd-2 / lch-1 or priCE.
• Such an inhibitor compound is produced in the usual way.
• the person skilled in the art will, for example, produce DCI or a derivative thereof e.g. to the work of Harper, J.W. et al., Biochemistry, 24, 1831-1841, (1985).
• YVAD-CHO or a derivative thereof e.g. the work of Thomberry, N.A. et al., Nature 356, 768-774, (1992).
• the above agent is used in particular to inhibit apoptosis in the case of a disease associated with an HIV infection.
• the agent proves to be particularly advantageous if it also contains one to all components of:
• TAT inhibitory or intercepting compound (b) a TAT inhibitory or intercepting compound, (c) a compound inhibiting the intracellular TAT receptor signaling pathway,
• a TAT receptor inhibitory compound includes any of the Inhibition of the TAT receptor suitable compound. This is preferably an anti-TAT receptor antibody. Such is produced in the usual way. One skilled in the art will ensure that the Fc portion of the antibody is not considered foreign in an individual. Methods which enable this are known to the person skilled in the art.
• a TAT analogue is to be mentioned as a further preferred “compound that inhibits the TAT receptor”. Such still binds to the TAT receptor, but no longer induces the intracellular TAT receptor signaling pathway. Such an analog is produced in the usual way, the person skilled in the art e.g. starting from the TAT described in Arya, S., K., et al., Science, Volume 229 (1985), pages 69-73.
• a TAT inhibitory compound includes any compound suitable for inhibiting or trapping TAT. This is preferably one of the following compounds: an anti-TAT antibody, a TAT receptor, an extracellular TAT receptor domain, a compound with at least one extracellular TAT receptor.
• a peptide having a TAT binding site a compound with at least one peptide having a TAT binding site and a carrier, - a TAT binding site based on nucleic acids, and a transdominant TAT mutant.
• TAT described in Arya, S., K. et al., Supra for the production of an anti-TAT antibody. He will take care that the Fc part of the antibody is not considered foreign in an individual. He is familiar with processes that make this possible. Furthermore, it is used for a TAT binding place on nucleic acid basis, for example, from the TAT-LTR sequence described in Feng, S. and Holland, E., C, Nature, Volume 334 (1 988), pages 1 65-167. Furthermore, the person skilled in the art with regard to a transdominant TAT mutant is described, for example, by the in Echetebu, C, O. and Rice, A., P., J. Acquir. Immune Def. Syndrome, Volume 6, (1993), pages 550-557.
• a compound which inhibits the intracellular TAT receptor signaling pathway includes any compound which is suitable for inhibiting the intracellular TAT receptor signaling pathway.
• a CD4 receptor inhibitory compound includes any compound useful for inhibiting the CD4 receptor. This is preferably an anti-CD4 receptor antibody.
• the production of such is carried out in a conventional manner, the skilled worker e.g. of that described in Capon, D., J. and Ward, R., H., R., Annu. Rev. Immunol. 9, (1991), pages 649-678, CD4 receptor described.
• One skilled in the art will ensure that the Fc portion of the antibody is not considered foreign in an individual. Methods which enable this are known to the person skilled in the art.
• a further preferred “compound which inhibits the CD4 receptor” is a gp 120
• analog Such still binds to the CD4 receptor, but no longer induces the intracellular CD4 receptor signaling pathway.
• Such an analog is prepared in a conventional manner, the person skilled in the art e.g. from the gp 120 described in Capon, D., J. and Ward, R., H., R., supra.
• a gp 120 inhibiting or intercepting compound includes any compound suitable for inhibiting or intercepting gp 120. This is preferably one of the following compounds: an anti-gp 120 antibody, a CD4 receptor, an extracellular CD4 receptor domain, a compound having at least one extracellular CD4 receptor domain and a carrier, a peptide having a gp 120 binding site, and a compound having at least one peptide having a gp 120 binding site and a carrier.
• a compound having at least one extracellular CD4 receptor domain or a peptide having at least one gp 120 binding site and a carrier can be produced in an analogous manner, as follows for a compound having at least one extracellular APO-1 domain and described a carrier.
