KR20070017320A - Methods of treating skin disorders - Google Patents

Abstract

Methods of treating skin diseases are provided.

Description

Methods of treating skin disorders

Related Applications

This application claims the priority of US Provisional Application No. 60 / 542,311, the entire contents of which are incorporated herein by reference.

(alefacept)는 건선을 치료하기 위한 용도로 인정된 생물학 물질이다.Psoriasis affects about 4.5 million American adults. AMEVIVE

alefacept is a biological substance recognized for use in treating psoriasis.

Summary of the Invention

The present invention provides a variety of conditions, including T-cell mediated conditions, such as memory T-cell-mediated conditions, such as psoriasis, atopic dermatitis, cutaneous T cell lymphoma, allergy and irritant contact dermatitis, squamous Provided are methods for treating skin conditions such as tachycardia, alopecia areata, necrosus, vitiligo, ocular cicatricial pemphigoid, UV damage and hives. The methods described herein can be used to inhibit inhibitors of LFA-3 / CD2 interactions, such as soluble LFA-3 polypeptides such as AMEVIVE. and multiple cycles of soluble LFA-3-immunoglobulin (Ig) fusion protein, such as (alefacept) (hereafter AMEVIVE). Treatment of multiple cycles with these materials has been found to provide more pronounced results (eg, significantly longer remission periods) than single cycle or dual cycle treatments without the risk of obvious additional side effects. In a preferred embodiment, the methods described herein relate to the treatment of psoriasis.

Thus, in one embodiment, the present invention is characterized by a method of treating a condition, such as a skin condition such as psoriasis or other skin conditions described herein. In one embodiment, the condition is mediated by memory effector T cells. The method comprises administering to the subject a plurality of courses of treatment (preferably at least three cycles of treatment) of the soluble CD2-binding LFA-3 polypeptide.

Preferably, the soluble CD2-binding LFA-3 polypeptide is an LFA-3 fusion protein, eg, an LFA-3 / immunoglobulin (Ig) fusion protein. Typical LFA-3 / Ig fusion proteins are soluble CD2 binding LFA-3 fused to all or part of the Fc region of an IgG, eg, all or part of an IgG heavy chain hinge region and all or part of a heavy chain constant region. Polypeptides. In a preferred embodiment, the Ig fusion protein comprises 92 amino acids of the mature LFA-3 amino acid, 10 C-terminal amino acids of the human IgGl hinge, CH2 region of human IgGI heavy chain, and CH3 region of human IgGl heavy chain, or At least in part. One such fusion protein is AMEVIVE. AMEVIVE is encoded by an insert contained in plasmid pSAB152 deposited in the American Type Culture Collection under accession number ATCC 68720. AMEVIVE is described in more detail herein below.

The multiple course of treatment includes at least three cycles of treatment, each cycle comprising (a) a dosing period in which the therapeutic agent is administered at least two times, followed by (b) a rest period during which no therapeutic agent is administered, the rest period being a cycle Is substantially longer than the interval IA between administrations, preferably at least as long as the administration period. In certain embodiments, the plurality of procedures include at least 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 12 cycles or more. The administration period of each cycle of the plurality of procedures may be preselected or determined, for example, by a healthcare provider for a particular patient. Typically, the dosing period is long enough to induce a therapeutic response, for example, to induce a selected level of relief measured by clinical measurements, such as a PASI score. In certain embodiments, the administration period is at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 20 weeks or more, but typically 4 to 24 weeks. In a preferred embodiment, each cycle consists of 12 weeks of dosing once a week of administration of the polypeptide followed by 12 weeks of resting period at which the patient is evaluated at least once for the effects of the substance, eg, therapeutic effect or side effects. In a preferred embodiment, the rest period of each successive cycle of the plurality of processes is longer than the rest period of the previous cycle in the plurality of processes. In certain embodiments, the rest period of the final cycle of the plurality of processes can be at least 2 years, at least 18 months, at least 3 years, 4 years, 5 years or more.

The soluble CD2 binding LFA-3 polypeptide may be administered at a dosage of about 0.001 to about 50 mg binding agent per kg of body weight. In one embodiment, the polypeptide is administered systemically, preferably by the intramuscular (IM) or intravenous (IV) route. The administration period typically includes periodic administration of the polypeptide, eg, once a week, twice a week, twice a week, or once a month. The polypeptide is typically a unit dose of 2-15 mg (eg 7.5 mg IV bolus) when administered by the IV route and 2-30 mg (eg 10 or 15 mg when administered by the IM route) IM injection).

In one embodiment, the method comprises evaluating the subject for the effect of the soluble CD2-binding LFA-3 polypeptide during one or both of the administration period and the resting period of each cycle in a plurality of procedures.

