Peptide inhibitors of LFA-1/ICAM-1 interaction

Details Peptide inhibitors of LFA-1/ICAM-1 interaction Peptide inhibitors of LFA-1/ICAM-1 interaction

2000-01-14

2003-11-18

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S300000

Reexamination Certificate

active

06649592

ABSTRACT:

BACKGROUND
1. Field of the Invention
The present invention relates to the field of biologically active peptide containing compositions for use in the prevention and treatment of hematopoietic neoplastic diseases, particularly leukemia.
2. Description of Related Art
LFA-1 (lymphocyte function associated antigen-1) is an integrin &agr;&bgr; heterodimer (Carlos and Harlan, 1994; Springer, 1994; Larson and Springer, 1990; McEver, 1990; Picker and Butcher, 1992). Although three other integrins restricted in expression to leukocytes share the same &bgr; subunit and have homologous &agr; subunits (Mac-1, p150,95, and alpha d), only LFA-1 is expressed on normal and leukemia T cells (Larson and Springer, 1990). LFA-1 binds ICAM-1 (intracellular adhesion molecule), and although LFA-1 is constitutively expressed on all leukocytes, LFA-1 binding to ICAM-1 requires cellular activation. Activation, in part, results in conformational changes in LFA-1 that affect its avidity for ICAM-1. In contrast, ICAM-1 is constitutively avid and expressed on a wide array of cell types including leukocytes, endothelium, stromal cells, and fibroblasts. In a model developed by the present inventor, a stromal cell derived soluble factor cooperates with LFA-1 on the surface of T lineage acute lymphoblastic leukemia (T-ALL) cells (Winter et al., 1998). The LFA-1 on T-ALL cells results in bone marrow (BM) stromal cell binding via ICAM-1 that leads to enhanced leukemia cell survival. Furthermore, aberrant LFA-1/ICAM-1 dependent interaction between circulating leukemia cells and endothelial cells lining blood vessels promotes extravasation of leukemia cells into tissue as seen in the life-threatening therapeutic complication of acute leukemia, retinoic acid syndrome (Brown et al., 1999). Hence, the development of effective in vivo inhibitors of LFA-1/ICAM interaction would be useful in the therapy of acute leukemia and prevention of therapeutic complications.
The present inventor has shown, for example, that inhibition of LFA-1/ICAM-1 dependent stromal cell binding with mAbs decreases survival of T-ALL cell lines and T-ALL cells isolated from patients. In one study, a representative sample from a patient with T-ALL showed that survival of T-ALL cells is augmented by BM stromal cells and that survival is inhibited by mAbs directed against LFA-1 (mAb TSI/22,5 &mgr;g/ml) or its ligand ICAM-1 (mAb 84H10, 10 &mgr;g/ml). This observation has been replicated for T-ALL cell lines Jurkat and Sup TI as well as a subset of patients with T-ALL. However, even though in vivo use of mAbs against LFA-1 or ICAM-1 blocks LFA-1 function in a number of disease models, unfortunately anaphylactic reactions and secondary physiologic effects have hampered this approach (McMuray, 1996; DeMeester: et al., 1996; Jackson et al., 1997; Cuthbertson et al., 1997; Gundel et al., 1992; Haming et al., 1993; Nakano et al., 1995).
Another means to interfere with protein-protein interactions is through the use of small peptide inhibitors. In fact, small peptide inhibitors to adhesion molecules structurally-related to LFA-1 have recently been approved for clinical use in coagulopathies (Ohman et al., 1995; Adgey et al., 1998; Leficovis and Topol, 1995). Short linear peptides (<30 amino acids) have also been described that prevent or interfere with integrin dependent firm adhesion using sequences derived from integrin or their ligands. In particular, these peptides have been derived from a number of integrin receptors: the &bgr;2 and &bgr;3 subunits of integrins, and the &agr;
iib
subunit of ICAM-1, and VCAM-1 (Murayama et al., 1996; Jacobsson and Frykberg, 1996; Zhang and Plow, 1996; Budnik et al., 1996; Vanderslice et al, 1997; Suehiro et al., 1996; Endemann et al., 1996). However, the clinical applicability of these linear peptides is limited. The half maximal inhibitory concentration (IC
50
; concentration at which aggregation is inhibited 50%) for most of these peptides is 10
