CA2417885A1 - Cell internalized peptide-drug conjugates - Google Patents
Cell internalized peptide-drug conjugates Download PDFInfo
- Publication number
- CA2417885A1 CA2417885A1 CA002417885A CA2417885A CA2417885A1 CA 2417885 A1 CA2417885 A1 CA 2417885A1 CA 002417885 A CA002417885 A CA 002417885A CA 2417885 A CA2417885 A CA 2417885A CA 2417885 A1 CA2417885 A1 CA 2417885A1
- Authority
- CA
- Canada
- Prior art keywords
- peptide
- icam
- drug
- conjugate
- binding
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Peptides which bind to leukocytes are conjugated with drugs. Advantageously, the peptide-drug conjugates interfere with ICAM-1/LFA-1 interactions and are internalized by leukocytes, whereby the drug portion of the conjugate exerts potent toxic side effects against targeted cells with minimal adverse side effects. Preferred conjugates include 4-30 mer peptides derived from ICAM-1 or LFA-1 coupled with drugs effective against leukocyte-related diseases.</SDOA B>
Description
CELL INTERNALIZED PEPTIDE-DRUG CONJUGATES
SEQUENCE LISTING
A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a CRROM.
BACKGROUND OF THE INVENTION
Field of the Invention The present invention is concerned with treating diseases involve ICAM-1 and proteins such as leukemia, Chron's disease, inflammation, asthma, rheumatoid arthritis, and other leukocyte related diseases. More particularly, the present invention is concerned with improving efficacy and reducing toxicity of drugs normally used to treat leukemia, Chron's disease, inflammation, asthma, rheumatoid arthritis, and other leukocyte related diseases. Still more particularly, the present invention is concerned with the conjugation of drugs with peptides which bind to cell adhesion receptors on cell surfaces of leukocytes, endothelial cells, and epithelial cells and are internalized by these cells. Finally, and most particularly, the invention is concerned with the conjugation of drugs with intercellular adhesion molecule-1 (ICAM-1) peptides and lymphocyte function-associated antigen-1 (LFA-1 ) peptides for drug delivery to the cytoplasmic domain of cells expressing ICAM-1 and LFA-1.
Description of the Prior Art Leukocyte-related diseases often result from aberrant immune responses including reactions of leukocytes on "self' antigens. Such reactions contribute to autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis.
Similarly, organtransplantation rej ection results from leukocyte attack, specifically fiom T-cells.
Accordingly inhibition of T-cell actions and their subsequent destruction aids in combating such diseases.
One wayto modulate leukocyte immune response utilizes inhibitors ofthe ICAM-1 receptor interaction. For example, monoclonal antibodies (mAbs) to ICAM-1 and LFA-1 have _2_ been utilized to generate tolerance in immune response disorders such as allograft rej ection (Nato et al., 1996; Nakamura et al., 1996), rheumatoid arthritis (Davis et al., 1995), and autoimmune encephalomyelitis (Willenborg et a1.,1996). Despite the encouraging clinical results in inducing tolerance, such mAbs may be potentially immunogenic and trigger an effectiveness-limiting immunity. In addition, the formulation of antibodies is challenging and costly. Another way to modulate immune response utilizes small peptide fragments derived from ICAM-1 and LFA-1 sequences which inhibit ICAM-1/LFA-1 interaction (Ross et al., 1992; Fecondo et al., 1993;
Benedict et al., 1994; Siahaan et al., 1996). These peptides may have a better physicochemical stability than antibodies and may not possess any immunogenic properties. It has also been shown that a cyclic peptide (cIBR) derived from the sequence of ICAM-1 inhibits ICAM-1 /LFA-1 interactions (Siahaan et al., 1996).
Furthermore, despite the ability to inhibit ICAM-1/LFA-1 interactions and attendant leukocyte-related diseases through treatment with antibodies, such treatments are typically ineffective over the long term due to their transient nature. Additionally, once the mAbs invoke an immune response, their effectiveness is severely limited.
When toxic drugs are used to kill the leukocytes and combat leukocyte-related diseases, many adverse side effects are encountered. These side effects include the non-selective killing of cells in addition to targeted cells as well as the suppression of the proliferation of healthy cells.
Therefore, new methods which selectively target drugs to cells involved in the disease process will be beneficial to patients. For example, selectively targeting cytotoxic drugs to leukocytes will reduce drug toxicity and increase drug efficacy.
SUMMARY OF THE INVENTION
The present invention overcomes the problems inherent in the prior art and provides peptides derived from ICAM-1 and LFA-1 sequences which bind to receptors on leukocytes.
These peptides are conjugated with drugs which are effective in treating leukocyte-related diseases. Such peptides and their conjugates are subsequently internalized by the leukocyte wherein the drug portion of the conjugate exerts toxic effects. Thus, the present invention provides an effective means of drug delivery to the cytoplasmic domain of leukocytes with improved efficacy and reduced toxicity in comparison to conventional methods of treatment.
Because these peptides bind to specific receptors on the surface of the leukocytes, drug delivery is "targeted" to cells having such receptors. This greatly reduces toxic effects on other cells which, due to the non-selectivity of the drugs utilized, generally accompanies treatment of leukocyte-related diseases.
Similarly, these drug-peptide conjugates can be used to treat diseases related to increased expression of ICAM-1 on endothelial and epithelial cells such as inflammation, astlnna, allergies and Chron's disease.
Peptides useful in the present invention include peptides which bind to LFA-1 or ICAM
receptors on leukocytes. Preferably, the peptides are derived from ICAM-1 and LFA-1 sequences and from about 4-30 amino acid residues in length. Still more preferably, such sequences will include from about 8-15 amino acid residues. Most preferably, such sequences will include from about 10-12 amino acid residues. Such sequences are linear and cyclic peptides derived from I-domain of LFA-1, including LAB, cLAB.L, cLAB.C, and cLAB.R which inhibit homotypic aggregation of a certain type of leukocyte, namely T-cells. These sequences are listed herein as SEQ ID Nos. 1-4. Other sequences include sequences derived from an ICAM-1 sequence, IB, listed herein as SEQ ID No. 5. The sequences derived from ICAM sequences are cyclized and include cIBL, cIBC, and cIBR, listed herein as SEQ ID Nos. 6-8, respectively.
Sequences including or having a sequence which has at least about 50% sequence identity with any one of SEQ ID Nos. 1-8 and which exhibits similar internalization properties are within the scope of the present invention. Preferably, such sequences will have at least about 60%
sequence identity with any one of SEQ ID Nos. 1-8 and still more preferably at least about 75%
sequence identity. Alternatively, sequences including or having a sequence which has at least about 50% sequence homology with any one of SEQ ID Nos. 1-8 and which exhibits similar internalization properties are embraced in the present invention. More preferably, such sequences will have at least about 60% sequence homologywith any one of SEQ ID
Nos. 1-8 and still more preferably at least about 75% sequence homology. Additionally, sequences which differ from any one of SEQ ID Nos. 1-8 due to a mutation event but which still exhibit similar properties are also embraced in the present invention. Such mutation events include but are not limited to point mutations, deletions, insertions and rearrangements.
Furthermore, as it is well known in the art, peptidomimetics may be developed which have the same modulation properties as the preferred peptides detailed herein. As these peptidomimetics require no more than routine skill in the art to produce, such peptidomimetics are embraced within the present application.
Notably, the side chains of these peptidomimetics will be very similar in structure to the side chains of the preferred peptides herein, however, their peptide backbone may be very different or even entirely dissimilar.
One major complementary signal for sustaining leukocyte activation is the interaction between ICAM-1(ICAM-1, also known as CD54)and LFA-1 (LFA-1, made up of a dimer comprising CDlla/CD18). Furthermore, the combination of ICAM-1/LFA-1 and complexes modulates the duration and amplitude of leukocyte activation. These complexes induce the movement of actin molecules in the cytoskeleton, thereby producing the accumulation of surface receptors such as LFA-l and ICAM-1 at the interface between antigen presenting cells (APC) and T-cells (Wiilfmg and Davis, 1998). When LFA-1 interacts with ICAM-2 (predominantly expressed on resting endothelium), its interaction with LFA-1 is more important for non-activated circulating T-cells. Meanwhile ICAM-3 is expressed by monocytes and resting lymphocytes and plays a major role in the initiation of the immune response (de Fougerolles et al., 1992).
LFA-lIICAM-1 interaction requires the activation of LFA-1 (Dustin and Springer,1989) which can be triggered by CD2, CD3, phorbol esters (i.e., phorbol 12-myristate-13 acetate or (PMA)) and MHC class II molecules by stimulating protein kinase C (PKC) mechanisms (Rothlein and Springer, 1986; van Kooylc, et al., 1993). PMA activates LFA-1 molecules by directly activating PKC while anti-CD2 and anti-CD3 antibodies activate PKC by stimulating inositol phospholipid metabolisms (Fidgor et al., 1990). Divalent cations contribute to LFA-1/ICAM-1 interactions by enhancing the functional activity of the adhesion molecules (van Kooyk et al., 1993), and the ion type requirement relates to a specific domain of LFA-1 and ICAM-1 (Stanley and Hogg, 1998).
SEQ ID No. 2 was synthesized as a 12-residue peptide containing 10 amino acid residues (I1e23' - G1y246) from the "insert" (I)-domain of LFA-1 which is known to contain residues for ICAM-1 binding (Benedict et al., 1994). Penicillamine (Pen) and cysteine (Cys) residues were then added to the N- and C-termini (Benedict et al., 1994) to form cyclic peptides via a disulfide bond between the Penl and Cysl2 residues. The formation of this cyclic peptide restricts the peptide conformation to produce a conformational stability, thereby providing better selectivity for cell surface receptors than its linear counterpart (Siahaan et al., 1996).
The resulting peptide, cLAB.L, inhibits T-cell aggregation by inhibiting ICAM-lILFA-1 interaction (Siahaan et al., 1996). There are two potential inhibitory mechanisms performed by this cyclic peptide: (a) inhibition of T-cell adhesion by binding to ICAM-1 and/or (b) disruption of a- and p-subunit dimerization by binding to the (3-subunit of LFA-1.
Because the heterodimeric formation of a- and (3-subunits of LFA-1 is necessary for ICAM
binding, disruption of this heterodimeric formation results in an inhibition of the interaction.
Investigations leading to the present invention have also determined and characterized the cLAB.L binding sites. It was discovered that cLAB.L binds to the D1-domain of ICAM-1 as well as to rCAM-3 on the surface of T-cells. Binding of the cLAB.L to the D
1-domain occurs more efficiently than its binding to ICAM-3. The binding characteristics of cLAB.L were evaluated using the FITC-labeled cLAB.L (FTIC-cLAB.L) on activated Molt-3 cells. The FITC-cLAB.L binding to the cell surface-receptor is inhibited by the unlabeled-cLAB.L suggesting that the FITC-cLAB.la bound to the same receptor as unlabeled-cLAB.L. Moreover, the binding of FITC-cLAB.L exhibited bimodal cell-distribution suggesting the occurrence of multiple and dynamic states of activated ICAM receptors. Binding of the peptide to ICAMs (e.g. ICAM-1, ICAM-2, ICAM-3) was enhanced by the presence of a Caz+ and Mg2+ mixture, thereby suggesting the involvement of divalent canons during peptide-ICAM interactions.
Additionally, it was discovered that this peptide was internalized by ICAM receptors on T-cells.
Proof of this internalization was supported by the marked differences in peptide binding between 4°C and 37°C as well as by the presence of the peptide in the cytoplasm when observed by confocal microscopy. This internalization of the peptide, when coupled with drug conjugation, permits delivery of drugs to the cytoplasmic domain of cells having ICAM receptors.
Thus, the invention provides effective treatment of leukocyte-related diseases, because leukocytes have ICAM
receptors.
It is known that the cyclic peptide (cIBR) derived from the sequence of ICAM-1 inhibits ICAM-1 /LFA-1 interactions (Siahaan et al., 1996). It has now been discovered that this peptide binds to and is internalized by LFA-1 surface receptors of T-cells.
Furthermore, it has also been determined that the conformation of this peptide plays an important role in its selectivity for the receptor. Such receptor internalization is believed to be a complementary mechanism of the inhibition of cell-cell adhesion.
Due to the internalization of peptides which bind to receptors on leukocytes, one such peptide, SEQ ID No. 8, was selected for conjugation experiments. To perform the conjugation experiments SEQ ID No. 8 was conjugated with drugs in order to evaluate resultant toxicity to targeted and non-targeted cells. Conjugation was accomplished by dissolving a quantity of drug in water and adding in an amount of peptide prior to storing the resultant solution overnight.
Such conjugates bound to leukocytes expressing LFA-1 thereby interfering with interactions. Binding to the LFA-1 receptors resulted in internalization of the peptide-drug conjugate into the cytoplasmic domain of cells wherein the drug portion of the conjugate exerted potent toxic affects. Advantageously, these toxic affects were localized to cells expressing LFA-1 on their surfaces, thereby delivering these drugs to a specific type of cell. Because only leukocytes express LFA-1 on their surfaces, drugs can be specifically delivered to the surfaces of leukocytes and internalized before exerting their toxic affects and without significant toxicity to other cells. The present invention therefore finds great utility in the treatment of leukocyte-related diseases. Preferably, diseases treatable by such peptide-drug conjugates include Chron's disease, asthma, inflammations, lupus, rheumatoid arthritis, multiple sclerosis, ulceritive colitis, pemphigus vulgaris, pemphigoid, allergies, HIV infections, and epidermolysis.
Thus, by conjugating peptides which are internalized with drugs, internalization of the conjugate by leukocytes contributes to targeted drug delivery with a high degree of specificity and toxicity for targeted cells and decreased toxicity for non-targeted cells.
In the case of peptides derived from LFA-1 sequences which are conjugated with drugs, such conjugates are internalized by leukocytes and epithelial and endothelial cells which have ICAM receptors on their surface. In the case of peptides derived from ICAM sequences which are conjugated with drugs, such conjugates are internalized by leukocytes which have LFA-1 receptors on their surface. Preferably, the peptides are conjugated with drugs effective in destroying leukocytes.
Still more preferably, the conjugated peptides are LFA-1 peptides which can modulate the functions of epithelial and endothelial cells via mechanisms in the cytoplasm such as the mechanisms used by the asthma drugs albuterol and propidium. Preferably, the drug is selected from the group consisting of drugs classified as antiinflammatory agents, antitumor agents (i.e., intercalating agents, tubulin assembly inhibitors, alkylating agents), oligonucleotides, cytokines, enzyme inhibitors (i.e., ACE inhibitors, HIV-protease inhibitors; viral protease inhibitors), and vasoregulator agents. Still more preferably, the drug is selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, propidium. Therefore, the resultant peptide-drug conjugates both interfere with ICAM-1/LFA-1 interactions and destroy leukocytes once the conjugates are internalized. Furthermore, the internalized conjugates may regulate functions of epithelial and endothelial cells such as those present in long epithelial cells and vascular endothelial cells. This ability to regulate functions is due to the presence of ICAM-1 on epithelial and endothelial cells which bind to and internalize LFA-1 peptides.
Notably, the present invention reduces the toxicity of anti-tumor and imrnunosuppressive, and other drugs by increasing the selectivity of the drugs to the target cells. This selectivity also results in increased potency at lower dosage levels. As an example, a drug currently used to treat leukemia, methotrexate (MTX), was conjugated to the N-terminal of the cIBR
peptide to yield a MTX-cIBR conjugate. The conjugate's potency and selectivity was then compared to that of MTX alone. The conjugate was shown to be more potent against leukemic cells (which have LFA-1 receptors) than MTX alone. Additionally, the conjugate was not effective in killing Madin-Darby Canine Kidney (MDCK) cells, which do not have LFA-1 receptors. The potency and selectivity of the conjugates represent a distinct advance in the state of the art as current treatments using MTX have severe side effects including suppression of the proliferation of other healthy cells. Due to the conjugates' increased potency and selectivity, patients will be able to receive lower dosage levels of drugs with fewer adverse side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
The filing of this patent contains at least one drawing executed in color.