In one embodiment, the method further comprises additional therapeutic or prophylactic agents, such as UV radiation (eg UVB radiation), cyclosporin A, prednisone, FK506, methotrexate, steroid, retinoid, Interferon and administering the nitrogen mustard to the subject. The additional agent may be administered during one or more cycles during the administration period, during the rest period, or both.

The subject is preferably a human. Preferred subjects are T cell-mediated skin diseases such as psoriasis, for example, dermal cell proliferation, increased red plaque formation, scalyness, itching, cracking, stinging, burns , Or subjects with symptoms of bleeding plaque, and subjects diagnosed with psoriasis.

In another aspect, the invention features a method of treating a subject having psoriasis. The method comprises (a) selecting a subject having at least two cycles, eg, selecting a subject based on having at least two cycles of treatment with a soluble CD2-binding LFA-3 polypeptide, and (b) Subjects are administered additional cycles of treatment of soluble CD2-binding LFA-3 polypeptides, eg, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more cycles. It includes.

In another aspect, the invention features a method of treating a subject, or advising or counseling a subject to be treated using the soluble CD2-binding LFA-3 polypeptides described herein. The method includes instructing or providing instructions to a subject or another individual, such as a healthcare provider, such as a physician, nurse, hospital staff, HMO or other entity providing healthcare, Here the subject must be administered treatment of a plurality of processes described herein.

The methods described herein can be used to treat any condition mediated by memory effector T cells. The methods described herein can be used to treat skin conditions such as psoriasis and scleroderma and non-skin conditions such as inflammatory bowel disease, uveitis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, and scleroderma.

Detailed description of the invention

The methods described herein generally relate to the treatment of multiple processes with soluble CD2 binding LFA-3 polypeptides for the treatment of T cell-mediated diseases (eg, psoriasis). Multiple course treatments have been surprisingly found to provide significantly longer periods of remission than single or double cycle treatments without the apparent risk of additional side effects.

Multiple course therapy

As used herein, a "cycle" of treatment includes (a) a period of administration in which the therapeutic agent is administered at least twice (the interval between administrations is referred to as IA) followed by (b) a rest period during which no therapeutic agent is administered. The rest period is substantially longer than the longest IA, for example at least 4-5 times longer, preferably at least as long as the administration period. During the cycle's administration period, the substance can often be administered (preferably regularly) at least 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 times. Typically, the dosing period is sufficiently long for patients exhibiting an improvement in a preselected level of disease, eg, a preselected PASI score, eg, PASI 50 or PASI 75. The rest period may include monitoring the patient for response to the therapeutic agent (eg, a therapeutic effect or side effect). In a preferred cycle, the substance is administered once a week for a 12 week dosing period followed by a 12 week rest period during which the patient is evaluated by the healthcare provider at least once.

In a preferred embodiment, there will be less than 50, 40, 30, 20 or 15 administrations during the administration period. In a preferred embodiment, the longest interval (IA) between any two adjacent doses in the dosing period of the cycle is less than 30 days, less than 20 days, less than 15 days, or less than 10 days. In a preferred embodiment, the interval between administrations is about 1 week. The dosing period of the cycle will vary depending on the dosing strategy. For example, if the dosing period is measured in weeks or months, the dosing period is monthly, weekly, two weeks, half a week, or during a specified number of weeks, as measured by a healthcare practitioner for a particular patient, or Daily administration may be included. Preferred administration periods for the cycle include about 6-24 administrations with an IA of 3-15 days. More preferably, the dosing period of the cycle comprises about 10-14 administrations wherein the IA is 5-9 days.

In certain cases, the period of rest is as long as, or longer than, the period during which the substance has a substantial relieving effect on the patient, as measured by standard clinical measurements. For example, the duration of rest for a psoriasis patient during a treatment cycle may include a designated psoriasis area severity index (PASI) score (eg, PASI 50 or PASI 75) or a designated physician comprehensive assessment (PGA) score (eg, PGA " It may be a period or longer periods in which "clear" or "almost clear" is maintained. However, at least a rest period as long as the relaxation period is typically 1 to 10 years, for example 2 to 10 years, for example 2 to 5 years. In certain embodiments, the rest period is at least 1 year, preferably at least 18 months, 2 years, 30 months, 36 months, 42 months, 48 months or longer.