• Compound includes any compound suitable for inhibiting the intracellular CD4 receptor signaling pathway.
• an above agent may have one to more compounds of a single component.
• carrier encompasses any compound to which one or more extracellular APO-1 domains can be bound.
• the carrier is a protein, e.g. Serum albumin, hemoglobin, fibrinogen, collagen or an Fc part of an antibody, the latter being preferred.
• the compound according to the invention is a fusion protein.
• a compound according to the invention can be produced in the usual way.
• the following manufacturing process proves to be favorable:
• Primers are used, whereby an amplified DNA fragment containing both DNAs in fusion is obtained, and
• the binding region of (a) and (b) is an antibody hinge region or a part thereof. It can also be a thrombin be gap.
• DNAs are amplified by the usual PCR technique.
• a DNA coding for at least one extracellular APO-1 domain the person skilled in the art will e.g. use the DNA described in EP-92 107 060.3, supra as the basis.
• the person skilled in the art will e.g. use the DNA described in Dübel, S., et al., Methods in Molecular and Cellular Biology, Volume 3 (1992), pages 47-52.
• the protein carrier e.g. Fc part of a human antibody coding DNA is known to the person skilled in the art e.g. also use the DNA described in Dübel, S. et al., supra.
• the amplified DNA fragment is expressed in conventional vectors, such as pCDN83, pCEV4 and pCDM8 for expression in animal cells, pGEMEX and pUC for expression in E. coli., And pY100 and YCpADI for expression in yeast.
• L, COS and CHO cells are particularly suitable as animal cells, while E. coli strains in particular are to be mentioned as prokaryotic microorganisms and those of Saccharom ⁇ ces and Pichia pastoris are particularly to be mentioned as yeast cells.
• a compound according to the invention is very suitable for inhibiting apoptosis. It can be used alone or in combination with one or all components of: (a) an APO-1 inhibiting compound,
• a compound according to the invention is particularly suitable for inhibiting apoptosis in the case of a disease associated with an HIV infection.
• the compound according to the invention can be used alone or in combination with one or all
• the present invention opens up a new way of treating diseases in which apoptosis of special cells plays an important role.
• the present invention represents a breakthrough in particular for the therapy of AIDS.
• FIG. 1 shows the inhibition of apoptosis by hu APO-1 -lg or anti-APO- 1 F (ab ') 2
• FIG. 2a shows the stimulation of the proteolytic activity of ICE by an anti-APO-1 antibody
• Fig. 2c shows the inhibition of apoptosis by YVAD-CHO
• 2d shows the inhibition of apoptosis by anti-sense ICE or CrmA cDNA.
• Example 1 Preparation of a fusion protein in which an extracellular
• APO-1 domain is fused to an Fc part of a human antibody via an antibody hinge region.
• a cDNA encoding an extracellular APO-1 domain (cf. Oehm, A. et al., Supra) and a cDNA encoding an Fc part of a human antibody (cf. Dübel, S. et al., supra) subjected to a conventional PCR amplification.
• GTT AGA TCT GGA TCC TTC 3 ' which codes for the 3' end of the extracellular APO-1 domain and the first 18 bp of the hinge region.
• PCR products obtained were separated in a "low melting point” agarose gel and the gel pieces containing DNA molecules of the correct size were combined. A further PCR amplification was carried out on a sample thereof. Only those above were used as primers which corresponded to the 5 'end of the extracellular APO-1 domain or the 3' end of the Fc part, which made it possible to access the extracellular APO-1 domain via the common hinge region to fuse the Fc portion, a "fusion" DNA fragment was obtained.
• This fragment was cleaved with Hindlll and Xbal, purified on an agarose gel and cloned in the vector pCDM8.
• the sequence of the pCDM8 insert was determined by dideoxynucleotide sequencing.