Copies of this patent with color drawings) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
Figure 1 is a graph illustrating blocking by cLAB.L peptide on the binding of FITC-conjugate antibodies to ICAM-1, ICAM-3 and LFA-1;
Fig. 2 is a graph illustrating the distribution of Molt-3 cell-population in response to the binding to FITC-cLAB.L whereby cells were preactivated with PMA for 1 hour (a), and cells were preactivated with PMA for 48 hours (b), while distribution of Molt-3 cell population in response to the binding of FITC-conjugate antibody to domain Dl of ICAM-1 is shown in (c);
_g-Fig. 3 is a graph illustrating the binding specificity of FITC-cLAB.L on population-1 and -2 of Molt-3 cells, indicated by the saturation profile of the FITC-cLAB.L
binding (a), and the ability of unlabeled cLAB.L to block the binding of FITC-cLAB.L in concentration-dependent manner (b);
Fig. 4a is a graph illustrating proportions of cell population-1 and -2 in response to the duration of PMA activation;
Fig. 4b is a graph illustrating the effect of cell activation on the binding of 25 ~M FITC-cLAB.L to cell population-1;
Fig. 5a is a graph illustrating time-temperature dependence-profiles of 25 gM
FITC-cLAB.L binding on population-1;
Fig. 5b is a graph illustrating the population-2 of Molt-3 cells;
Fig. 5c is a graph illustrating the effect of cation addition on the intensity of FITC-cLAB.L binding to PMA-activated Molt-3 cells;
Fig. 6a is a confocal microscopy photograph illustrating microscopy of FITC-cLAB.L
binding and internalization on a Molt-3 cell clump at 37°C;
Fig. 6b is a confocal microscopyphotograph ofFITC-cLAB.L binding and internalization on a single Molt-3 cell at 37°C;
Fig. 6c is a confocal microscopyphotograph ofFITC-cLAB.L binding and internalization on a single Molt-3 cell at 4°C' Fig. 6d is a confocal microscopy photograph of surface projection of Molt-3 cell following 37°C incubation;
Fig. 6e is a confocal miscoscopy photograph of the same area as in Fig. 6d and showing the distribution of the FITC-cLAB.L in the cytoplasm;
Fig. 6f illustrates a single confocal section showing a minimal distribution of the FITC-cLAB.L on the cell peripheral;
Fig. 7 is a schematic of two different forms of an MTX-cIBR conjugate in accordance with the present invention;
Fig. 8 is a schematic illustration ofthe internalizationprocess of an MTX-cIBR
conjugate in accordance with the present invention;
Fig. 9 is a graph of an MTT assay comparing the toxicities of different concentration of MTX-cIBR conjugates and MTX alone in accordance with the present invention;
and Fig. 10 is a comparative graph illustrating metabolic activity after treatment with MTX, cIBR, and MTX-cIBR conjugates.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
As used herein, the following definitions will apply: "Sequence Identity" as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences.
Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity.
Sequence identity can be readily calculated by known methods, including but not limited to, those described in: Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York ( 1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et al., eds., M.
Stockton Press, New York (1991); and Carillo, H., et al. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but axe not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.
Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, "sequence homology", as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary.
However, in contrast to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with S another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
A "conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
"Isolated" means altered "by the hand of man" from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. "Drug" means any natural or artificially made chemical for use in the diagnosis, cure, mitigation, treatment, or prevention of illness or disease.
"Targeted cell" means a cell of a specific type or class which exhibits certain physical or functional characteristics and which is of interest due to these characteristics. In the case of medical treatment, when certain types of cells are involved in a disease-type process, treatment can be improved if such cells can be selectively treated without significantly affecting other cells which are not involved in the disease process. Thus, offending cells would be "targeted" and subject to selective treatment while other cells were unaffected.
Similarly, a "non-targeted cell" means a cell which does not have a physical or functional characteristic of a "targeted cell." Preferably, non-targeted cells are not significantly affected during medical treatment.
-12_ EXAMPLES
The following examples set forth preferred embodiments of the present invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
This Example confirmed that a peptide derived from LFA-1 inhibited ICAM-1/LFA-interaction by binding to ICAM sequences. Furthermore, it was determined that this peptide was internalized by leukocytes after binding to ICAM. Thus, peptides such as cLAB.L may be used as a convenient shuttle source to the interior of the leukocyte, thereby providing new methods of treating leukocyte-related diseases. In addition, these peptides can also be used to shuttle drugs to the other cells expressing ICAM-1 such as endothelial and epithelial cells.
Materials and Methods:
Cell Culture and Cell activation. Molt-3 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in suspension in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 mg/L of penicillin/streptomycin. Cells were grown or activated in 75-cm2 tissue culture flasks (Corning) at 37°C in a saturating humidified atmosphere of 95% air and 5% CO2. For the purpose of cell activation, phorbol 12-myristate-13-acetate (PMA) from Sigma (St. Louis, MO) or anti-CD3 antibody from Chemicon (Temecula, CA) was used in concentrations of 0.2 ~,M and 10 ~g/mL, respectively. Incubation with PMA was varied (1, 2, 4, 16, 24, 40 and 48 hours) to study the affect of time on receptor activation. In other cases, periods of PMA activation for 4 hours and 48 hours were compared in their effects on cLAB.L binding to the cells.
Peptide Labeling. 50 mg of cLAB.L peptide was dissolved in a minimal amount of Milli-Q water and added with FITC at two times the molarity of the peptide.
The FITC is reacted to the N-terminus of cLAB.L. The pH mixture was adjusted to 9.0 with 1 N NaOH
and the reaction was run for 1 hour. The pH was then brought to 7.0 by adding 10%
acetic acid. The mixture was lyophilized and purified using preparative reversed-phase (RP) HPLC. The molecular weight of the fraction containing FITC-cLAB.L was confirmed by fast atom bombardment (FAB) mass spectrometry to give M+1=1586.
Peptide Binding Experiment. Activated or non-activated cells were centrifuged at 2000 rpm for 5 minutes and resuspended in serum-free medium to reach a concentration of 3.5 X
106/mL. Peptide stock solution was prepared in phosphate buffer saline (PBS).
Serum-free medium of RPMI1640 was used for the binding experiment in a 48-well cell culture cluster (Corning) into which the mixture of cell suspension, medium, and peptide solution was 400 ~L/well. The mixture was incubated at either 4°C or 37°C. In time-temperature dependence experiments, a sample was taken every 30 minutes for up to 4 hours incubation time. In a concentration-dependence experiment, incubation was carried out for 2 hours.
In experiments to block FITC-cLAB.L binding, cLAB.L was added to the cell suspension followed by incubation for 1 hour at 4°C. The FITC-cLAB.L was then added followed by another hour of incubation.
Cell suspensions without peptide addition were used as controls throughout the experiments.
At the end of incubation, cell suspensions were centrifuged at 3000 g for 3 minutes before being decanted and rinsed with 10 mM Hepes/PBS. The cell pellet was fixed using 4%
paraformaldehyde/PB S for 20 minutes at room temperature. The mixture was then washed twice with 10 mM Hepes/PBS and resuspended in PBS. Samples were analyzed with a flow cytometer (Becton Dickinson). As many as 10,000 cells were counted for every sample, and each experiment was done at least in triplicate. Peptide binding affinity was represented by the average of median values of fluorescence intensity (FI), or in some cases, by its ratio to the relevant standard condition/reference.
Antibody Binding Experiment. A one step-directmethod using FITC-labeled anti-LFA-1, anti-ICAM-1 or anti-ICAM-3 was applied to examine the ability of cLAB.L to block antibody binding to Molt-3 cells. The mAbs to LFA-1 tested were anti-human CD1 la clone 38 and anti-human CD18 clone IB4 from Ancell {Bayport, MN). Another pair of clones (DF1524 and YFCl 18.3 from Accurate (Westbury, NY)) of anti-LFA-1 mAb that have been proven to inhibit aggregation and adhesion were also tested. Clone 15.2 and clone 8.4A6 of anti-ICAM-1 mAbs (Ancell), that recognize Dl- and D2-domain of ICAM-1, respectively, were also used in this study. For ICAM-3, clone 186-2G9 which recognized the D1-domain was used to identify the peptide-ICAM-3 recognition. Peptide solution was incubated with Molt-3 cells for 1 hour prior to the addition of FITC-antibody. The flow cytometer analysis was carried out as described for the peptide binding experiment. A cyclic Arg-Gly-Asp (RGD) peptide (cyclo-(2,10)-Ac-Gly-Pen-Gly-His-Arg-Gly-Asp-Leu-Arg-Cys-Ala-NHZ) (SEQ ID No. 9) was used as a control peptide throughout the antibody binding experiments because LFA-1 does not recognize the tripeptide sequence Arg-Gly-Asp (SEQ ID No. 10) unlike other integrins (i.e., (33 integrins: a~(33, a"b(33 and (31 integrins). Furthermore, ICAM-1 does not contain an RGD sequence (Detmer and Wright, 1988).
Fluorescence and Confocal Microscopy Studies. Samples from peptide-binding experiments at 4°C and 37°C were photographed using fluorescence microscope Nikon Eclipse TE300 and confocal microscope (Biorad MRC100 Laser Scanning Confocal Imaging System connected to Nikon Diaphot 2000 microscope).
Inhibition of Antibody Binding to LFA-1, ICAM-1 and ICAM-3 by cLAB.L. To determine whether the peptide binds to LFA-1, ICAM-1 or ICAM-3, the ability of the cLAB.L
peptide to inhibit binding of FITC-labeled mAbs to LFA-1, ICAM-1 and ICAM-3 was evaluated and a cyclic RGD peptide was used as a negative control (Fig. 1 ). Two peptide concentrations, 80 and 160 wM, were used in this inhibition study. Two anti-ICAM-1 mAbs directed toward the D 1- and D2-domains and an anti-ICAM-3 antibody directed towaxd the D 1-domain of ICAM-3 were used to evaluate the binding properties of cLAB.L. Two pairs of mAbs to LFA-1 were also tested in this experiment. One pair of mAbs was anti-CD 11 a clone 38 and anti-CD-18 clone IB4, neither of which inhibit cell adhesion. The other pair was anti-CDlla clone DF1524 and anti-CD 18 clone YFC 118.3, which both inhibit cell adhesion.
Fig. 1 shows the results for these studies. For a negative control, cyclic RGD
peptide at 80 and 160 qM had no effect on the binding of all the tested mAbs (see controls as the representatives). In the figure, LFA-1 sp indicates the use of antibody specifically described by the manufacturer as an inhibitor of cell adhesion/aggregation. Fluorescence data were normalized betweenfluorescence of FITG-antibodies in the presence of cyclic-RGD peptide (control peptide) and the fluorescence of cells only. Both control peptide and cLAB.L were tested at two different concentrations shown on Fig. 1 as * and * * for 80 and 160 ~M respectively.
The data shown represent the mean ~ S.E. of six determinations. The use of blank bars is to show that no blocking was observed. Binding of anti-ICAM-1 mAbs to D1-domain was blocked by cLAB.L
peptide in a concentration dependent manner. However, the cLAB.L peptide did not inhibit the binding of anti-ICAM-1 antibody to the D2-domain. This result indicates that the cLAB.L
peptide binds to the D1-domain but not D2-domain of ICAM-1. Interestingly, the cLAB.L
peptide can also bind to the D1-domain of ICAM-3 because it can block the binding of anti-ICAM-3 binding to D1-domain. The cLAB.L peptide at a concentration of 160 ~M
blocks the binding of anti-ICAM-1 (D1) and anti-ICAM-3 (D1) 59% and 74%, respectively.
Thus, cLAB.L
prefers binding to ICAM-1 over ICAM-3 thereby indicating that the peptide's major mechanism of activity is inhibition of the ICAM-lILFA-1 interaction. The cLAB.L peptide can weakly inhibit the binding of anti-CD 11 a DF 1524, presumably due to the recognition of cLAB.L by this anti-CDlla antibody. Furthermore, the cLAB.L peptide prevents the binding of antibody to CD 11 a on T-cells. In other words, the same sequence as cLAB.L in CD-11 a is recognized by this anti-CDlla DF1524 antibody. The cLAB.L peptide did not block the binding of other anti-CD 11 a and anti-CD-18 antibodies. This result indicates that cLAB.L may not bind to CD-18 to disrupt the CD 11 a-CD 18 dimerization.
Binding Specificity of FITC-cLAB.L. As illustrated in Figs. 2a and 2b, the flow cytometry experiments show that FITC-cLAB.L binds to two populations (population-1 and -2) of Molt-3 cells. For these experiments, the peptide concentration was 25 ~.M
and the antibody concentration was used at the dilution suggested by the manufacturer. In contrast, FITC-labeled mAbs to ICAM-1 or LFA-1 show binding to only one cell population. This is illustrated in Fig.
2c. For FITC-cLAB.L peptide, the first population (population-1 ) has a high number of cells with low fluorescence intensity while the second population (population-2) has low number of cells but high fluorescence intensity (Figs. 2a and b). This indicates that the receptors in population-2 have a higher affinity than those in population-1.
In order to characterize receptor-mediated binding specificity of FITC-cLAB.L, two different experiments were performed. The first experiment tested concentration-dependent blocking of FITC-cLAB.L binding by the unlabeled cLAB.L. The second experiment tested FITC-cLAB.L binding saturation to the receptors on Molt-3 cells. Results of these experiments are given in Figs. 3 a and 3b. The FITC-cLAB.L binding on 4 hour-preactivated cells approached saturation at around 400 ~,M and 800 ~.M on cell populations -l and -2, respectively. These results are given in Fig. 3a. To examine the blocking ability of unlabeled cLAB.L, two different preactivated cells (i.e., 4 hour and 48 hour activation) were used. These results are shown in Fig.
3b. In Fig. 3a, a comparison was made between cells preactivated with PMA for 4 hours and 48 hours. The percentage of blocking by unlabeled peptide in Fig. 3b was taken as the relative fluorescence values to the binding of 25 ~M FITC-cLAB.L without the addition of unlabeled peptide (ratio of unlabeled and labeled peptide = 0). Each value in Fig. 3a represents the mean of fluorescence values relative to the reference (binding of 3.12 p,M FITC-cLAB.L) ~ S.E. Four determinations were carried out for both experiments which are illustrated in Figs. 3a and 3b.
Error bars are missing when less than the size of a symbol. A blocking experiment was carried out by incubating the cells with unlabeled cLAB.L prior to the addition of FITC-cLAB.L. This reaction was run at 4°C to miW mize peptide internalization (see below). The results show that the unlabeled cLAB.L inhibits the binding of FITC-cLAB.L on cell populations -1 and -2 in a concentration-dependent fashion (Fig. 3b). For population-l, both cell activation times at 4 hours and 48 hours produce comparable inhibitory results. On the other hand, the population-2 of 48 hour-preactivated cells were more prone to blocking by unlabeled peptide than those of 4 hour preactivation. These results indicate that the 4 hour activation provides a higher affinity state of the receptors.
Effect of Activation Time and Activators on FITC-cLAB.L Binding. The number of cells and fluorescence intensities between the two populations were changed at various time of PMA activation. The fluorescence intensity in population-1 increased sharply in the first four hours of PMA activation, and then reached a steady state at 40 hours time of activation. At the same time, the cell number of population-2 increased with increasing activation time while the cell number in population-1 decreased proportionately (Fig. 4a). Each value shown in Fig. 4a represents the mean ~ S.E. of four determinations. The fluorescence intensity from the binding of FITC-cLAB.L (50 E.iM) to PMA-activated cells was three- to six-fold higher than binding to the non-activated cells. Such a difference indicates that cell activation is crucial for peptide binding.
The effect of temperature and different activators (i.e., PMA, anti-CD3 and both Ca2+ and Mg2+) on FITC-cLAB.L binding to the receptors on the activated Molt-3 cells was evaluated. The results forpopulation-1 are shown in Fig. 4b and similar observations were found for population-2. Binding of the FITC-cLAB.L to non-activated Molt-3 cells at 4°C and 37°C was used as a negative control. The binding experiment was carried out in the presence of activating molecules anti-CD3, PMA, or divalent cations (Ca2++ Mgz~ at final concentration of 1.5 mM) at 4°C or 37°C. Peptide binding on non-activated cells at 4°C was used as reference. The data shown represent the mean of fluorescence values relative to the reference ~ S.E. of four determinations.
Error bars are missing when less than the size of symbol. As shown in Figs. 5a and 5b, peptide binding at 4°C and 37°C was compared using cells preactivated with PMA for 4 hours or 48 hours. Again, each value represents the mean of fluorescence value ~ S.E. of four determinations and error bars are missing when less than the size of symbol. The binding reactions were monitored over a four hour period. PMA and CD3 activated Molt-3 cells bind to the peptide with higher affinity than the non-activated cells at 4°C and 37°C. The fluorescence intensities increased over the four hour time period when the experiments were performed at 37°C.