"Treatment of a plurality of processes" means at least three cycles of treatment. The cycles within the treatment of multiple processes may be the same, but need not be the same, eg, dosing strategy for a dosing period; The duration of IA, dosing period, rest period, or the length of both may be different. For example, the treatment of a plurality of processes may comprise (a) a first cycle consisting of 12 weeks of weekly administration of AMEVIVE followed by 12 weeks of rest, and (b) 12 weeks of weekly administration of AMEVIVE. (C) a second cycle consisting of a one-year rest period with a substantial relieving effect on this patient, followed by (c) a third cycle consisting of two weeks of rest followed by ten weeks of half-week administration of the therapeutic agent, and (d) any It may comprise a continuous cycle of, for example, an additional fourth, fifth, sixth, seventh, eighth cycle or more.

In a preferred embodiment, the rest period and the alleviating effect increase with increasing number of cycles during the treatment of a plurality of processes. The increase in duration of rest or remission of effect with increasing cycles of treatment is preferably at least 10%, more preferably at least 15% or 20%, more preferably at least 25%, 30%, 40%, 50 % Or more. In certain embodiments, the rest period and alleviation effect for a third cycle (and / or subsequent cycles) in the treatment of multiple processes is at least 18 months, preferably at least 2 years, more preferably at least 30 months, 36 Months, 42 months, 48 months or more.

CD2 : LFA Inhibitors of -3 Interactions

Any inhibitor of the CD2: LFA-3 interaction is useful in the methods of the invention. The inhibitor may be a soluble LFA-3 polypeptide, an anti-LFA-3 antibody analog, an anti-CD2 antibody analog, a soluble CD2 polypeptide, a small molecule (e.g., a chemical having a molecular weight of less than 2500 Da, preferably less than 1500 Da, Chemicals such as organic small molecules, such as products of combinatorial libraries, LFA-3 and CD2 mimetic agents and derivatives thereof.

Preferred inhibitors for use in the methods described herein are soluble CD2 binding LFA-3 polypeptides.

Availability CD2  And LFA -3 polypeptide

Soluble LFA-3 polypeptides or soluble CD2 polypeptides that inhibit the interaction of LFA-3 and CD2 are useful in the methods of the invention. Preference is given to soluble LFA-3 polypeptides, in particular soluble LFA-3 / Ig fusions.

As used herein, a “soluble CD2-binding LFA-3 polypeptide” is a polypeptide which comprises at least the CD2-binding domain of LFA-3 (SEQ ID NO: 2) and is incapable of attaching itself to the membrane. Such soluble polypeptides include, for example, LFA-3 polypeptides that lack sufficient portions of the transmembrane domain to attach the polypeptide or that have been modified such that the transmembrane domain is nonfunctional. Soluble CD2-binding LFA-3 polypeptides include soluble fusion proteins comprising at least the CD2-binding domain of LFA-3 fused to a heterologous polypeptide. In one embodiment, the heterologous polypeptide is an Fc region of immunoglobulin (eg, an IgGl hinge region and a CH2-CH3 domain) or a substantial portion thereof.

Soluble LFA-3 polypeptides may be derived from the transmembrane form of LFA-3, in particular from the extracellular domain (eg, amino acids 1-187 of SEQ ID NO: 2 of US 6,162,432, incorporated herein by reference). ). Such polypeptides are described in US Pat. No. 4,956,281 and US Pat. No. 6,162,432, which are incorporated herein by reference. Preferred soluble LFA-3 polypeptides comprise polypeptides comprising residues 1-92 of SEQ ID NO: 2, residues 1-80 of SEQ ID NO: 2, residues 50-65 of SEQ ID NO: 2 and residues 20-80 of SEQ ID NO: 2, SEQ ID NO: 2 is shown in US Pat. No. 6,162,432. A vector comprising a DNA sequence encoding the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 1) is deposited in the American Type Culture Collection (Rockville, Maryland) under Accession No. 75107, SEQ ID NOs: 1 and 2 are shown in US Pat. No. 6,162,432.

Soluble LFA-3 polypeptides can also be derived from PI-linked forms of LFA-3, such as those described in PCT patent application serial number WO 90/02181. A vector comprising a DNA sequence encoding a PI-linked LFA-3 (SEQ ID NO: 3) is deposited in the American Type Culture Collection (Rockville, Maryland) under Accession No. 68788. It should be understood that the PI-linked form of LFA-3 and the transmembrane form of LFA-3 have the same amino acid sequence throughout the entire extracellular domain. Thus, preferred PI-linked LFA-3 polypeptides are identical to the transmembrane form of LFA-3.

(alefacept)이다.Most preferred soluble CD2 binding LFA-3 polypeptides for use in the present invention are LFA-3 / Ig fusion proteins. One example of such a fusion protein is AMEVIVE

(alefacept).

