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

2001-01-16

2003-10-07

Chan, Christina (Department: 1644)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S317000, C530S321000

Reexamination Certificate

active

06630447

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 T I 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.
As a part of the present invention, phage display has been used to identify peptide sequences that bind ICAM-1 and block LFA-1/ICAM interaction. Phage that specifically bound ICAM-1 were identified by repeated selection from a cysteine-constrained heptapeptide phage display library. The peptide sequences expressed on ICAM-1 binding phage were determined by nucleotide sequencing. A consensus sequence, CLLRMRSIC (SEQ ID NO:1) was derived from the analysis of the most frequently occurring sequences. Analysis of less frequently recurring amino acids of ICAM-1 binding-phage identified variants of SEQ ID NO: 1 wherein 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 asparagine (SEQ ID NO: 9), or in which the 7
th
amino acid is leucine (SEQ ID NO: 3), or in which the 8
th
amino acid is arginine (SEQ ID NO: 2), or any combination of these substitutions.
Another aspect of the present disclosure is the application of an alanine screening technique to the consensus sequence, SEQ ID NO: 1. An alanine was substituted at each position in the hepatapeptide LLRMRSI of the consensus sequence SEQ ID NO: 1, and each peptide was examined in for its ability to inhibit LFA-1/ICAM-1 mediated cell aggregation. Alanine substitution of the isoleucine at position 8 of SEQ ID NO: 1, i.e., CLLRMRSAC (SEQ ID NO: 40), resulted in a more potent antagonist. Loss of inhibitory function resulted from alanine substitution of the leucines in positions 2 (SEQ ID NO: 34) and 3 (SEQ ID NO: 35), methionine in position 5 (SEQ ID NO: 37), and arginine in position 6 (SEQ ID NO: 38) of SEQ ID NO: 1, indicating that these amino acids are important to the antagonistic activity of the peptide. Substitution of the serine in position 7 (SEQ ID NO: 39) had no significant effect on the inhibitory activity of the peptide. Substitution of the arginine in position 4 (i.e., SEQ ID NO: 36) of SEQ ID NO: 1 was not soluble in aqueous solution at 1 nm and was not tested.
A further aspect of the present invention is the use of conservative amino acid substitutions of the key amino acid residues identified by the alanine screen. Sequences that exhibit greater inhibition of LFA-1/ICAM-1 mediated cell inhibition as co

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