However, the binding of the peptide was suppressed at 4°C thereby indicating that at 37°C, the peptide was internalized by the receptors. In contrast, addition Ca2+ and Mg2+
mixture to the non-activated cells was unable to promote peptide binding. These results indicate that the divalent cations only affected binding of the peptide to the activated cells as shown in Fig. 5c. For this experiment, the final concentrations of Caz+, Mga+, and Ca2++ Mgz+ were 1.5 mM. The control was compared to the intensity of FITC-cLAB.L binding to PMA-activated cells without added canons. The data shown represent the mean of fluorescence values relative to the control ~ S.E.
of three determinations. Error bars are not shown due to very small values.
Effects of Temperature and Divalent Cations on FITC-cLAB.L Binding Affinity.
The effect of activation times and temperature on the FITC-cLAB.L binding to the Molt-3 cells was also evaluated in order to study the relationship of the activated state of the receptors (i.e., ICAM-1) and activation time. The cells were preactivated with PMA for 4 hours and 48 hours.
Binding of FITC-cLAB.L to the cells was sampled every 30 minutes for 240 minutes at 4°C and 37°C. Fluorescence intensities of binding for 30 minutes and 240 minutes incubation time are shown in Figs.Sa and Sb. There are two consistent patterns shown by both population-1 and -2.
First, the fluorescence intensity of peptide binding was lower at 4°C
than at the 37°C incubation.
This result was also confirmed by fluorescence microscopy (Figs. 6a-c). For each of the photos in Fig. 6a-c, the fluorescence and confocal-fluorescence microscopes were used to observe surface binding and internalization respectively. Fig. 6a shows the fluorescence microscopy of Molt-3 cell-clump after incubation at 37°C. Cell population-1 was more susceptible to suppression by low temperature than is cell population-2. Furthermore, flow cytometry of samples taken every 30 minutes showed a more noticeable onset of saturation of binding in population-1 than population-2. These results provide support for the idea that the peptide is internalized by a receptor-mediated process, and population-1 is more sensitive to the energy-dependent internalization than population-2. Additionally, the peptide has a better affinity to both populations at 4 hour activation compared to the 48 hour activation, indicating that the activated state of the receptors diminished with time.
ICAM-1/LFA-1-mediated cell adhesion is dependent on divalent cations, temperature and an intact microfilamentous cytoskeleton (Detmer and Wright, 1988). Therefore, the effect of divalent cations on binding of FITC-cLAB.L to the PMA-activated cells was evaluated. These results are shown in Fig. 5c. A mixture of Mgz+ and Ca2+ improved binding of FITC-cLAB.L 3.4-fold and 1.8-fold to population-1 and -2, respectively, thereby demonstrating that divalent cations have higher influence on the FITC-cLAB.L binding to the receptors in cell population-1 than the receptors in population-2. Similarly, addition of Ca2+ cations alone caused a 1.5-fold binding enhancement of FITC-cLAB.L on population-l, while no effect was detected on population-2.
No peptide binding was observed with the addition of Mg2+ alone. However, Mg2+
addition did increase the cell numbers of population-2 (data not shown). These results demonstrate that binding of cLAB.L to the surface receptor is influenced by the synergistic effects of Ca2+ and Mg2+ canons.
Microscopy of Peptide Binding and Internalization. In addition to flow cytometer analysis, binding and internalization of FITC-cLAB.L at 4°C and 37°C was examined using fluorescence microscope (Figs. 6a-c) and confocal-fluorescence microscope (Figs. 6d-f). Surface binding of FITC-cLAB.L on an aggregate of Molt-3 cells is indicated by the green fluorescence with fluorescence microscope (Fig. 6a), and by the white to grey color with confocal-fluorescence microscope (Fig. 6d). Observation on single cells clearly showed a higher fluorescence on the cell from the 37°C binding experiment (Fig. 6b) than that of the 4°C binding experiment (Fig.
6c). Sectional images of cells from the two different temperatures indicate that peptide internalization took place at 37°C (Fig. 6e), but was almost negligible at 4°C (Fig. 6f).
Discussion These experiments evaluated the mechanisms of action of cLAB.L peptide [cyclo-(1,12)-Pen-Ile-Thr-Asp-Gly-Glu-Ala-Thr-Asp-Ser-Gly-Cys-OH] (SEQ ID No. 2) in inhibiting ICAM-1/LFA-1-mediated homotypic T-cell adhesion. As mentioned above, there are two possible mechanisms of action for this peptide in inhibiting homotypic T-cell adhesion.
One possibility is that the cLAB.L binds to ICAMs (ICAM-1 and ICAM-3) to inhibit ICAMs/LFA-1 interaction.
The second possibility is that the cLAB.L peptide can also bind to the (3-subunit (CD 18) of LFA-1, thereby disrupting the integrity of the LFA-1 and inhibiting the ICAMs/LFA-1 interactions.
From these experiments, it was determined that cLAB.L peptide primarily binds to the D1 domain of ICAM-1. To a lesser extent, the cLAB.L peptide also binds to the D1 domain of ICAM-3 (Fig. 1 ). These results are supported by the ability of this peptide to inhibit anti-ICAM-1 and anti-ICAM-3 antibodies binding to the D 1 domain but not D2 domain. A
weaker blocking effect of cLAB.L for binding of anti-ICAM-3 compared to anti-ICAM-1 may be due to the higher sequence and/or conformational selectivity of cLAB.L for ICAM-1 than ICAM-3.
Although 52%
homology between ICAM-1 and ICAM-3 is largely contributed by domain D2 (77%), many studies have pointed out that the important residues for LFA-1 binding to ICAM-3 is in domain Dl (Hotness, 1995; Bell et al., 1998). Thus, it is the non-conserved residues in D1-domain of ICAM-3 which contribute to integrin binding. These results also emphasize that the ICAM-1/LFA-1 interaction serves a different function than the ICAM-3/LFA-1 interaction.
Results with cLAB.L were also consistent with the previous study of a 9-amino acid linear peptide (TDGEATDSG), provided herein as SEQ ID No. 11. This peptide has sequence similarity with cLAB.L and inhibited binding of MgZ+/EGTA-activated, but not phorbol 12, 13-dibutyrate-activated T-cells, to ICAM-1 soluble protein. This peptide was also the most potent peptide found from the I-domain (McDowall et al., 1998). This linear peptide was shown to reduce binding of T-cells to ICAM-1 to 70% with 2 niM of linear peptides which is higher than the concentrations (80 and 160 ~M) of cLAB.L used to inhibit the binding of ICAM-1-antibody in this work. This indicates that the conformational rigidity of the cyclic peptide cLAB.L
contributes to its binding selectivity (Benedict et al., 1994) to ICAM-1.
The cLAB.L peptide was also found to inhibit binding of anti-LFA-1 antibody to the I-domain (Fig. 1 ). This was due to the recognition of cLAB.L by the anti-LFA-1 antibody because this peptide was derived from the I-domain of the LFA-1. In contrast, this peptide cannot inhibit antibody binding to the [3-subunit and other a-subunit of LFA-1 (Fig. 1).
These results suggest that the primary mechanism of binding of this peptide is via the ICAM-1 D1 domain while weakly binding via the ICAM-3 D1 domain but not to LFA-1.
The characteristics ofblocking ofunlabeled cLAB.L (Fig. 3a), saturationprofile (Fig. 3b), and marked differences in binding between 4°C and 37°C (Fig. 5a and Sb) of FITC-cLAB.L
support the validity of FITC-cLAB.L as a model to study the binding of cLAB.L.
The binding of FITC-cLAB.L featured a two-population distribution and responded to the duration of cell activation (Figs. 2 and 4a), thus suggesting the occurrence of multiple and dynamic states of ICAM-1 and/or ICAM-3. A similar bimodal expression was observed as a result of activation on T-cells with a population ratio 2.5-to-1 (Kurzinger et al., 1981; Sanders et al., 1988). In another case of x.5(31 integrin, integrin activation produces two or more different conformational states reflected in distinct stages in adhesion signaling (Fault et al., 1993; Garcia et a1.,1998). A similar explanation may be used for FITC-cLAB.L binding to ICAMs. The short (4 hour) and long (48 hour) activation times produced two different activated states of ICAMs with different affinities for FITC-cLAB.L. Longer PMA activation time (48 hour) gave a higher number of cell population-2 than a 4 hour PMA activation time. The activated state of ICAMs in population-2 has higher affinity than that shown in population-1.
Population-2 may have an activated ICAM state which forms clusters with high affinities for FITC-cLAB.L
peptide. On the other hand, population-1 has activated ICAMs which spread out through the cell surface with lower affinities for FITC-cLAB.L than for that of in population-2. Thus, a longer activation time gave a change for membrane receptor and cytoskeleton rearrangements.
There are at least two possible explanations for why PMA activation modulates the binding of LFA-1 peptide (cLAB.L) in this case despite the suggested constitutive-avidity of ICAM-1 to LFA-1. The first possible explanation is an indirect mechanism of activation based on the facts that cell-cell adhesion via adhesion molecules consists of a multistep process involving a cascade of recognition and conformational states. In relation to this event, it is apparent that PMA activation of Molt-3 cells triggers other cellular mechanisms such as redistribution of some components ofplasma membrane or release ofreceptors from cytoskeleton which subsequently potentiate the binding of cLAB.L to ICAM-1. Reference may be made to the observed redistribution of lipid packing of the plasma membrane (Smith et a1.,1993; Del Buono et al., 1996) and the postulation on the release of receptors from cytoskeletal constraint (Kucik et al., 1996) due to cell activation. The second possible explanation is that an active LFA-1, resulting from PMA stimulation, directly triggers a more avid ICAM-1 in Molt-3 cells. This explanation may be justified by the generality that T-cell stimulation could lead to induction of ICAM-1 on antigen-presenting cells. The use of PMA has an effect in increasing the binding of JY and SKW3 cells to purified LFA-1 within 0-200 sites Emi z of LFA-1 (Dustin and Springer, 1989), thus indicating a response of ICAM-1 on the surface of T-cells to PMA
stimulation.
Others have also used PMA to induce ICAM-1 expression in epithelial cells (Bloemen et al., 1993) and shown that PMA stimulation may cause a rapid and transient phosphorylation on serine residue of ICAM-3 (Lozano et al., 1992).
Temperature and other activators can influence binding properties of FITC-cLAB.L
peptide to T-cells. For example, anti-CD3 antibody produced the same effect as PMA. However, divalent cations alone did not activate binding between FITC-cLAB.L and ICAMs.
For PMA and anti-CD3 antibody, the fluorescence intensity of FITC-cLAB.L is higher at 37°C than at 4°C in both populations. Thus, FITC-cLAB.L was internalized by ICAM receptors because low temperature (4°C) suppressed the ATP-dependent receptor-internalization. Using fluorescence microscopy, the binding of the labeled peptide is higher at 37°C than at 4°C (Figs. 6a-cs).
Furthermore, internalization of this peptide was confirmed by the sectional images from confocal microscopy (Figs. 6d-f). The internalization of ligand by ICAM receptors may be a process of receptor recycling to control the receptor activity on the cell surface.
Therefore, the receptor-ligand binding followed by endocytosis is one of the mechanisms for the termination of receptor activity (Almenar-Queralt et a1.,1995). This receptor-ligand internalization process is also found in other cell adhesion molecules such as integrin (Raub and I~uentzel, 1989) and VCAM-1 (Ricard et al., 1998).
Binding of FITC-cLAB.L to the Dl domain of ICAM-1 on both populations of PMA
activated Molt-3 T-cells was enhanced dramatically by the presence of a Ca2+
and Mg'+ ion mixture (Fig. 5c). However, Caz+ or Mg2+ alone gave a less dramatic or no change in binding of FITC-cLABLto ICAMs, respectively. Ca2+ improved binding of FITC-cLAB.L to population-1 better than population-2. These results were supported by the suggestion that a section of I-domain binds to the ICAM-1 via divalent cations. Important residues on I-domain of LFA-1 for ICAM-1 binding have been proposed including Met'4°, Glu'46, Thrz43 and Serz45 (Huang and Springer, 1995), and recently Leu2os and Gluzø' (Edwards et al., 1998).
Meanwhile, there are distinct but partially overlapping binding sites in the I-domain of LFA-1 for ICAM-1 and ICAM-3 (Binnerts et al., 1996) but none of the reported residues are part of the cLAB.L sequence. It was shown using CD, NMR and molecular modeling that cLAB.L can bind to Ca2+ and Mg2~. The binding properties of cLAB.L were also evaluated using docking experiments between cLAB.L
and the D 1-domain of ICAM-1 in the presence and absence of divalent cations.
The peptide can bind to ICAM-1 D1-domain via residue numbers 1 (Pen), 2 (Ile), 6 (Glu), and 10 (Ser).
Conclusion In conclusion, direct binding between cLAB.L and ICAM-1 on the surface of Molt-cells is primarily via binding to ICAM-1, and to a lesser extent, via ICAM-3.
The FITC-labeled peptide binding is expressed in bimodal distribution of cell population and it is saturable. The FITC-labeled peptide can be inhibited by the unlabeled cLAB.L, thereby indicating a receptor mediated binding and no interference of FITC conjugation on the selectivity of peptide binding to the receptors. The peptide binding is influenced by a Ca2~ and Mg2+mixture, and the peptide may also be internalized by ICAMs. Accordingly receptor-mediated internalization of cLAB.L, combined with other methods such as drug conjugation, may have useful applications in targeting leukocyte-related diseases.
This Example confirmed that peptides derived from ICAM-1 could be conjugated with drugs and bind with cells expressing LFA-1 surface receptors. Furthermore, the conjugate was internalized and cells which internalized the conjugate were killed in a concentration-dependent manner. Due to the peptide portion of the conjugate and its affinity for LFA-1 receptors, the drug portion of the conjugate was ineffective at killing cells which did not have LFA-1 receptors.
Thus, peptide-drug conjugates were toxic toward selected targeted cells (i.e.
cells expressing LFA-1 receptors) while the conjugate rendered the drug less toxic to .non-targeted cells.
Accordingly, similar conjugates will be effective in treating diseases related to LFA-1 expressing cells (T-cell-related disorders).
Materials and Methods:
Synthesis of MTX-cIBR conjugate:
Methotrexate (10 mg, 0.02 mmol, Sigma A6770) was dissolved in milliQ water and pH
of the solution was adjusted to 7Ø EDC (0.02 mmol). Next, 10 mg (0.008 mmol) cIBR peptide (SEQ ID No. 8) (0.008 mmol) was added into the MTX solution and stored overnight at room temperature. The reaction was concentrated in vacuo and the crude product was purified with reversed-phase HPLC with C-18 column (12 ~,M, 300 ~, 25 cm ~ 21.5 mm i.d.) and UV
detection at 220 nm. A flow rate of 10 ml/min was used using a gradient run using solvent A
which consisted of 0.1% TFA in HZO:acetonitrile (95:5) and solvent B which consisted of 100%
acetonitrile. Fractions were collected and subjected to analytical HPLC in order to determine product purity. Fractions containing the desired product were then pooled, concentrated and lyophilzed. The identity of the conjugate (MTX-cIBR) was confirmed by mass spectrometry (M+1 = 1611.4) and NMR.
Toxicity studies:
Toxicity studies were done by MTT assay, using both activated and non-activated MOLT-3 T-cells. Activation of the MOLT-3 T-cells was accomplished by using PMA
(phorbol 12-myristate-13-acetate) at a final concentration of 0.2~uM. Aliquots of 200~L of cell suspensions containing 2.92 x 105 cells/ml nonactivated, or 2.47 x 105 cells/ml activated MOLT-3 cells with 80% viability were plated onto a 48-well plate. Graded amounts of MTX, MTX-cIBR, and/or cIBR dissolved in PBS were then added to the plates and incubated for 48 hours at 37°C in a humidified 5% COZ/95% air atmosphere. As a negative control, a treatment with lOmM
iodoacetamide (IAA) for 48 hours was used to determine the minimal value of metabolic activity.
A positive control of cells only was used to determine maximum metabolic activity. At the end of the incubation period, 40u1 MTT (Sigma, St. Louis, MO) solution (Smg/ml PBS) was added to each well and further incubated for 2 hours at 37°C. Following incubation, cell suspensions were transferred to microcentrifuge tubes and centrifuged for 3minutes at 3,000 RPM before removing the supernatants. The formazan crystals produced were solubilized by the addition of lml of 0.04 N HCl in isopropanol and subsequently sonicated for 7-10 minutes.
