Drug – bio-affecting and body treating compositions – Conjugate or complex of monoclonal or polyclonal antibody,... – Conjugated via claimed linking group – bond – chelating agent,...
Reexamination Certificate
1999-08-12
2002-02-12
Gambel, Phillip (Department: 1644)
Drug, bio-affecting and body treating compositions
Conjugate or complex of monoclonal or polyclonal antibody,...
Conjugated via claimed linking group, bond, chelating agent,...
C424S130100, C424S133100, C424S135100, C424S141100, C424S143100, C424S144100, C424S153100, C424S173100, C424S178100, C424S183100, C530S387100, C530S387300, C530S388100, C530S388200, C530S388220, C530S388700, C530S388730, C530S391100, C530S391700
Reexamination Certificate
active
06346248
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to compositions and methods of treating diseases of the immune system. In particular, this invention relates to methods of preventing allograft rejection and methods of treating autoimmune diseases and various malignancies of lymphoid origin.
BACKGROUND OF THE INVENTION
Immunotoxins
Immunotoxins (IT's) are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually a monoclonal antibody (Mab) that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells. Unlike chemotherapeutic molecules, these toxins kill both resting and dividing cells. The toxins share a number of common features: (i) they are synthesized as single chain proteins and are processed either post translationally or in the target cell to which they are delivered into two-chain molecules with interchain disulfide bonds; (ii) the disulfide bond linking the two chains is critical for cytotoxicity; and (iii) all toxins have separate subunits or domains devoded to binding to cells, translocation across membranes, and the destruction of protein synthesis in the target cell. These domains can be separated or genetically manipulated to delete those that are unwanted.
The most widely used plant toxins ricin and abrin, consist of two disulfate-linked polypeptides A and B (Olsnes et al., in
Molecular Action of Toxins and Viruses
p51-105 (1982)). Another group of plant-derived toxins used in immunotoxins are the ribosome inactivating proteins (RIPs). These molecules are single-chain proteins frequently found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri
FEBS
195:1 (1986)). The cross-linker used to join the Mab and the toxin must remain stable extracellularly, but labile intracellularly so that the toxin fragment can be released in the cytosol. The choice of cross-linker depends on whether intact toxins, A-chains or RIPs are used. A-chains and RIPs are generally coupled to the Mab using linkers that introduce a disulfide bond between the ligand and the A-chain (Myers et al.,
J. Immunol. Meth.
136:221 (1991)). Bonds that cannot be reduced render these immunotoxins much less toxic or nontoxic, probably because the A-chain must be released from the ligand by reduction to be cytotoxic. Intact toxins are usually linked to ligands using non-reducible linkages (such as thioether) to prevent release of the active free toxin in vivo.
RIPs, efficiently inhibit eukaryotic protein synthesis. Gelonin is a type I RIP (single catalytic chain), which has an advantage above type II RIPs in that type II RIPs have in addition to the catalytic chain, a cell-binding lectin-like B-chain. Because gelonin has no cell-binding lectin-like B-chain, it is unable to bind to cell membranes in the absence of a targeting agent and therefore has a low nonspecific toxicity. Even in comparison with another type I RIP (saporin), LD50 studies in mice have shown that native gelonin is approximately 10-fold less toxic than saporin, and thus may be particularly suitable for therapeutic applications. Moreover, immuno-conjugates with gelonin when targeted to cells have low IC50 values, inhibit a greater percentage of target cells and require less exposure time in comparison to other toxins. For these reasons, the low native toxicity and the high specific toxicity, the therapeutic window is very high for gelonin. Gelonin is among the most promising toxins used for the construction of ITs. In direct comparison experiments, gelonin was superior to two of the most popular toxins, ricin A chain and Pseudomonas exotoxin A (Fishwild et al
Clin Exp Immunol
97:10 (1994)). The cDNA of gelonin was recently isolated (Better et al
J Biol Chem
270:14,951 (1995)), allowing the construction of single chain antibody-toxin fusion proteins (ScFv-IT). A complete Mab consists of two complete heavy and two complete light chains and has a molecular weight of 150 kDa. An immunotoxin molecule based on a whole antibody will have a molecular weight in the range of 200 kDa, depending on the type of toxin and the amount of toxin molecules coupled per antibody. A single chain antibody fragment (ScFv) however, consists of only the variable part of the heavy and light chain coupled via a short linker and has a molecular weight of approximately 25 kDa. When a toxin molecule is directly fused to a ScFv molecule by genetic engineering, the size of the ScFv-immunotoxin molecule thus obtained, will be a factor 4 smaller when compared to a complete antibody-immunotoxin molecule. Since tumor penetration is mainly dependent on size (the smaller the IT the better the tumor penetration), it is preferred to use ScFv-IT molecules. In addition, the serum half-live of a ScFv-IT is much shorter when compared to a complete antibody-immunotoxin molecule, thus reducing the non-specific systemic toxicity.