−4
M with purified receptor-ligand pairs (univalent interactions) and they are ineffective at inhibiting multivalent interactions, during cell-cell adhesion. In addition, linear peptides have short serum half-lives because of proteolysis. Therefore, prohibitively high concentrations of peptide would have to be administered in a clinical setting and a biologic effect would not necessarily occur.
Longer peptides, ranging in length from 25-200 residues, have also been reported to block &bgr;1, &bgr;2, and &bgr;3 integrin dependent adhesion (Zhang and Plow, 1996; Budnik et al., 1996; Vanderslice et al, 1997; Suehiro et al., 1996; Endeman et al., 1996). In general, these peptide inhibitors may have higher affinities or slower off-rates than short peptides and, therefore, are better inhibitors. However, they are still susceptible to proteolysis.
Therefore, a need exists to develop novel and specific classes of pharmaceutical agents to inhibit the binding of LFA-1 and ICAM-1 and to be useful in the treatment of hematopoietic neoplastic diseases as well as other diseases that involve emigration of leukocytes from blood into tissue, such as myocardial infarction, radiation injury, asthma, rheumatoid arthritis, and lymphoma metastasis.
SUMMARY
The present invention addresses the problems in the art by providing compositions that include cyclic peptide inhibitors of binding interactions between the integrin, lymphocyte function associated antigen-1 (LFA-1) expressed on leukocytes, including leukemic T-cells, and intracellular adhesion molecule 1 (ICAM-1), expressed on a variety of cell types. As stated above, this binding of activated LFA-1 is implicated in a variety of diseases and inhibition of this binding interaction with a cyclic peptide inhibitor will have implications in the treatment or management of those diseases.
The present invention may be described, therefore, in certain aspects as a composition comprising a cyclic peptide inhibitor of LFA-1/ICAM-1 interaction, wherein the composition has the amino acid sequence, CLLRMRSIC (SEQ ID NO:3) or a conservative variant thereof. Conservative variants are described elsewhere herein, and include the exchange of an amino acid for another of like charge, size, or hydrophobicity, for example. The present disclosure also include variants of the sequence disclosed above in which the 3
rd
and 4
th
amino acids remain the same and other amino acids of the sequence are substituted and tested empirically for their ability to inhibit the LFA-1/ICAM-1 interaction. The amino acid sequence is numbered in the conventional sense, in that the first amino acid on the N-terminus is cysteine, followed by 7 amino acids and then a carboxy-terminal cysteine. In the practice of the invention, the two terminal cysteines may form a disulfide bonded cystine residue resulting in a cyclic peptide as is well known in the art.
Based on the empirical data obtained by the inventor and disclosed herein, a cyclic peptide of the invention may have the sequence of SEQ ID NO:3, or it may be a derivative of that sequence in which the second amino acid is methionine (SEQ ID NO:17), or in which the 5
th
amino acid is proline (SEQ ID NO:5), or in which the 6
th
amino acid is asparagines (SEQ ID NO:9), or in which the 7
th
amino acid is leucine (SEQ ID NO:2), or in which the 8
th
amino acid is arginine (SEQ ID NO:1), or any combination of these substitutions, or even conservative variants of any of these substitutions. In the preferred embodiments of the invention, the peptides or peptide mimetics of the invention exhibit an inhibition constant (IC
50
) for the binding interaction of LFA-1/ICAM-1 of from about 10 &mgr;M to about 900 &mgr;M for cell aggregation or from about 10 to about 250 nM for monovalent LFA-1/ICAM-1 binding. The term “IC
50
” is well known in the art, and means the half maximal inhibitory concentration, or concentration at which aggregation is inhibited by 50%.
Any of the compositions described herein may be formulated for pharmacological or therapeutic administration either to a mammal, or more preferably to a human. As such, the compo

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