The microcentrifuge tubes were again spun for 3 minutes at 3,000 RPM and 100uL
aliquots of supernatant placed onto a 96 well plate and the absorbance at 540-nm wavelength was immediately measured using a Titertek Multiskan MCC/340. Metabolic activity was calculated as follows, where O.D. is the measured optical density at 540nm.
Metabolic activity = - (O.D.~-O.D.Samp~e)/( O.D.MTT - O.D.,,,~,) Toxicity studies using MDCK cells proceeded as follows. To a 48 well plate, 300~,L aliquots of cell suspension (8 x lO5ce11/ml) were grown to a confluent monolayer. Once a complete monolayer was reached, treatment with graded amounts of MTX, or MTX-cIBR were added and incubated as described. MTT assay was followed as above with one distinction:
the cell monolayer, which remained attached to the plate after treatment, was scraped off and transferred to the microcentrifuge tube.
Results:
Results from toxicity studies show that the MTX-cIBR conjugate is toxic in a concentration dependent manner and is approximately 19 fold more toxic than MTX alone at a similar concentration. To demonstrate that this increase in toxicity was due to the cIBR binding to MOLT-3, it was shown that the toxicity of MTX-cIBR can be inhibited in a concentration dependent manner by adding increasing amounts of cIBR.
The specificity of this drug-peptide conjugate for cells expressing LFA-1 was demonstrated by comparing the toxicity in MDCK epithelial cells which do not express LFA-1.
Treating MDCK cells with concentrations of MTX and MTX-cIBR which produced nearly completely toxic effects in T-cells showed that MTX-cIBR was approximately 5 times less toxic than MTX alone. Without LFA-1 receptors, the conjugate is not carried inside of the cells (e.g.
it is not internalized). Therefore, this binding specificity provides a great deal of selectivity because only T-cells express LFA-1. Accordingly, T-cell-related diseases can be treated with such conjugates without the adverse side effects which generally accompanytreatment with drugs effective against such diseases. Additionally, dosage levels may be lower due to the high potency of the drugs after internalization by the targeted cells. .
Thus, the present invention can be used to conjugate drugs with peptides in order to treat leukocyte-related diseases such as asthma, inflammations, Chron's Disease, rheumatoid arthritis, multiple sclerosis, ulcerative collitis, pemphigus vulgaris, pephigoid, allergies, HIV-infections, and epidermolysis. Additionally, drugs effective in treating such diseases can be made less toxic to non-targeted cells as well as more potent against targeted cells by conjugating them with the peptides of the present invention. Another use of the present invention involves delivering HIV
protease inhibitors to T-cells. This method would help to avoid drug resistance against HIV
protease inhibitors, which has been rising in patients with AIDS. Because the replication of HIV
is in T-cells, the use of peptides targeted to T-cells (i.e. peptides which bind to LFA-1 receptors) is appropriate for suppressing HIV replication. Additionally, the efflux pump receptors may be entirely avoided using the methods and peptide-drug conjugates of the present invention.
Procedures and teachings of the following references are hereby incorporated by reference:
Almenar-Queralt A, et al., Apical topography and modulation of ICAM-1 expression on activated endothelium. Am. J. Pathol. 147:1278-1288 (1995).
Bell ED, et al., The leukocyte function-associated antigen-1 (LFA-1)-binding site on ICAM-3 comprises residues on both faces of the first immunoglobulin domain. J.
Immunol.
161:1363-1370 (1998).
Benedict SH, et al., ICAM-1/LFA-1 short-chain peptides and method of using same. USPateht 229, 531 (1994).
Binnerts ME, et al., Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CDlla I domain. J. Biol. Chem. 271:9962-9968 (1996).
Bloernen PGM, et al., Expression and modulation of adhesion molecules on human bronchial epithelial cells. AnZ. J. Respi~. Cell Mol. Biol. 9:586-593 (1993).
Buckley CD, et al., Role of ligands in the activation of LFA-1. Nature 397:534-539 (1999).
Davis LS, et al., Induction of persistent T cell hyporesponsiveness ih vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis. J. Immu~ol. 154:3525-(1995).
de Fougerolles AR, et al., Characterization of the function of intercellular adhesion molecule .
(ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses. JExp Med 179:619-629 (1994).
Del Buono BJ, et al., Alterations in plasma membrane lipid organization during lymphocyte differentiation. J. Cell. Physiol.126:379-388 (1996).
Detmer PA and Wright SD, Adhesion-promoting receptors on leukocytes. Curr. Op.
Immurcol.
1: 10-15 (1988).
Dustin ML and Springer TA, T cell receptor crosslinking transiently stimulates adhesiveness through LFA-1. Nature (Lohd.) 341:619-624 (1989).
Edwards CP, et al., Mapping the intercellular adhesion molecule-l and -3 binding site on the inserted domain of leukocyte function-associated antigen-1. J. Biol. Chem.
273:28937-28944 (1998).
Faull RJ, et al., J. Cell. Biol. 121:155-162 (1993).
Fecondo JV, et al., Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. AIDS Res. Hum. Retrovirus 9:733-740 (1993).
Fidgor CG, et al., On the mode of action of LFA-1. In2munol. Today 11:277-280 (1990).
Garcia AJ, et al., Two-stage activation for a5 (3, integrin binding to surface-adsorbed fibronectin.
J. Biol. Chem. 273:34710-34715 (1998).
Giirsoy RN and Siahaan TJ, Binding and internalization of an ICAM-1 peptide by the surface receptors of T-cells. J. Peptide. Res. 53:414-421 (1999a).
Gursoy RN and Siahaan TJ, Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1,12)-Pen-Pro-Arg-Gly-Gly-Ser-Val Leu-Val-Thr-Gly-Cys-OH. J. Peptide. Res. 53:432-441 (1999b).
Hotness CL, et al., Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin function-associated antigen 1. J. Biol. Chem. 270:877-884 (1995).
Kato Y, et al., Specific acceptance of fetal bowel allograft in mice after combined treatment with anti-intercellular adhesion molecule-l and leukocyte function-associated antigen-1 antibodies. Ann. Surg. 223:94-100 (1996).
Kucik DF, et al., Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin. Invest. 97:2139-2144 (1996).
Kurzinger K, et al., A novel lymphocyte function-associated antigen (LFA-1):
cellular distribution, quantitative expression, and structure. J. Immunol. 127:596-602 (1981).
Lozano F, et al., Effect of protein-kinase C activators on the phosphorylation and the surface expression of the CD50 leukocyte antigen. Eur. J. Biochem. 203:321-326 (1992).
McDowall A, et al., The I domain of integrin leulcocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1). J. Biol. Chem. 273:27396-27403 (I998).
Nakamura EK, et al., Long-term survival of solid organ allografts by brief anti-lympocyte function-associated antigen-1 monoclonal monotherapy. Transplantation 62:547-(1996).
Raub, TJ and Kuentzel SL, Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using anti-fibronectic receptor beta subunit monoclonal antibody. Exp. Cell Res. 184:407-426 (1989).
Ricaxd I, Payet MD and Dupuis G, VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its a,4(31 integrin counter-receptor remains associated with the plasma membrane in human lymphocytes. Eur. J. Immunol. 28:1708-1718 (1998).
Ross L, et al., Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. J. Biol. Chem. 267:8537-8543 (1992).
Rothlein R and Springer TA, The requirement for lymphocytes function associated antigen-1 in homotypic leukocyte adhesion stimulated by phorbol ester. ,I. Exp.
Med.163:1132-1149 (1986).
Sanders ME, et al., Molecular pathways of adhesion in spontaneous rosetting of T-lymphocytes to the Hodgkin's cell line L428. J. In2munol. 140:1401-1407 (1988).
Siahaan TJ, et al., Counter receptor binding domains that block or enhance binding to LFA-1 or ICAM-1, in Peptides: Chemistry, Structure And Biology (Kaumaya PTP and Hodges RS
eds) pp 792-793, Mayflower Scientific, England (1996).
Smith DM, et al., Plasma membrane lipid packing and leukocyte function-associated antigen-1-dependent aggregation of lymphocytes. J. Cell. Physiol. 156:182-188 (1993).
Stanley P and Hogg N, The I-domain of integrin LFA-1 interacts with ICAM-1 domain-1 at residue glu-34 but not gln-73. J. Biol. Chem. 273:3358-3362 (1998).
van Kooyk Y, et al., Critical amino acids in the lymphocyte function-associated antigen-1 I
domain mediate intercellular adhesion molecule 3 binding and immune function.
Cell Adhesion C019Zm. 1:21-32 (1993).
Willenborg DO, et al., Short teatment with soluble neuroantigen and anti-CDlla (LFA-1) protects rats against autoimmune encephalomyelitis: treatment abrogates autoimmune disease but not autoimmunity. J. Immunol. 157:1973-1980 (1996).
Wiilfmg C and Davis MM, A receptor/cytoslceletal movement triggered by costimulation during T cell activation. Science 282:2266-2269 (1998).
30406.ST25.txt SEQUENCE LISTING
<110> SIAHAAN, TERUNA
YUSUF-MAKAGIANSAR, HELENA
ANDERSON, MEAGAN
XU, BONG
<120> CELL-INTERNALIZED PEPTIDE-DRUG CONJUGATES
<130> 30406 <140> 09/629,719 <141> 2000-08-O1 <160> 11 <170> Patentln version 3.0 <210>1 <211>24 <212>PRT
<213>Homo sapiens <400> 1 Ile Thr Asp Gly Glu Ala Thr Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Ile Ile Tyr Ile Ile Gly Ile <210>2 <211>12 <212>PRT
<213>Homo sapiens 30406.5T25.txt <220>
<221> misc_feature <223> xaa is penicillamine <400> 2 iaa Ile Thr Asp 51y Glu Ala Thr Asp i0er Gly Cys <210> 3 <21I> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 3 xaa Asp ser Gly Asn Ile Asp Ala Ala Lys Asp Cys <210> 4 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 4 Xaa Ala Leu Asp Ile Ile Tyr Ile Ile Gly Ile Cys <210> 5 <211> 21 Page Z
30406.sT25.txt <212> PRT
<213> Homo Sapiens <400> 5 Gln Thr Ser Val Ser Pro ser Lys Val Iie Leu Pro Arg Gly Giy ser Val Leu Val Thr Gly <210>6 <211>12 <212>PRT
<213>Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 6 Xaa Gln Thr Ser Val ser Pro ser Lys val Ile Cys <210> 7 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 7 Xaa Pro ser Lys Val Ile Leu Pro Arg Gly Gly Cys <210> 8 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Xaa is penicillamine 30406.ST25.txt <400> 8 iaa Pro Arg Gly 51y ser val Leu val lhOr Gly cys <210> 9 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Xaa is penicillamine <400> 9 Gly Xaa Gly His Arg Gly Asp Leu Arg Cys Ala <210> 10 <211> 3 <212> PRT
<213> Homo Sapiens <400> 10 Arg Gly Asp <210> 11 <211> 9 <212> PRT
30406.ST25.txt <213> Homo Sapiens <400> 11 Thr Asp Gly Glu Ala Thr Asp Ser Gly
SEQUENCE LISTING
A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a CRROM.
BACKGROUND OF THE INVENTION
Field of the Invention The present invention is concerned with treating diseases involve ICAM-1 and proteins such as leukemia, Chron's disease, inflammation, asthma, rheumatoid arthritis, and other leukocyte related diseases. More particularly, the present invention is concerned with improving efficacy and reducing toxicity of drugs normally used to treat leukemia, Chron's disease, inflammation, asthma, rheumatoid arthritis, and other leukocyte related diseases. Still more particularly, the present invention is concerned with the conjugation of drugs with peptides which bind to cell adhesion receptors on cell surfaces of leukocytes, endothelial cells, and epithelial cells and are internalized by these cells. Finally, and most particularly, the invention is concerned with the conjugation of drugs with intercellular adhesion molecule-1 (ICAM-1) peptides and lymphocyte function-associated antigen-1 (LFA-1 ) peptides for drug delivery to the cytoplasmic domain of cells expressing ICAM-1 and LFA-1.
Description of the Prior Art Leukocyte-related diseases often result from aberrant immune responses including reactions of leukocytes on "self' antigens. Such reactions contribute to autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis.
Similarly, organtransplantation rej ection results from leukocyte attack, specifically fiom T-cells.
Accordingly inhibition of T-cell actions and their subsequent destruction aids in combating such diseases.
One wayto modulate leukocyte immune response utilizes inhibitors ofthe ICAM-1 receptor interaction. For example, monoclonal antibodies (mAbs) to ICAM-1 and LFA-1 have _2_ been utilized to generate tolerance in immune response disorders such as allograft rej ection (Nato et al., 1996; Nakamura et al., 1996), rheumatoid arthritis (Davis et al., 1995), and autoimmune encephalomyelitis (Willenborg et a1.,1996). Despite the encouraging clinical results in inducing tolerance, such mAbs may be potentially immunogenic and trigger an effectiveness-limiting immunity. In addition, the formulation of antibodies is challenging and costly. Another way to modulate immune response utilizes small peptide fragments derived from ICAM-1 and LFA-1 sequences which inhibit ICAM-1/LFA-1 interaction (Ross et al., 1992; Fecondo et al., 1993;
Benedict et al., 1994; Siahaan et al., 1996). These peptides may have a better physicochemical stability than antibodies and may not possess any immunogenic properties. It has also been shown that a cyclic peptide (cIBR) derived from the sequence of ICAM-1 inhibits ICAM-1 /LFA-1 interactions (Siahaan et al., 1996).
Furthermore, despite the ability to inhibit ICAM-1/LFA-1 interactions and attendant leukocyte-related diseases through treatment with antibodies, such treatments are typically ineffective over the long term due to their transient nature. Additionally, once the mAbs invoke an immune response, their effectiveness is severely limited.
When toxic drugs are used to kill the leukocytes and combat leukocyte-related diseases, many adverse side effects are encountered. These side effects include the non-selective killing of cells in addition to targeted cells as well as the suppression of the proliferation of healthy cells.
Therefore, new methods which selectively target drugs to cells involved in the disease process will be beneficial to patients. For example, selectively targeting cytotoxic drugs to leukocytes will reduce drug toxicity and increase drug efficacy.
SUMMARY OF THE INVENTION
The present invention overcomes the problems inherent in the prior art and provides peptides derived from ICAM-1 and LFA-1 sequences which bind to receptors on leukocytes.
These peptides are conjugated with drugs which are effective in treating leukocyte-related diseases. Such peptides and their conjugates are subsequently internalized by the leukocyte wherein the drug portion of the conjugate exerts toxic effects. Thus, the present invention provides an effective means of drug delivery to the cytoplasmic domain of leukocytes with improved efficacy and reduced toxicity in comparison to conventional methods of treatment.
Because these peptides bind to specific receptors on the surface of the leukocytes, drug delivery is "targeted" to cells having such receptors. This greatly reduces toxic effects on other cells which, due to the non-selectivity of the drugs utilized, generally accompanies treatment of leukocyte-related diseases.
Similarly, these drug-peptide conjugates can be used to treat diseases related to increased expression of ICAM-1 on endothelial and epithelial cells such as inflammation, astlnna, allergies and Chron's disease.
Peptides useful in the present invention include peptides which bind to LFA-1 or ICAM
receptors on leukocytes. Preferably, the peptides are derived from ICAM-1 and LFA-1 sequences and from about 4-30 amino acid residues in length. Still more preferably, such sequences will include from about 8-15 amino acid residues. Most preferably, such sequences will include from about 10-12 amino acid residues. Such sequences are linear and cyclic peptides derived from I-domain of LFA-1, including LAB, cLAB.L, cLAB.C, and cLAB.R which inhibit homotypic aggregation of a certain type of leukocyte, namely T-cells. These sequences are listed herein as SEQ ID Nos. 1-4. Other sequences include sequences derived from an ICAM-1 sequence, IB, listed herein as SEQ ID No. 5. The sequences derived from ICAM sequences are cyclized and include cIBL, cIBC, and cIBR, listed herein as SEQ ID Nos. 6-8, respectively.
Sequences including or having a sequence which has at least about 50% sequence identity with any one of SEQ ID Nos. 1-8 and which exhibits similar internalization properties are within the scope of the present invention. Preferably, such sequences will have at least about 60%
sequence identity with any one of SEQ ID Nos. 1-8 and still more preferably at least about 75%
sequence identity. Alternatively, sequences including or having a sequence which has at least about 50% sequence homology with any one of SEQ ID Nos. 1-8 and which exhibits similar internalization properties are embraced in the present invention. More preferably, such sequences will have at least about 60% sequence homologywith any one of SEQ ID
Nos. 1-8 and still more preferably at least about 75% sequence homology. Additionally, sequences which differ from any one of SEQ ID Nos. 1-8 due to a mutation event but which still exhibit similar properties are also embraced in the present invention. Such mutation events include but are not limited to point mutations, deletions, insertions and rearrangements.