CD80/CD86 Costimulatory Molecules
CD80 (B7.1) is a monomeric transmembrane glycoprotein with an apparent molecular mass of 45-65 kDa and is a member of the immunoglobulin superfamily (Freeman et al.
J. Immunol.
143:2714, (1989)). It was initially reported that the expression of the CD80 molecule was restricted to activated B cells (Freeman et al.,
J. Immunol.
143:2714, (1989)) and monocytes stimulated with IFN-&ggr; (Freedman et. al.,
Cell. Immunol.
137:429, (1991)). More recently, CD80 expression has also been found on cultured peripheral blood dendritic cells (Young et al.
J. Clin. Invest.
90: 229 (1992)). The expression of the CD80 molecule in a number of normal and pathological tissues has been examined by immunohistochemistry using an anti-CD80 monoclonal antibody (Vandenberghe et al.,
Int. Immunology
5:317 (1993)). In addition to the staining of activated B cells, it was shown that the CD80 molecule is constitutively expressed in vivo on dendritic cells in both lymphoid and non-lymphoid tissue. Monocytes/macrophages were only found to be positive under inflammatory conditions and endothelial cells were always negative. Interestingly, the number of CD80 positive cells in skin lesions of patients with acute GVHD was strongly increased when compared to normal skin. This expression pattem of CD80 on different antigen presenting cells (APCs), strongly suggests an important costimulatory role in T-cell activation.
It has recently been demonstrated that CD80 is a member of a family of closely related molecules molecules, that can functionally interact with CD28 (Hathcock et al.
Science
262:905 (1993); Freeman et al.
Science
262:907 (1993); Azuma et al.
Nature
366:76 (1993)). The second member of this family, B7.2 or CD86, is also a transmembrane glycoprotein, with an apparent molecular mass of approximately 70 KDa and is also a member of the immunoglobulin superfamily (Freeman et al.
Science
262:907 (1993); Azuma et al.
Nature
366:76 (1993)). The CD86 molecule seems to have a very similar distribution pattern as CD80, with the exception that induction of cell-surface expression seems to be faster and that it is present on freshly isolated monocytes.
Transplant Rejection
Incompatibility for the histocompatibility antigens, both major (MHC) and minor antigens, is the cause for graft rejection. Both CD4+ helper T cells (Th) and CD8+ cytotoxic T cells (CTL) are involved in the rejection process. Activation of T cells after transplantation is the result of ligand-receptor interactions, when the TcR/CD3 complex recognizes its specific alloantigen in the context of the appropriate MHC molecule. To induce proliferation and maturation into effector cells, T cells need a second signal in addition to the one mediated by the TcR/CD3 complex. Intercellular signaling after TcR/MHC-peptide interaction in the abse
De Boer Mark
De Gast G. C.
Bierman, Muserlian and Lucas
Gambel Phillip
Innogenetics N.V.
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