• Example 2 Inhibition of apoptosis by hu APO-1-lg or anti-APO-
• SKW 6.4 cells are apoptosis-positive cells, i.e. they can induce apoptosis, e.g. by binding an anti-APO-1 antibody.
• SKW 6.4 cells were treated for 24 h with an anti-APO-1 antibody in the presence of different amounts of the fusion protein from Example 1 (hu APO-1-Ig) or from anti-APO-1-F (ab) 2 (cf. above ) incubated. The inhibition of apoptosis was determined (see FIG. 1).
• Example 3 Inhibition of apoptosis by DCI, YVAD-CHO, anti-sense
• L 929-APO-1 cells (cf. Schulze-Osthoff, K. et al., EMBO J. 13, 4587-459, (1994)), like SKW 6.4 cells from Example 2, are apoptosis-positive.
• L 929-APO-1 cells (o) and SKW 6.4 cells (•) were cultivated and treated with anti-APO-1 antibody (1 ⁇ g / ml) for the times shown in FIG. 2a. 10 minutes before the cells were harvested, they were permeabilized with 0.05% digitonin and incubated with 20 / M of the fluorogenic ICE substrate DABCYL-YVADAP-EDANS (Bachern, Bubendorf, Switzerland). The cells were harvested and analyzed by FACS analysis using an excitation wavelength of 360 nm and an emission wavelength of 488 nm.
• L 29-APO-1 cells were incubated with DCI (• 45 M, A 15 / M) or without DCI (o). After one hour, anti-APO-1 antibody was added in the amounts shown in FIG. 2b and left on the cells for 7 hours. The percent cell death was determined by formazan production from diphenyl tetrazolium salt (MTT assay).
• L 29-APO-1 cells (o) and SKW6.4 cells (•) were permeabilized by a short hypothonic shock and incubated with the concentrations of YVAD-CHO shown in FIG. 2c for 30 minutes. The cells were incubated with anti-APO-1 antibody (1 ⁇ g / ml) for 60 minutes. Apoptosis was determined by DNA fragmentation using the procurement dye 33342
• a mouse ICE cDNA was isolated by RT-PCR using EL-4 / c mRNA and oligo dT primer for the first strand synthesis.
• CGGCCTCGAGCATCATCTAAGGAAGTATTGGC-3 'became a 1 322 bp PCR product obtained which was used as a sample for screening an E14 / 13 cDNA expression gene bank cloned in pCAGGS (cf. Niwa, H. et al., Gene 108, 193-200 (1994)).
• a 1387 bp ICE cDNA clone was obtained, the sequence of which was determined by DNA sequencing. This clone was named pCAGGS-ICE.
• a 320 bp EcoRI fragment of the above ICE cDNA was cloned in reverse orientation in pCAGGS, the fragment containing 48 bp of the 5 'UTR and the first 255 bp of the ICE ORF.
• the expression plasmid pCAGGS anti-sense ICE was obtained.
• a vaccine coding for CrmA was obtained by using the primer pair 5'-GC-GAAGCTTACACGACCAATATCGATTACTA-3 'and 5'-CGCCATGGTTAA-CAATTAGTTGTCGGAGAG-3'.
• This cDNA was cloned into the known plasmid pSV25S as a HindIII / KpnI fragment.
• the expression plasmid pSV25S-CrmA was obtained.
• the above expression plasmids were used to transfect L 929-APO-1 cells.
• the cells were taken up in TBS buffer and equilibrated on ice for 10 minutes.
• the cells were each transfected with 20 ⁇ g of the above expression plasmids using a Biorad electroporator (960 FD, 220 V). After electroporation, the cells were kept on ice for a further 30 minutes before they were sown in cell culture plates. Dead cells were washed away after 16 hours. Living cells were treated with anti-APO-1 antibodies (1 / g / ml) for the times indicated in FIG. 2d. Apoptosis was determined by FACS analysis as described above, where the Hoechst dye 33342 was used. Data were determined as a percentage of cell death from double experiments.
• anti-APO-1 antibody-induced apoptosis can be inhibited by anti-sense ICE or CrmA.

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