Furthermore, as it is well known in the art, peptidomimetics may be developed which have the same modulation properties as the preferred peptides detailed herein. As these peptidomimetics require no more than routine skill in the art to produce, such peptidomimetics are embraced within the present application.
Notably, the side chains of these peptidomimetics will be very similar in structure to the side chains of the preferred peptides herein, however, their peptide backbone may be very different or even entirely dissimilar.
One major complementary signal for sustaining leukocyte activation is the interaction between ICAM-1(ICAM-1, also known as CD54)and LFA-1 (LFA-1, made up of a dimer comprising CDlla/CD18). Furthermore, the combination of ICAM-1/LFA-1 and complexes modulates the duration and amplitude of leukocyte activation. These complexes induce the movement of actin molecules in the cytoskeleton, thereby producing the accumulation of surface receptors such as LFA-l and ICAM-1 at the interface between antigen presenting cells (APC) and T-cells (Wiilfmg and Davis, 1998). When LFA-1 interacts with ICAM-2 (predominantly expressed on resting endothelium), its interaction with LFA-1 is more important for non-activated circulating T-cells. Meanwhile ICAM-3 is expressed by monocytes and resting lymphocytes and plays a major role in the initiation of the immune response (de Fougerolles et al., 1992).
LFA-lIICAM-1 interaction requires the activation of LFA-1 (Dustin and Springer,1989) which can be triggered by CD2, CD3, phorbol esters (i.e., phorbol 12-myristate-13 acetate or (PMA)) and MHC class II molecules by stimulating protein kinase C (PKC) mechanisms (Rothlein and Springer, 1986; van Kooylc, et al., 1993). PMA activates LFA-1 molecules by directly activating PKC while anti-CD2 and anti-CD3 antibodies activate PKC by stimulating inositol phospholipid metabolisms (Fidgor et al., 1990). Divalent cations contribute to LFA-1/ICAM-1 interactions by enhancing the functional activity of the adhesion molecules (van Kooyk et al., 1993), and the ion type requirement relates to a specific domain of LFA-1 and ICAM-1 (Stanley and Hogg, 1998).
SEQ ID No. 2 was synthesized as a 12-residue peptide containing 10 amino acid residues (I1e23' - G1y246) from the "insert" (I)-domain of LFA-1 which is known to contain residues for ICAM-1 binding (Benedict et al., 1994). Penicillamine (Pen) and cysteine (Cys) residues were then added to the N- and C-termini (Benedict et al., 1994) to form cyclic peptides via a disulfide bond between the Penl and Cysl2 residues. The formation of this cyclic peptide restricts the peptide conformation to produce a conformational stability, thereby providing better selectivity for cell surface receptors than its linear counterpart (Siahaan et al., 1996).
The resulting peptide, cLAB.L, inhibits T-cell aggregation by inhibiting ICAM-lILFA-1 interaction (Siahaan et al., 1996). There are two potential inhibitory mechanisms performed by this cyclic peptide: (a) inhibition of T-cell adhesion by binding to ICAM-1 and/or (b) disruption of a- and p-subunit dimerization by binding to the (3-subunit of LFA-1.
Because the heterodimeric formation of a- and (3-subunits of LFA-1 is necessary for ICAM
binding, disruption of this heterodimeric formation results in an inhibition of the interaction.
Investigations leading to the present invention have also determined and characterized the cLAB.L binding sites. It was discovered that cLAB.L binds to the D1-domain of ICAM-1 as well as to rCAM-3 on the surface of T-cells. Binding of the cLAB.L to the D
1-domain occurs more efficiently than its binding to ICAM-3. The binding characteristics of cLAB.L were evaluated using the FITC-labeled cLAB.L (FTIC-cLAB.L) on activated Molt-3 cells. The FITC-cLAB.L binding to the cell surface-receptor is inhibited by the unlabeled-cLAB.L suggesting that the FITC-cLAB.la bound to the same receptor as unlabeled-cLAB.L. Moreover, the binding of FITC-cLAB.L exhibited bimodal cell-distribution suggesting the occurrence of multiple and dynamic states of activated ICAM receptors. Binding of the peptide to ICAMs (e.g. ICAM-1, ICAM-2, ICAM-3) was enhanced by the presence of a Caz+ and Mg2+ mixture, thereby suggesting the involvement of divalent canons during peptide-ICAM interactions.
Additionally, it was discovered that this peptide was internalized by ICAM receptors on T-cells.
Proof of this internalization was supported by the marked differences in peptide binding between 4°C and 37°C as well as by the presence of the peptide in the cytoplasm when observed by confocal microscopy. This internalization of the peptide, when coupled with drug conjugation, permits delivery of drugs to the cytoplasmic domain of cells having ICAM receptors.
Thus, the invention provides effective treatment of leukocyte-related diseases, because leukocytes have ICAM
receptors.
It is known that the cyclic peptide (cIBR) derived from the sequence of ICAM-1 inhibits ICAM-1 /LFA-1 interactions (Siahaan et al., 1996). It has now been discovered that this peptide binds to and is internalized by LFA-1 surface receptors of T-cells.
Furthermore, it has also been determined that the conformation of this peptide plays an important role in its selectivity for the receptor. Such receptor internalization is believed to be a complementary mechanism of the inhibition of cell-cell adhesion.
Due to the internalization of peptides which bind to receptors on leukocytes, one such peptide, SEQ ID No. 8, was selected for conjugation experiments. To perform the conjugation experiments SEQ ID No. 8 was conjugated with drugs in order to evaluate resultant toxicity to targeted and non-targeted cells. Conjugation was accomplished by dissolving a quantity of drug in water and adding in an amount of peptide prior to storing the resultant solution overnight.
Such conjugates bound to leukocytes expressing LFA-1 thereby interfering with interactions. Binding to the LFA-1 receptors resulted in internalization of the peptide-drug conjugate into the cytoplasmic domain of cells wherein the drug portion of the conjugate exerted potent toxic affects. Advantageously, these toxic affects were localized to cells expressing LFA-1 on their surfaces, thereby delivering these drugs to a specific type of cell. Because only leukocytes express LFA-1 on their surfaces, drugs can be specifically delivered to the surfaces of leukocytes and internalized before exerting their toxic affects and without significant toxicity to other cells. The present invention therefore finds great utility in the treatment of leukocyte-related diseases. Preferably, diseases treatable by such peptide-drug conjugates include Chron's disease, asthma, inflammations, lupus, rheumatoid arthritis, multiple sclerosis, ulceritive colitis, pemphigus vulgaris, pemphigoid, allergies, HIV infections, and epidermolysis.
Thus, by conjugating peptides which are internalized with drugs, internalization of the conjugate by leukocytes contributes to targeted drug delivery with a high degree of specificity and toxicity for targeted cells and decreased toxicity for non-targeted cells.
In the case of peptides derived from LFA-1 sequences which are conjugated with drugs, such conjugates are internalized by leukocytes and epithelial and endothelial cells which have ICAM receptors on their surface. In the case of peptides derived from ICAM sequences which are conjugated with drugs, such conjugates are internalized by leukocytes which have LFA-1 receptors on their surface. Preferably, the peptides are conjugated with drugs effective in destroying leukocytes.
Still more preferably, the conjugated peptides are LFA-1 peptides which can modulate the functions of epithelial and endothelial cells via mechanisms in the cytoplasm such as the mechanisms used by the asthma drugs albuterol and propidium. Preferably, the drug is selected from the group consisting of drugs classified as antiinflammatory agents, antitumor agents (i.e., intercalating agents, tubulin assembly inhibitors, alkylating agents), oligonucleotides, cytokines, enzyme inhibitors (i.e., ACE inhibitors, HIV-protease inhibitors; viral protease inhibitors), and vasoregulator agents. Still more preferably, the drug is selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, propidium. Therefore, the resultant peptide-drug conjugates both interfere with ICAM-1/LFA-1 interactions and destroy leukocytes once the conjugates are internalized. Furthermore, the internalized conjugates may regulate functions of epithelial and endothelial cells such as those present in long epithelial cells and vascular endothelial cells. This ability to regulate functions is due to the presence of ICAM-1 on epithelial and endothelial cells which bind to and internalize LFA-1 peptides.
Notably, the present invention reduces the toxicity of anti-tumor and imrnunosuppressive, and other drugs by increasing the selectivity of the drugs to the target cells. This selectivity also results in increased potency at lower dosage levels. As an example, a drug currently used to treat leukemia, methotrexate (MTX), was conjugated to the N-terminal of the cIBR
peptide to yield a MTX-cIBR conjugate. The conjugate's potency and selectivity was then compared to that of MTX alone. The conjugate was shown to be more potent against leukemic cells (which have LFA-1 receptors) than MTX alone. Additionally, the conjugate was not effective in killing Madin-Darby Canine Kidney (MDCK) cells, which do not have LFA-1 receptors. The potency and selectivity of the conjugates represent a distinct advance in the state of the art as current treatments using MTX have severe side effects including suppression of the proliferation of other healthy cells. Due to the conjugates' increased potency and selectivity, patients will be able to receive lower dosage levels of drugs with fewer adverse side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
The filing of this patent contains at least one drawing executed in color.
Copies of this patent with color drawings) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
Figure 1 is a graph illustrating blocking by cLAB.L peptide on the binding of FITC-conjugate antibodies to ICAM-1, ICAM-3 and LFA-1;
Fig. 2 is a graph illustrating the distribution of Molt-3 cell-population in response to the binding to FITC-cLAB.L whereby cells were preactivated with PMA for 1 hour (a), and cells were preactivated with PMA for 48 hours (b), while distribution of Molt-3 cell population in response to the binding of FITC-conjugate antibody to domain Dl of ICAM-1 is shown in (c);
_g-Fig. 3 is a graph illustrating the binding specificity of FITC-cLAB.L on population-1 and -2 of Molt-3 cells, indicated by the saturation profile of the FITC-cLAB.L
binding (a), and the ability of unlabeled cLAB.L to block the binding of FITC-cLAB.L in concentration-dependent manner (b);
Fig. 4a is a graph illustrating proportions of cell population-1 and -2 in response to the duration of PMA activation;
Fig. 4b is a graph illustrating the effect of cell activation on the binding of 25 ~M FITC-cLAB.L to cell population-1;
Fig. 5a is a graph illustrating time-temperature dependence-profiles of 25 gM
FITC-cLAB.L binding on population-1;
Fig. 5b is a graph illustrating the population-2 of Molt-3 cells;
Fig. 5c is a graph illustrating the effect of cation addition on the intensity of FITC-cLAB.L binding to PMA-activated Molt-3 cells;
Fig. 6a is a confocal microscopy photograph illustrating microscopy of FITC-cLAB.L
binding and internalization on a Molt-3 cell clump at 37°C;
Fig. 6b is a confocal microscopyphotograph ofFITC-cLAB.L binding and internalization on a single Molt-3 cell at 37°C;
Fig. 6c is a confocal microscopyphotograph ofFITC-cLAB.L binding and internalization on a single Molt-3 cell at 4°C' Fig. 6d is a confocal microscopy photograph of surface projection of Molt-3 cell following 37°C incubation;
Fig. 6e is a confocal miscoscopy photograph of the same area as in Fig. 6d and showing the distribution of the FITC-cLAB.L in the cytoplasm;
Fig. 6f illustrates a single confocal section showing a minimal distribution of the FITC-cLAB.L on the cell peripheral;
Fig. 7 is a schematic of two different forms of an MTX-cIBR conjugate in accordance with the present invention;
Fig. 8 is a schematic illustration ofthe internalizationprocess of an MTX-cIBR
conjugate in accordance with the present invention;
Fig. 9 is a graph of an MTT assay comparing the toxicities of different concentration of MTX-cIBR conjugates and MTX alone in accordance with the present invention;
and Fig. 10 is a comparative graph illustrating metabolic activity after treatment with MTX, cIBR, and MTX-cIBR conjugates.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
As used herein, the following definitions will apply: "Sequence Identity" as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences.
Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity.
Sequence identity can be readily calculated by known methods, including but not limited to, those described in: Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York ( 1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et al., eds., M.
Stockton Press, New York (1991); and Carillo, H., et al. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but axe not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.
Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, "sequence homology", as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary.
However, in contrast to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with S another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
A "conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
"Isolated" means altered "by the hand of man" from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. "Drug" means any natural or artificially made chemical for use in the diagnosis, cure, mitigation, treatment, or prevention of illness or disease.
"Targeted cell" means a cell of a specific type or class which exhibits certain physical or functional characteristics and which is of interest due to these characteristics. In the case of medical treatment, when certain types of cells are involved in a disease-type process, treatment can be improved if such cells can be selectively treated without significantly affecting other cells which are not involved in the disease process. Thus, offending cells would be "targeted" and subject to selective treatment while other cells were unaffected.
Similarly, a "non-targeted cell" means a cell which does not have a physical or functional characteristic of a "targeted cell." Preferably, non-targeted cells are not significantly affected during medical treatment.
-12_ EXAMPLES
The following examples set forth preferred embodiments of the present invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
This Example confirmed that a peptide derived from LFA-1 inhibited ICAM-1/LFA-interaction by binding to ICAM sequences. Furthermore, it was determined that this peptide was internalized by leukocytes after binding to ICAM. Thus, peptides such as cLAB.L may be used as a convenient shuttle source to the interior of the leukocyte, thereby providing new methods of treating leukocyte-related diseases. In addition, these peptides can also be used to shuttle drugs to the other cells expressing ICAM-1 such as endothelial and epithelial cells.
Materials and Methods:
Cell Culture and Cell activation. Molt-3 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in suspension in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 mg/L of penicillin/streptomycin. Cells were grown or activated in 75-cm2 tissue culture flasks (Corning) at 37°C in a saturating humidified atmosphere of 95% air and 5% CO2. For the purpose of cell activation, phorbol 12-myristate-13-acetate (PMA) from Sigma (St. Louis, MO) or anti-CD3 antibody from Chemicon (Temecula, CA) was used in concentrations of 0.2 ~,M and 10 ~g/mL, respectively. Incubation with PMA was varied (1, 2, 4, 16, 24, 40 and 48 hours) to study the affect of time on receptor activation. In other cases, periods of PMA activation for 4 hours and 48 hours were compared in their effects on cLAB.L binding to the cells.
Peptide Labeling. 50 mg of cLAB.L peptide was dissolved in a minimal amount of Milli-Q water and added with FITC at two times the molarity of the peptide.
The FITC is reacted to the N-terminus of cLAB.L. The pH mixture was adjusted to 9.0 with 1 N NaOH
and the reaction was run for 1 hour. The pH was then brought to 7.0 by adding 10%
acetic acid. The mixture was lyophilized and purified using preparative reversed-phase (RP) HPLC. The molecular weight of the fraction containing FITC-cLAB.L was confirmed by fast atom bombardment (FAB) mass spectrometry to give M+1=1586.
Peptide Binding Experiment. Activated or non-activated cells were centrifuged at 2000 rpm for 5 minutes and resuspended in serum-free medium to reach a concentration of 3.5 X
106/mL. Peptide stock solution was prepared in phosphate buffer saline (PBS).
Serum-free medium of RPMI1640 was used for the binding experiment in a 48-well cell culture cluster (Corning) into which the mixture of cell suspension, medium, and peptide solution was 400 ~L/well. The mixture was incubated at either 4°C or 37°C. In time-temperature dependence experiments, a sample was taken every 30 minutes for up to 4 hours incubation time. In a concentration-dependence experiment, incubation was carried out for 2 hours.
In experiments to block FITC-cLAB.L binding, cLAB.L was added to the cell suspension followed by incubation for 1 hour at 4°C. The FITC-cLAB.L was then added followed by another hour of incubation.
Cell suspensions without peptide addition were used as controls throughout the experiments.
At the end of incubation, cell suspensions were centrifuged at 3000 g for 3 minutes before being decanted and rinsed with 10 mM Hepes/PBS. The cell pellet was fixed using 4%
paraformaldehyde/PB S for 20 minutes at room temperature. The mixture was then washed twice with 10 mM Hepes/PBS and resuspended in PBS. Samples were analyzed with a flow cytometer (Becton Dickinson). As many as 10,000 cells were counted for every sample, and each experiment was done at least in triplicate. Peptide binding affinity was represented by the average of median values of fluorescence intensity (FI), or in some cases, by its ratio to the relevant standard condition/reference.
Antibody Binding Experiment. A one step-directmethod using FITC-labeled anti-LFA-1, anti-ICAM-1 or anti-ICAM-3 was applied to examine the ability of cLAB.L to block antibody binding to Molt-3 cells. The mAbs to LFA-1 tested were anti-human CD1 la clone 38 and anti-human CD18 clone IB4 from Ancell {Bayport, MN). Another pair of clones (DF1524 and YFCl 18.3 from Accurate (Westbury, NY)) of anti-LFA-1 mAb that have been proven to inhibit aggregation and adhesion were also tested. Clone 15.2 and clone 8.4A6 of anti-ICAM-1 mAbs (Ancell), that recognize Dl- and D2-domain of ICAM-1, respectively, were also used in this study. For ICAM-3, clone 186-2G9 which recognized the D1-domain was used to identify the peptide-ICAM-3 recognition. Peptide solution was incubated with Molt-3 cells for 1 hour prior to the addition of FITC-antibody. The flow cytometer analysis was carried out as described for the peptide binding experiment. A cyclic Arg-Gly-Asp (RGD) peptide (cyclo-(2,10)-Ac-Gly-Pen-Gly-His-Arg-Gly-Asp-Leu-Arg-Cys-Ala-NHZ) (SEQ ID No. 9) was used as a control peptide throughout the antibody binding experiments because LFA-1 does not recognize the tripeptide sequence Arg-Gly-Asp (SEQ ID No. 10) unlike other integrins (i.e., (33 integrins: a~(33, a"b(33 and (31 integrins). Furthermore, ICAM-1 does not contain an RGD sequence (Detmer and Wright, 1988).
Fluorescence and Confocal Microscopy Studies. Samples from peptide-binding experiments at 4°C and 37°C were photographed using fluorescence microscope Nikon Eclipse TE300 and confocal microscope (Biorad MRC100 Laser Scanning Confocal Imaging System connected to Nikon Diaphot 2000 microscope).
Inhibition of Antibody Binding to LFA-1, ICAM-1 and ICAM-3 by cLAB.L. To determine whether the peptide binds to LFA-1, ICAM-1 or ICAM-3, the ability of the cLAB.L
peptide to inhibit binding of FITC-labeled mAbs to LFA-1, ICAM-1 and ICAM-3 was evaluated and a cyclic RGD peptide was used as a negative control (Fig. 1 ). Two peptide concentrations, 80 and 160 wM, were used in this inhibition study. Two anti-ICAM-1 mAbs directed toward the D 1- and D2-domains and an anti-ICAM-3 antibody directed towaxd the D 1-domain of ICAM-3 were used to evaluate the binding properties of cLAB.L. Two pairs of mAbs to LFA-1 were also tested in this experiment. One pair of mAbs was anti-CD 11 a clone 38 and anti-CD-18 clone IB4, neither of which inhibit cell adhesion. The other pair was anti-CDlla clone DF1524 and anti-CD 18 clone YFC 118.3, which both inhibit cell adhesion.
Fig. 1 shows the results for these studies. For a negative control, cyclic RGD
peptide at 80 and 160 qM had no effect on the binding of all the tested mAbs (see controls as the representatives). In the figure, LFA-1 sp indicates the use of antibody specifically described by the manufacturer as an inhibitor of cell adhesion/aggregation. Fluorescence data were normalized betweenfluorescence of FITG-antibodies in the presence of cyclic-RGD peptide (control peptide) and the fluorescence of cells only. Both control peptide and cLAB.L were tested at two different concentrations shown on Fig. 1 as * and * * for 80 and 160 ~M respectively.
The data shown represent the mean ~ S.E. of six determinations. The use of blank bars is to show that no blocking was observed. Binding of anti-ICAM-1 mAbs to D1-domain was blocked by cLAB.L
peptide in a concentration dependent manner. However, the cLAB.L peptide did not inhibit the binding of anti-ICAM-1 antibody to the D2-domain. This result indicates that the cLAB.L
peptide binds to the D1-domain but not D2-domain of ICAM-1. Interestingly, the cLAB.L
peptide can also bind to the D1-domain of ICAM-3 because it can block the binding of anti-ICAM-3 binding to D1-domain. The cLAB.L peptide at a concentration of 160 ~M
blocks the binding of anti-ICAM-1 (D1) and anti-ICAM-3 (D1) 59% and 74%, respectively.
Thus, cLAB.L
prefers binding to ICAM-1 over ICAM-3 thereby indicating that the peptide's major mechanism of activity is inhibition of the ICAM-lILFA-1 interaction. The cLAB.L peptide can weakly inhibit the binding of anti-CD 11 a DF 1524, presumably due to the recognition of cLAB.L by this anti-CDlla antibody. Furthermore, the cLAB.L peptide prevents the binding of antibody to CD 11 a on T-cells. In other words, the same sequence as cLAB.L in CD-11 a is recognized by this anti-CDlla DF1524 antibody. The cLAB.L peptide did not block the binding of other anti-CD 11 a and anti-CD-18 antibodies. This result indicates that cLAB.L may not bind to CD-18 to disrupt the CD 11 a-CD 18 dimerization.
Binding Specificity of FITC-cLAB.L. As illustrated in Figs. 2a and 2b, the flow cytometry experiments show that FITC-cLAB.L binds to two populations (population-1 and -2) of Molt-3 cells. For these experiments, the peptide concentration was 25 ~.M
and the antibody concentration was used at the dilution suggested by the manufacturer. In contrast, FITC-labeled mAbs to ICAM-1 or LFA-1 show binding to only one cell population. This is illustrated in Fig.
2c. For FITC-cLAB.L peptide, the first population (population-1 ) has a high number of cells with low fluorescence intensity while the second population (population-2) has low number of cells but high fluorescence intensity (Figs. 2a and b). This indicates that the receptors in population-2 have a higher affinity than those in population-1.
In order to characterize receptor-mediated binding specificity of FITC-cLAB.L, two different experiments were performed. The first experiment tested concentration-dependent blocking of FITC-cLAB.L binding by the unlabeled cLAB.L. The second experiment tested FITC-cLAB.L binding saturation to the receptors on Molt-3 cells. Results of these experiments are given in Figs. 3 a and 3b. The FITC-cLAB.L binding on 4 hour-preactivated cells approached saturation at around 400 ~,M and 800 ~.M on cell populations -l and -2, respectively. These results are given in Fig. 3a. To examine the blocking ability of unlabeled cLAB.L, two different preactivated cells (i.e., 4 hour and 48 hour activation) were used. These results are shown in Fig.
3b. In Fig. 3a, a comparison was made between cells preactivated with PMA for 4 hours and 48 hours. The percentage of blocking by unlabeled peptide in Fig. 3b was taken as the relative fluorescence values to the binding of 25 ~M FITC-cLAB.L without the addition of unlabeled peptide (ratio of unlabeled and labeled peptide = 0). Each value in Fig. 3a represents the mean of fluorescence values relative to the reference (binding of 3.12 p,M FITC-cLAB.L) ~ S.E. Four determinations were carried out for both experiments which are illustrated in Figs. 3a and 3b.
Error bars are missing when less than the size of a symbol. A blocking experiment was carried out by incubating the cells with unlabeled cLAB.L prior to the addition of FITC-cLAB.L. This reaction was run at 4°C to miW mize peptide internalization (see below). The results show that the unlabeled cLAB.L inhibits the binding of FITC-cLAB.L on cell populations -1 and -2 in a concentration-dependent fashion (Fig. 3b). For population-l, both cell activation times at 4 hours and 48 hours produce comparable inhibitory results. On the other hand, the population-2 of 48 hour-preactivated cells were more prone to blocking by unlabeled peptide than those of 4 hour preactivation. These results indicate that the 4 hour activation provides a higher affinity state of the receptors.
Effect of Activation Time and Activators on FITC-cLAB.L Binding. The number of cells and fluorescence intensities between the two populations were changed at various time of PMA activation. The fluorescence intensity in population-1 increased sharply in the first four hours of PMA activation, and then reached a steady state at 40 hours time of activation. At the same time, the cell number of population-2 increased with increasing activation time while the cell number in population-1 decreased proportionately (Fig. 4a). Each value shown in Fig. 4a represents the mean ~ S.E. of four determinations. The fluorescence intensity from the binding of FITC-cLAB.L (50 E.iM) to PMA-activated cells was three- to six-fold higher than binding to the non-activated cells. Such a difference indicates that cell activation is crucial for peptide binding.
The effect of temperature and different activators (i.e., PMA, anti-CD3 and both Ca2+ and Mg2+) on FITC-cLAB.L binding to the receptors on the activated Molt-3 cells was evaluated. The results forpopulation-1 are shown in Fig. 4b and similar observations were found for population-2. Binding of the FITC-cLAB.L to non-activated Molt-3 cells at 4°C and 37°C was used as a negative control. The binding experiment was carried out in the presence of activating molecules anti-CD3, PMA, or divalent cations (Ca2++ Mgz~ at final concentration of 1.5 mM) at 4°C or 37°C. Peptide binding on non-activated cells at 4°C was used as reference. The data shown represent the mean of fluorescence values relative to the reference ~ S.E. of four determinations.
Error bars are missing when less than the size of symbol. As shown in Figs. 5a and 5b, peptide binding at 4°C and 37°C was compared using cells preactivated with PMA for 4 hours or 48 hours. Again, each value represents the mean of fluorescence value ~ S.E. of four determinations and error bars are missing when less than the size of symbol. The binding reactions were monitored over a four hour period. PMA and CD3 activated Molt-3 cells bind to the peptide with higher affinity than the non-activated cells at 4°C and 37°C. The fluorescence intensities increased over the four hour time period when the experiments were performed at 37°C.
However, the binding of the peptide was suppressed at 4°C thereby indicating that at 37°C, the peptide was internalized by the receptors. In contrast, addition Ca2+ and Mg2+
mixture to the non-activated cells was unable to promote peptide binding. These results indicate that the divalent cations only affected binding of the peptide to the activated cells as shown in Fig. 5c. For this experiment, the final concentrations of Caz+, Mga+, and Ca2++ Mgz+ were 1.5 mM. The control was compared to the intensity of FITC-cLAB.L binding to PMA-activated cells without added canons. The data shown represent the mean of fluorescence values relative to the control ~ S.E.
of three determinations. Error bars are not shown due to very small values.
Effects of Temperature and Divalent Cations on FITC-cLAB.L Binding Affinity.
The effect of activation times and temperature on the FITC-cLAB.L binding to the Molt-3 cells was also evaluated in order to study the relationship of the activated state of the receptors (i.e., ICAM-1) and activation time. The cells were preactivated with PMA for 4 hours and 48 hours.
Binding of FITC-cLAB.L to the cells was sampled every 30 minutes for 240 minutes at 4°C and 37°C. Fluorescence intensities of binding for 30 minutes and 240 minutes incubation time are shown in Figs.Sa and Sb. There are two consistent patterns shown by both population-1 and -2.
First, the fluorescence intensity of peptide binding was lower at 4°C
than at the 37°C incubation.
This result was also confirmed by fluorescence microscopy (Figs. 6a-c). For each of the photos in Fig. 6a-c, the fluorescence and confocal-fluorescence microscopes were used to observe surface binding and internalization respectively. Fig. 6a shows the fluorescence microscopy of Molt-3 cell-clump after incubation at 37°C. Cell population-1 was more susceptible to suppression by low temperature than is cell population-2. Furthermore, flow cytometry of samples taken every 30 minutes showed a more noticeable onset of saturation of binding in population-1 than population-2. These results provide support for the idea that the peptide is internalized by a receptor-mediated process, and population-1 is more sensitive to the energy-dependent internalization than population-2. Additionally, the peptide has a better affinity to both populations at 4 hour activation compared to the 48 hour activation, indicating that the activated state of the receptors diminished with time.
ICAM-1/LFA-1-mediated cell adhesion is dependent on divalent cations, temperature and an intact microfilamentous cytoskeleton (Detmer and Wright, 1988). Therefore, the effect of divalent cations on binding of FITC-cLAB.L to the PMA-activated cells was evaluated. These results are shown in Fig. 5c. A mixture of Mgz+ and Ca2+ improved binding of FITC-cLAB.L 3.4-fold and 1.8-fold to population-1 and -2, respectively, thereby demonstrating that divalent cations have higher influence on the FITC-cLAB.L binding to the receptors in cell population-1 than the receptors in population-2. Similarly, addition of Ca2+ cations alone caused a 1.5-fold binding enhancement of FITC-cLAB.L on population-l, while no effect was detected on population-2.
No peptide binding was observed with the addition of Mg2+ alone. However, Mg2+
addition did increase the cell numbers of population-2 (data not shown). These results demonstrate that binding of cLAB.L to the surface receptor is influenced by the synergistic effects of Ca2+ and Mg2+ canons.
Microscopy of Peptide Binding and Internalization. In addition to flow cytometer analysis, binding and internalization of FITC-cLAB.L at 4°C and 37°C was examined using fluorescence microscope (Figs. 6a-c) and confocal-fluorescence microscope (Figs. 6d-f). Surface binding of FITC-cLAB.L on an aggregate of Molt-3 cells is indicated by the green fluorescence with fluorescence microscope (Fig. 6a), and by the white to grey color with confocal-fluorescence microscope (Fig. 6d). Observation on single cells clearly showed a higher fluorescence on the cell from the 37°C binding experiment (Fig. 6b) than that of the 4°C binding experiment (Fig.
6c). Sectional images of cells from the two different temperatures indicate that peptide internalization took place at 37°C (Fig. 6e), but was almost negligible at 4°C (Fig. 6f).
Discussion These experiments evaluated the mechanisms of action of cLAB.L peptide [cyclo-(1,12)-Pen-Ile-Thr-Asp-Gly-Glu-Ala-Thr-Asp-Ser-Gly-Cys-OH] (SEQ ID No. 2) in inhibiting ICAM-1/LFA-1-mediated homotypic T-cell adhesion. As mentioned above, there are two possible mechanisms of action for this peptide in inhibiting homotypic T-cell adhesion.
One possibility is that the cLAB.L binds to ICAMs (ICAM-1 and ICAM-3) to inhibit ICAMs/LFA-1 interaction.
The second possibility is that the cLAB.L peptide can also bind to the (3-subunit (CD 18) of LFA-1, thereby disrupting the integrity of the LFA-1 and inhibiting the ICAMs/LFA-1 interactions.
From these experiments, it was determined that cLAB.L peptide primarily binds to the D1 domain of ICAM-1. To a lesser extent, the cLAB.L peptide also binds to the D1 domain of ICAM-3 (Fig. 1 ). These results are supported by the ability of this peptide to inhibit anti-ICAM-1 and anti-ICAM-3 antibodies binding to the D 1 domain but not D2 domain. A
weaker blocking effect of cLAB.L for binding of anti-ICAM-3 compared to anti-ICAM-1 may be due to the higher sequence and/or conformational selectivity of cLAB.L for ICAM-1 than ICAM-3.
Although 52%
homology between ICAM-1 and ICAM-3 is largely contributed by domain D2 (77%), many studies have pointed out that the important residues for LFA-1 binding to ICAM-3 is in domain Dl (Hotness, 1995; Bell et al., 1998). Thus, it is the non-conserved residues in D1-domain of ICAM-3 which contribute to integrin binding. These results also emphasize that the ICAM-1/LFA-1 interaction serves a different function than the ICAM-3/LFA-1 interaction.
Results with cLAB.L were also consistent with the previous study of a 9-amino acid linear peptide (TDGEATDSG), provided herein as SEQ ID No. 11. This peptide has sequence similarity with cLAB.L and inhibited binding of MgZ+/EGTA-activated, but not phorbol 12, 13-dibutyrate-activated T-cells, to ICAM-1 soluble protein. This peptide was also the most potent peptide found from the I-domain (McDowall et al., 1998). This linear peptide was shown to reduce binding of T-cells to ICAM-1 to 70% with 2 niM of linear peptides which is higher than the concentrations (80 and 160 ~M) of cLAB.L used to inhibit the binding of ICAM-1-antibody in this work. This indicates that the conformational rigidity of the cyclic peptide cLAB.L
contributes to its binding selectivity (Benedict et al., 1994) to ICAM-1.
The cLAB.L peptide was also found to inhibit binding of anti-LFA-1 antibody to the I-domain (Fig. 1 ). This was due to the recognition of cLAB.L by the anti-LFA-1 antibody because this peptide was derived from the I-domain of the LFA-1. In contrast, this peptide cannot inhibit antibody binding to the [3-subunit and other a-subunit of LFA-1 (Fig. 1).
These results suggest that the primary mechanism of binding of this peptide is via the ICAM-1 D1 domain while weakly binding via the ICAM-3 D1 domain but not to LFA-1.
The characteristics ofblocking ofunlabeled cLAB.L (Fig. 3a), saturationprofile (Fig. 3b), and marked differences in binding between 4°C and 37°C (Fig. 5a and Sb) of FITC-cLAB.L
support the validity of FITC-cLAB.L as a model to study the binding of cLAB.L.
The binding of FITC-cLAB.L featured a two-population distribution and responded to the duration of cell activation (Figs. 2 and 4a), thus suggesting the occurrence of multiple and dynamic states of ICAM-1 and/or ICAM-3. A similar bimodal expression was observed as a result of activation on T-cells with a population ratio 2.5-to-1 (Kurzinger et al., 1981; Sanders et al., 1988). In another case of x.5(31 integrin, integrin activation produces two or more different conformational states reflected in distinct stages in adhesion signaling (Fault et al., 1993; Garcia et a1.,1998). A similar explanation may be used for FITC-cLAB.L binding to ICAMs. The short (4 hour) and long (48 hour) activation times produced two different activated states of ICAMs with different affinities for FITC-cLAB.L. Longer PMA activation time (48 hour) gave a higher number of cell population-2 than a 4 hour PMA activation time. The activated state of ICAMs in population-2 has higher affinity than that shown in population-1.
Population-2 may have an activated ICAM state which forms clusters with high affinities for FITC-cLAB.L
peptide. On the other hand, population-1 has activated ICAMs which spread out through the cell surface with lower affinities for FITC-cLAB.L than for that of in population-2. Thus, a longer activation time gave a change for membrane receptor and cytoskeleton rearrangements.
There are at least two possible explanations for why PMA activation modulates the binding of LFA-1 peptide (cLAB.L) in this case despite the suggested constitutive-avidity of ICAM-1 to LFA-1. The first possible explanation is an indirect mechanism of activation based on the facts that cell-cell adhesion via adhesion molecules consists of a multistep process involving a cascade of recognition and conformational states. In relation to this event, it is apparent that PMA activation of Molt-3 cells triggers other cellular mechanisms such as redistribution of some components ofplasma membrane or release ofreceptors from cytoskeleton which subsequently potentiate the binding of cLAB.L to ICAM-1. Reference may be made to the observed redistribution of lipid packing of the plasma membrane (Smith et a1.,1993; Del Buono et al., 1996) and the postulation on the release of receptors from cytoskeletal constraint (Kucik et al., 1996) due to cell activation. The second possible explanation is that an active LFA-1, resulting from PMA stimulation, directly triggers a more avid ICAM-1 in Molt-3 cells. This explanation may be justified by the generality that T-cell stimulation could lead to induction of ICAM-1 on antigen-presenting cells. The use of PMA has an effect in increasing the binding of JY and SKW3 cells to purified LFA-1 within 0-200 sites Emi z of LFA-1 (Dustin and Springer, 1989), thus indicating a response of ICAM-1 on the surface of T-cells to PMA
stimulation.
Others have also used PMA to induce ICAM-1 expression in epithelial cells (Bloemen et al., 1993) and shown that PMA stimulation may cause a rapid and transient phosphorylation on serine residue of ICAM-3 (Lozano et al., 1992).
Temperature and other activators can influence binding properties of FITC-cLAB.L
peptide to T-cells. For example, anti-CD3 antibody produced the same effect as PMA. However, divalent cations alone did not activate binding between FITC-cLAB.L and ICAMs.
For PMA and anti-CD3 antibody, the fluorescence intensity of FITC-cLAB.L is higher at 37°C than at 4°C in both populations. Thus, FITC-cLAB.L was internalized by ICAM receptors because low temperature (4°C) suppressed the ATP-dependent receptor-internalization. Using fluorescence microscopy, the binding of the labeled peptide is higher at 37°C than at 4°C (Figs. 6a-cs).
Furthermore, internalization of this peptide was confirmed by the sectional images from confocal microscopy (Figs. 6d-f). The internalization of ligand by ICAM receptors may be a process of receptor recycling to control the receptor activity on the cell surface.
Therefore, the receptor-ligand binding followed by endocytosis is one of the mechanisms for the termination of receptor activity (Almenar-Queralt et a1.,1995). This receptor-ligand internalization process is also found in other cell adhesion molecules such as integrin (Raub and I~uentzel, 1989) and VCAM-1 (Ricard et al., 1998).
Binding of FITC-cLAB.L to the Dl domain of ICAM-1 on both populations of PMA
activated Molt-3 T-cells was enhanced dramatically by the presence of a Ca2+
and Mg'+ ion mixture (Fig. 5c). However, Caz+ or Mg2+ alone gave a less dramatic or no change in binding of FITC-cLABLto ICAMs, respectively. Ca2+ improved binding of FITC-cLAB.L to population-1 better than population-2. These results were supported by the suggestion that a section of I-domain binds to the ICAM-1 via divalent cations. Important residues on I-domain of LFA-1 for ICAM-1 binding have been proposed including Met'4°, Glu'46, Thrz43 and Serz45 (Huang and Springer, 1995), and recently Leu2os and Gluzø' (Edwards et al., 1998).
Meanwhile, there are distinct but partially overlapping binding sites in the I-domain of LFA-1 for ICAM-1 and ICAM-3 (Binnerts et al., 1996) but none of the reported residues are part of the cLAB.L sequence. It was shown using CD, NMR and molecular modeling that cLAB.L can bind to Ca2+ and Mg2~. The binding properties of cLAB.L were also evaluated using docking experiments between cLAB.L
and the D 1-domain of ICAM-1 in the presence and absence of divalent cations.
The peptide can bind to ICAM-1 D1-domain via residue numbers 1 (Pen), 2 (Ile), 6 (Glu), and 10 (Ser).
Conclusion In conclusion, direct binding between cLAB.L and ICAM-1 on the surface of Molt-cells is primarily via binding to ICAM-1, and to a lesser extent, via ICAM-3.
The FITC-labeled peptide binding is expressed in bimodal distribution of cell population and it is saturable. The FITC-labeled peptide can be inhibited by the unlabeled cLAB.L, thereby indicating a receptor mediated binding and no interference of FITC conjugation on the selectivity of peptide binding to the receptors. The peptide binding is influenced by a Ca2~ and Mg2+mixture, and the peptide may also be internalized by ICAMs. Accordingly receptor-mediated internalization of cLAB.L, combined with other methods such as drug conjugation, may have useful applications in targeting leukocyte-related diseases.
This Example confirmed that peptides derived from ICAM-1 could be conjugated with drugs and bind with cells expressing LFA-1 surface receptors. Furthermore, the conjugate was internalized and cells which internalized the conjugate were killed in a concentration-dependent manner. Due to the peptide portion of the conjugate and its affinity for LFA-1 receptors, the drug portion of the conjugate was ineffective at killing cells which did not have LFA-1 receptors.
Thus, peptide-drug conjugates were toxic toward selected targeted cells (i.e.
cells expressing LFA-1 receptors) while the conjugate rendered the drug less toxic to .non-targeted cells.
Accordingly, similar conjugates will be effective in treating diseases related to LFA-1 expressing cells (T-cell-related disorders).
Materials and Methods:
Synthesis of MTX-cIBR conjugate:
Methotrexate (10 mg, 0.02 mmol, Sigma A6770) was dissolved in milliQ water and pH
of the solution was adjusted to 7Ø EDC (0.02 mmol). Next, 10 mg (0.008 mmol) cIBR peptide (SEQ ID No. 8) (0.008 mmol) was added into the MTX solution and stored overnight at room temperature. The reaction was concentrated in vacuo and the crude product was purified with reversed-phase HPLC with C-18 column (12 ~,M, 300 ~, 25 cm ~ 21.5 mm i.d.) and UV
detection at 220 nm. A flow rate of 10 ml/min was used using a gradient run using solvent A
which consisted of 0.1% TFA in HZO:acetonitrile (95:5) and solvent B which consisted of 100%
acetonitrile. Fractions were collected and subjected to analytical HPLC in order to determine product purity. Fractions containing the desired product were then pooled, concentrated and lyophilzed. The identity of the conjugate (MTX-cIBR) was confirmed by mass spectrometry (M+1 = 1611.4) and NMR.
Toxicity studies:
Toxicity studies were done by MTT assay, using both activated and non-activated MOLT-3 T-cells. Activation of the MOLT-3 T-cells was accomplished by using PMA
(phorbol 12-myristate-13-acetate) at a final concentration of 0.2~uM. Aliquots of 200~L of cell suspensions containing 2.92 x 105 cells/ml nonactivated, or 2.47 x 105 cells/ml activated MOLT-3 cells with 80% viability were plated onto a 48-well plate. Graded amounts of MTX, MTX-cIBR, and/or cIBR dissolved in PBS were then added to the plates and incubated for 48 hours at 37°C in a humidified 5% COZ/95% air atmosphere. As a negative control, a treatment with lOmM
iodoacetamide (IAA) for 48 hours was used to determine the minimal value of metabolic activity.
A positive control of cells only was used to determine maximum metabolic activity. At the end of the incubation period, 40u1 MTT (Sigma, St. Louis, MO) solution (Smg/ml PBS) was added to each well and further incubated for 2 hours at 37°C. Following incubation, cell suspensions were transferred to microcentrifuge tubes and centrifuged for 3minutes at 3,000 RPM before removing the supernatants. The formazan crystals produced were solubilized by the addition of lml of 0.04 N HCl in isopropanol and subsequently sonicated for 7-10 minutes.
The microcentrifuge tubes were again spun for 3 minutes at 3,000 RPM and 100uL
aliquots of supernatant placed onto a 96 well plate and the absorbance at 540-nm wavelength was immediately measured using a Titertek Multiskan MCC/340. Metabolic activity was calculated as follows, where O.D. is the measured optical density at 540nm.
Metabolic activity = - (O.D.~-O.D.Samp~e)/( O.D.MTT - O.D.,,,~,) Toxicity studies using MDCK cells proceeded as follows. To a 48 well plate, 300~,L aliquots of cell suspension (8 x lO5ce11/ml) were grown to a confluent monolayer. Once a complete monolayer was reached, treatment with graded amounts of MTX, or MTX-cIBR were added and incubated as described. MTT assay was followed as above with one distinction:
the cell monolayer, which remained attached to the plate after treatment, was scraped off and transferred to the microcentrifuge tube.
Results:
Results from toxicity studies show that the MTX-cIBR conjugate is toxic in a concentration dependent manner and is approximately 19 fold more toxic than MTX alone at a similar concentration. To demonstrate that this increase in toxicity was due to the cIBR binding to MOLT-3, it was shown that the toxicity of MTX-cIBR can be inhibited in a concentration dependent manner by adding increasing amounts of cIBR.
The specificity of this drug-peptide conjugate for cells expressing LFA-1 was demonstrated by comparing the toxicity in MDCK epithelial cells which do not express LFA-1.
Treating MDCK cells with concentrations of MTX and MTX-cIBR which produced nearly completely toxic effects in T-cells showed that MTX-cIBR was approximately 5 times less toxic than MTX alone. Without LFA-1 receptors, the conjugate is not carried inside of the cells (e.g.
it is not internalized). Therefore, this binding specificity provides a great deal of selectivity because only T-cells express LFA-1. Accordingly, T-cell-related diseases can be treated with such conjugates without the adverse side effects which generally accompanytreatment with drugs effective against such diseases. Additionally, dosage levels may be lower due to the high potency of the drugs after internalization by the targeted cells. .
Thus, the present invention can be used to conjugate drugs with peptides in order to treat leukocyte-related diseases such as asthma, inflammations, Chron's Disease, rheumatoid arthritis, multiple sclerosis, ulcerative collitis, pemphigus vulgaris, pephigoid, allergies, HIV-infections, and epidermolysis. Additionally, drugs effective in treating such diseases can be made less toxic to non-targeted cells as well as more potent against targeted cells by conjugating them with the peptides of the present invention. Another use of the present invention involves delivering HIV
protease inhibitors to T-cells. This method would help to avoid drug resistance against HIV
protease inhibitors, which has been rising in patients with AIDS. Because the replication of HIV
is in T-cells, the use of peptides targeted to T-cells (i.e. peptides which bind to LFA-1 receptors) is appropriate for suppressing HIV replication. Additionally, the efflux pump receptors may be entirely avoided using the methods and peptide-drug conjugates of the present invention.
Procedures and teachings of the following references are hereby incorporated by reference:
Almenar-Queralt A, et al., Apical topography and modulation of ICAM-1 expression on activated endothelium. Am. J. Pathol. 147:1278-1288 (1995).
Bell ED, et al., The leukocyte function-associated antigen-1 (LFA-1)-binding site on ICAM-3 comprises residues on both faces of the first immunoglobulin domain. J.
Immunol.
161:1363-1370 (1998).
Benedict SH, et al., ICAM-1/LFA-1 short-chain peptides and method of using same. USPateht 229, 531 (1994).
Binnerts ME, et al., Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CDlla I domain. J. Biol. Chem. 271:9962-9968 (1996).
Bloernen PGM, et al., Expression and modulation of adhesion molecules on human bronchial epithelial cells. AnZ. J. Respi~. Cell Mol. Biol. 9:586-593 (1993).
Buckley CD, et al., Role of ligands in the activation of LFA-1. Nature 397:534-539 (1999).
Davis LS, et al., Induction of persistent T cell hyporesponsiveness ih vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis. J. Immu~ol. 154:3525-(1995).
de Fougerolles AR, et al., Characterization of the function of intercellular adhesion molecule .
(ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses. JExp Med 179:619-629 (1994).
Del Buono BJ, et al., Alterations in plasma membrane lipid organization during lymphocyte differentiation. J. Cell. Physiol.126:379-388 (1996).
Detmer PA and Wright SD, Adhesion-promoting receptors on leukocytes. Curr. Op.
Immurcol.
1: 10-15 (1988).
Dustin ML and Springer TA, T cell receptor crosslinking transiently stimulates adhesiveness through LFA-1. Nature (Lohd.) 341:619-624 (1989).
Edwards CP, et al., Mapping the intercellular adhesion molecule-l and -3 binding site on the inserted domain of leukocyte function-associated antigen-1. J. Biol. Chem.
273:28937-28944 (1998).
Faull RJ, et al., J. Cell. Biol. 121:155-162 (1993).
Fecondo JV, et al., Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. AIDS Res. Hum. Retrovirus 9:733-740 (1993).
Fidgor CG, et al., On the mode of action of LFA-1. In2munol. Today 11:277-280 (1990).
Garcia AJ, et al., Two-stage activation for a5 (3, integrin binding to surface-adsorbed fibronectin.
J. Biol. Chem. 273:34710-34715 (1998).
Giirsoy RN and Siahaan TJ, Binding and internalization of an ICAM-1 peptide by the surface receptors of T-cells. J. Peptide. Res. 53:414-421 (1999a).
Gursoy RN and Siahaan TJ, Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1,12)-Pen-Pro-Arg-Gly-Gly-Ser-Val Leu-Val-Thr-Gly-Cys-OH. J. Peptide. Res. 53:432-441 (1999b).
Hotness CL, et al., Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin function-associated antigen 1. J. Biol. Chem. 270:877-884 (1995).
Kato Y, et al., Specific acceptance of fetal bowel allograft in mice after combined treatment with anti-intercellular adhesion molecule-l and leukocyte function-associated antigen-1 antibodies. Ann. Surg. 223:94-100 (1996).
Kucik DF, et al., Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin. Invest. 97:2139-2144 (1996).
Kurzinger K, et al., A novel lymphocyte function-associated antigen (LFA-1):
cellular distribution, quantitative expression, and structure. J. Immunol. 127:596-602 (1981).
Lozano F, et al., Effect of protein-kinase C activators on the phosphorylation and the surface expression of the CD50 leukocyte antigen. Eur. J. Biochem. 203:321-326 (1992).
McDowall A, et al., The I domain of integrin leulcocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1). J. Biol. Chem. 273:27396-27403 (I998).
Nakamura EK, et al., Long-term survival of solid organ allografts by brief anti-lympocyte function-associated antigen-1 monoclonal monotherapy. Transplantation 62:547-(1996).
Raub, TJ and Kuentzel SL, Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using anti-fibronectic receptor beta subunit monoclonal antibody. Exp. Cell Res. 184:407-426 (1989).
Ricaxd I, Payet MD and Dupuis G, VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its a,4(31 integrin counter-receptor remains associated with the plasma membrane in human lymphocytes. Eur. J. Immunol. 28:1708-1718 (1998).
Ross L, et al., Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. J. Biol. Chem. 267:8537-8543 (1992).
Rothlein R and Springer TA, The requirement for lymphocytes function associated antigen-1 in homotypic leukocyte adhesion stimulated by phorbol ester. ,I. Exp.
Med.163:1132-1149 (1986).
Sanders ME, et al., Molecular pathways of adhesion in spontaneous rosetting of T-lymphocytes to the Hodgkin's cell line L428. J. In2munol. 140:1401-1407 (1988).
Siahaan TJ, et al., Counter receptor binding domains that block or enhance binding to LFA-1 or ICAM-1, in Peptides: Chemistry, Structure And Biology (Kaumaya PTP and Hodges RS
eds) pp 792-793, Mayflower Scientific, England (1996).
Smith DM, et al., Plasma membrane lipid packing and leukocyte function-associated antigen-1-dependent aggregation of lymphocytes. J. Cell. Physiol. 156:182-188 (1993).
Stanley P and Hogg N, The I-domain of integrin LFA-1 interacts with ICAM-1 domain-1 at residue glu-34 but not gln-73. J. Biol. Chem. 273:3358-3362 (1998).
van Kooyk Y, et al., Critical amino acids in the lymphocyte function-associated antigen-1 I
domain mediate intercellular adhesion molecule 3 binding and immune function.
Cell Adhesion C019Zm. 1:21-32 (1993).
Willenborg DO, et al., Short teatment with soluble neuroantigen and anti-CDlla (LFA-1) protects rats against autoimmune encephalomyelitis: treatment abrogates autoimmune disease but not autoimmunity. J. Immunol. 157:1973-1980 (1996).
Wiilfmg C and Davis MM, A receptor/cytoslceletal movement triggered by costimulation during T cell activation. Science 282:2266-2269 (1998).
30406.ST25.txt SEQUENCE LISTING
<110> SIAHAAN, TERUNA
YUSUF-MAKAGIANSAR, HELENA
ANDERSON, MEAGAN
XU, BONG
<120> CELL-INTERNALIZED PEPTIDE-DRUG CONJUGATES
<130> 30406 <140> 09/629,719 <141> 2000-08-O1 <160> 11 <170> Patentln version 3.0 <210>1 <211>24 <212>PRT
<213>Homo sapiens <400> 1 Ile Thr Asp Gly Glu Ala Thr Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Ile Ile Tyr Ile Ile Gly Ile <210>2 <211>12 <212>PRT
<213>Homo sapiens 30406.5T25.txt <220>
<221> misc_feature <223> xaa is penicillamine <400> 2 iaa Ile Thr Asp 51y Glu Ala Thr Asp i0er Gly Cys <210> 3 <21I> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 3 xaa Asp ser Gly Asn Ile Asp Ala Ala Lys Asp Cys <210> 4 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 4 Xaa Ala Leu Asp Ile Ile Tyr Ile Ile Gly Ile Cys <210> 5 <211> 21 Page Z
30406.sT25.txt <212> PRT
<213> Homo Sapiens <400> 5 Gln Thr Ser Val Ser Pro ser Lys Val Iie Leu Pro Arg Gly Giy ser Val Leu Val Thr Gly <210>6 <211>12 <212>PRT
<213>Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 6 Xaa Gln Thr Ser Val ser Pro ser Lys val Ile Cys <210> 7 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> xaa is penicillamine <400> 7 Xaa Pro ser Lys Val Ile Leu Pro Arg Gly Gly Cys <210> 8 <211> 12 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Xaa is penicillamine 30406.ST25.txt <400> 8 iaa Pro Arg Gly 51y ser val Leu val lhOr Gly cys <210> 9 <211> 11 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Xaa is penicillamine <400> 9 Gly Xaa Gly His Arg Gly Asp Leu Arg Cys Ala <210> 10 <211> 3 <212> PRT
<213> Homo Sapiens <400> 10 Arg Gly Asp <210> 11 <211> 9 <212> PRT
30406.ST25.txt <213> Homo Sapiens <400> 11 Thr Asp Gly Glu Ala Thr Asp Ser Gly
Claims (34)
1. A conjugate comprising a drug coupled with an isolated peptide sequence selected from the group consisting of peptide sequences derived from ICAM-1 and LFA-1 and peptidomimetics thereof.
2. The conjugate of claim 1, said isolated peptide sequence having from about 4-30 amino acid residues.
3. The conjugate of claim 1 said isolated peptide sequence selected from the group consisting of SEQ ID Nos. 1-8 and peptidomimetics thereof.
4. The conjugate of claim 3, said peptide differing from that of said isolated peptide sequence selected from the group consisting of SEQ ID. Nos. 1-8 due to a mutation event.
5. The conjugate of claim 4, said mutation event being selected from the group consisting of point mutations, deletions, insertions and rearrangements.
6. The conjugate of claim 1, said drug selected from a class of drugs consisting of antiinflammatory agents, antitumor agents, oligonucleotides, cytokines, enzyme inhibitors, and vasoregulator agents.
7. The conjugate of claim 1 said drug selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, and propidium.
8. The conjugate of claim 1, said drug being methotrexate.
9. The conjugate of claim 3, said isolated peptide sequence having at least about 50% homology with at least one of said SEQ ID Nos. 1-8 and peptidomimetics thereof.
10. A method of delivering drugs to leukocytes comprising the steps of:
forming a conjugate comprising a drug and an isolated peptide sequence selected from the group consisting of peptide sequences derived from ICAM-1 and LFA-1 sequences and peptidomimetics thereof;
contacting a leukocyte, epithelial cell, or endothelial cell with said conjugate; and causing said conjugate to be internalized within the leukocyte, epithelial cell, or endothelial cell.
forming a conjugate comprising a drug and an isolated peptide sequence selected from the group consisting of peptide sequences derived from ICAM-1 and LFA-1 sequences and peptidomimetics thereof;
contacting a leukocyte, epithelial cell, or endothelial cell with said conjugate; and causing said conjugate to be internalized within the leukocyte, epithelial cell, or endothelial cell.
11. The method of claim 10, said drug selected from a class of drugs consisting of antiinflammatory agents, antitumor agents, oligonucleotides, cytolcines, enzyme inhibitors, and vasoregulator agents.
12. The method of claim 10, said drug being selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, and propidium.
13. The method of claim 10, said drug being selected from the group consisting of methotrexate and doxorubicin.
14. The method of claim 10, said isolated peptide sequence having from about 4-30 amino acid residues.
15. The method of claim 10, said isolated peptide sequence being selected from the group consisting of SEQ ID Nos. 1-8 and peptidomimetics thereof.
16. The method of claim 15, said isolated peptide sequence having at least about 50% homology with at least one of said SEQ ID Nos. 1-8 and peptidomimetics thereof.
17. In a method of administering a drug to cells wherein the improvement comprises reducing the toxicity of the drug to non-targeted cells, said method comprising the step of coupling said drug with a peptide.
18. The method of claim 17, said drug selected from a class of drugs consisting of antiinflammatory agents, antitumor agents, oligonucleotides, cytokines, enzyme inhibitors, and vasoregulator agents.
19. The method of claim 17, said drug being selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, and propidium.
20. The method of claim 17, said drug being selected from the group consisting of methotrexate and doxorubicin.
21. The method of claim 17, said peptide having from about 4-30 amino acid residues.
22. The method of claim 17, said peptide being selected from the group consisting of SEQ ID Nos. 1-8 and peptidomimetics thereof.
23. The method of claim 21, said peptide having at least about 50% sequence homology with at least one of said SEQ ID Nos. 1-8 and peptidomimetics thereof.
24. A method of treating leukocyte-related diseases comprising the steps of:
conjugating a drug with a peptide sequence derived from the sequence of ICAM-1 and peptidomimetics thereof in order to produce a peptide-drug conjugate, said peptide sequence being adapted to bind with LFA-1 receptors;
contacting said conjugate with a leukocyte;
causing said conjugate to be internalized by the leukocyte; and causing said drug to kill the leukocyte.
conjugating a drug with a peptide sequence derived from the sequence of ICAM-1 and peptidomimetics thereof in order to produce a peptide-drug conjugate, said peptide sequence being adapted to bind with LFA-1 receptors;
contacting said conjugate with a leukocyte;
causing said conjugate to be internalized by the leukocyte; and causing said drug to kill the leukocyte.
25. The method of claim 24, said peptide sequence having from about 4-30 amino acid residues.
26. The method of claim 24, said peptide sequence being selected from the group consisting of sequences having at least about 50% sequence homology with at least one of SEQ ID Nos. 1-8 and peptidomimetics thereof.
27. The method of claim 24, said drug selected from a class of drugs consisting of antiinflammatory agents, antitumor agents, oligonucleotides, cytolcines, enzyme inhibitors, and vasoregulator agents.
28. The method of claim 24, said drug selected from the group consisting of methotrexate, lovastatin, taxol, ajmalicine, vinblastine, vincristine, cyclophosphamide, fluorouracil, idarubicin, ifosfamide, irinotecan, 6-mercaptopurine, mytomycins, mitoxantrone, paclitaxel, taxol, pentostatin, plicamycin, topotecan, fludarabine, etoposide, doxorubicin, doxotaxel, danorubicin, albuterol, and propidium.
29. A method of treating an epithelial or endothelial cell-related disease comprising the steps of:
conjugating a drug with a peptide derived from LFA-1 and peptidomimetics thereof;
contacting said conjugate with a leukocyte, epithelial cell, or endothelial cell;
causing said conjugate to be internalized by the leukocyte, epithelial, or endothelial cell;
and causing said conjugate to modulate the function of the contacted leukocyte, epithelial, or endothelial cell.
conjugating a drug with a peptide derived from LFA-1 and peptidomimetics thereof;
contacting said conjugate with a leukocyte, epithelial cell, or endothelial cell;
causing said conjugate to be internalized by the leukocyte, epithelial, or endothelial cell;
and causing said conjugate to modulate the function of the contacted leukocyte, epithelial, or endothelial cell.
30. The method of claim 29, said disease being selected from the group consisting of asthma, inflammations, Chron's Disease, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, pemphigus vulgaris, pephigoid, allergies, HIV-infections, and epidermolysis.
31. The method of claim 29, said disease being related to an increased expression of ICAM-1.
32. The method of claim 29, said peptide being adapted to bind with ICAM-1 receptors.
33. The method of claim 29, said peptide having from about 4-30 amino acid residues.
34. The method of claim 29, said peptide being selected from the group consisting of sequences having at least about 50% sequence homology with at least one of Sequence ID Nos. 1-8 and peptidomimetics thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62971900A | 2000-08-01 | 2000-08-01 | |
US09/629,719 | 2000-08-01 | ||
PCT/US2001/024088 WO2002009649A2 (en) | 2000-08-01 | 2001-08-01 | Cell internalized peptide-drug conjugates |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2417885A1 true CA2417885A1 (en) | 2002-02-07 |
Family
ID=24524196
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002417885A Abandoned CA2417885A1 (en) | 2000-08-01 | 2001-08-01 | Cell internalized peptide-drug conjugates |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1311280A4 (en) |
JP (1) | JP2004505029A (en) |
AU (1) | AU2001278113A1 (en) |
CA (1) | CA2417885A1 (en) |
WO (1) | WO2002009649A2 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1866416A2 (en) * | 2005-03-22 | 2007-12-19 | Medstar Health Inc. | Delivery systems and methods for diagnosing and treating cardiovascular diseases |
WO2012105813A2 (en) * | 2011-02-02 | 2012-08-09 | Medipost Co., Ltd. | Use of icam-1 for prevention or treatment of neurological diseases |
EP3497452A4 (en) | 2016-08-12 | 2020-01-22 | Immunowork, LLC | Diagnosis, prevention, and/or treatment of autoimmune diseases |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5183805A (en) * | 1990-08-13 | 1993-02-02 | Board Of Regents, The University Of Texas System | Bioactive egf peptides for promotion of tissue regeneration and cancer therapy |
JPH09512021A (en) * | 1994-04-19 | 1997-12-02 | ザ ユニバーシティ オブ カンサス | ICAM-1 / LFA-1 short chain peptides and methods of using the peptides |
CA2252468A1 (en) * | 1996-04-26 | 1997-11-06 | The University Of Kansas | Peptide compositions which induce immune tolerance and methods of use |
-
2001
- 2001-08-01 AU AU2001278113A patent/AU2001278113A1/en not_active Abandoned
- 2001-08-01 WO PCT/US2001/024088 patent/WO2002009649A2/en not_active Application Discontinuation
- 2001-08-01 EP EP01956077A patent/EP1311280A4/en not_active Withdrawn
- 2001-08-01 JP JP2002515204A patent/JP2004505029A/en not_active Withdrawn
- 2001-08-01 CA CA002417885A patent/CA2417885A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
AU2001278113A1 (en) | 2002-02-13 |
WO2002009649A2 (en) | 2002-02-07 |
EP1311280A4 (en) | 2005-02-23 |
EP1311280A2 (en) | 2003-05-21 |
JP2004505029A (en) | 2004-02-19 |
WO2002009649A8 (en) | 2002-08-01 |
WO2002009649A3 (en) | 2002-04-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0758383B1 (en) | Lag-3 protein soluble polypeptide fractions, method of production, therapeutic composition and anti-idiotype antibody | |
JP5670961B2 (en) | Composition comprising ShK toxin | |
Kupfer et al. | Small splenic B cells that bind to antigen-specific T helper (Th) cells and face the site of cytokine production in the Th cells selectively proliferate: immunofluorescence microscopic studies of Th-B antigen-presenting cell interactions. | |
Ross | Regulation of the Adhesion versus Cytotoxic Functions of the Mac-1/CR3/α M β 2-lntegrin Glycoprotein | |
JPH09503511A (en) | Cell and serum protein anchors and conjugates | |
AU677772B2 (en) | Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA -3 interaction | |
Moingeon et al. | Complementary roles for CD2 and LFA‐1 adhesion pathways during T cell activation | |
Seaman et al. | Inositol trisphosphate is generated by a rat natural killer cell tumor in response to target cells or to crosslinked monoclonal antibody OX-34: possible signaling role for the OX-34 determinant during activation by target cells. | |
Lagaudrière‐Gesbert et al. | Signaling through the tetraspanin CD82 triggers its association with the cytoskeleton leading to sustained morphological changes and T cell activation | |
Rao et al. | Potent costimulation of effector T lymphocytes by human collagen type I | |
HU217176B (en) | Novel intercellular adhesion molecule and its binding ligands, compositions containing thereof and process for producing thereof | |
Lundblad et al. | The interaction of thrombin with blood platelets | |
AU751151B2 (en) | T-cell membrane protein (TIRC7), peptides and antibodies derived therefrom and uses thereof | |
Gürsoy et al. | Binding and internalization of an ICAM‐1 peptide by the surface receptors of T cells | |
Farries et al. | Competition for binding sites on C3b by CRI, CR2, MCP, factor B and factor H | |
Jenkins et al. | Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells. | |
DE69529515T2 (en) | THE REGULATION OF THE ACTIVITY OF CYTOTOXIC T CELL LYMPHOCYTES WITH MHC CLASS I PEPTIDES | |
US5645837A (en) | Peptides that inhibit T cell activation and methods of using the same | |
Yusuf-Makagiansar et al. | Inhibition of the adherence of T-lymphocytes to epithelial cells by a cyclic peptide derived from inserted domain of lymphocyte function-associated antigen-1 | |
CA2417885A1 (en) | Cell internalized peptide-drug conjugates | |
Gonzalez et al. | Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis | |
JP2003128579A (en) | Method of improving allograft or xenograft tolerance by administration of lfa-3 or cd2 binding protein of specific species | |
IL147123A (en) | Use of a composition consisting of at least one soluble form of hla- g for preparing a medicinal product for treating inflammatory pathological skin conditions | |
Yusuf-Makagiansar et al. | Synergistic inhibitory activity of α-and β-LFA-1 peptides on LFA-1/ICAM-1 interaction | |
CA2220098A1 (en) | Cd6 ligand |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Dead |