( alefacept ) AMEVIVE

( alefacept )

AMEVIVE is a fusion protein comprising the first extracellular domain (CD58) of human LFA-3 fused to the Fc portion of human IgGl. In particular, AMEVIVE mature LFA-3 of the amino terminal 92 amino acids, the chain between the two cysteine residues to 10 amino acids C- terminal of the human IgGl hinge region containing thought to be participating in a disulfide bond, and a human IgG _l heavy chain constant Substantial portions of the C _H 2 and C _H 3 sites of the domain (eg, SEQ ID NO: 8). The protein is a glycosylated disulfide linked dimer having a molecular weight of about 112 kD under a PAGE non-reduced state. The constant region of AMEVIVE has C-terminal variability corresponding to the splice variant form of the full length fusion polypeptide.

PSAB152, a plasmid encoding AMEVIVE, was deposited in the American Type Culture Collection (Rockville, Maryland) under the accession number ATCC 68720.

pMDR (92) Ig-3 is an example of an expression vector that can be used to prepare AMEVIVE. pMDR (92) Ig-3 comprises the following components: (a) fragment of pBR322 comprising the ColEl origin and beta lactamase expression cassette (GenBank Accession No. J01749); (b) DHFR expression cassette consisting of: SV40 early promoter (part of GenBank Accession No. J02400) deleted with enhancer, murine DHFR cDNA (GenBank Accession No. L26316), SV40 poly A site and small t intron (GenBank Accession No Part of J02400), and human gastrin transcription terminator sequence, 3'UTR (Sato et al. (1986) Mol Cell Biol 6: 1032-1043); (c) AMEVIVE expression cassette, preferably comprising the following in sequence: SV40 early promoter / enhancer (GenBank Accession No. J02400), adenovirus major late promoter and splice donor and intron sequences (GenBank Accession No. J01917) ), A tripartite leader comprising a murine Ig heavy chain variable region intron sequence and a splice acceptor (Kaufman and Sharp (1982) Mol Cell Biol. 2: 1304-1319), (optionally) cloning linker, human fibroblast The first 92 amino acids of the LFA-3 gene, cloning linker (optionally), poly A, isolated from the human tonsil cDNA library fused in frame to the nucleic acid encoding the hinge CH2 and CH3 sites of the human IgGl gene isolated from the genomic DNA library. MIS 3′UT site comprising the site (GenBank Accession No. K03474), and SV40 polyA site and small t intron (GenBank Accession No. J02400); And fragments of pBR327 (GenBank Accession No. L08856).

Host cell lines that can be used to prepare AMEVIVE can be derived from CHO-DUkX-Bl cells. In one embodiment, the DHFR (-) mutant of the cell line can be transfected with the vector pMDR (92) Ig-3, wherein the DHFR (+) transformant is selected from a selection medium (eg, 25 nM methotrexate (MTX). May be cultured). Positive transformants may receive increasing concentrations of MTX (eg, 50 nM), followed by selection of colonies that produce high levels of AMEVIVE.

Production of AMEVIVE can be performed as follows: CHO host cells are thawed, scaled up to 2000 L of culture, maintained in culture for 6-7 days under pH control and nutrient supply (48 hours, 96 hours) And 120 hours), after which the conditioned medium is harvested via microfiltration. MTX is preferably present in the culture medium. AMEVIVE can be recovered from the conditioned medium by performing the following steps: (i) Protein A chromatography, (ii) ceramic hydroxyapatite chromatography, (iii) virus inactivation at low pH, (iv) hydrophobic interactions Chromatography followed by (v) a second concentration step of obtaining concentration, diafiltration, virus filtration and fusion products.

Another way of making AMEVIVEs for use in the methods of the present invention is described in co-pending commonly assigned US patent application Ser. No. 07 / 770,967. In general, the regulated culture medium of COS7 or CHO cells transfected with pSAB152 was concentrated using the AMICON S1Y30 spiral cartridge system (AMICON, Danvers, Massachusetts) and protein A-Sepharose 4B (Sigma, St. Louis). , Missouri). Bound proteins were eluted and subjected to Superose-12 (Pharmacia / LKB, Piscataway, New Jersey) gel filtration chromatography.

Measured by SDS-PAGE gel and Western blot analysis (eg, Towbin et al., Proc. Natl. Acad. Sci. USA, 74, pp. 4440-54 (1979); Antibodies: A Laboratory Manual, pp .474-510 (see Cold Spring Harbor Laboratory (1988)), Superose-12 fractions containing AMEVIVE with the least amount of contaminating protein were collected and concentrated in YM30 Centricon (AMICON). Detected on Western blot using a detectably labeled goat anti-rabbit IgG followed by 3 polyclonal antiserum, purified AMEVIVE of COS7 or CHO cells were two monomeric LFA-3-Ig fusion proteins linked by disulfide bonds. Was a dimer.

LFA-3-Ig fusion activity can be tested using the following bioassays: (1) CD32 / 64 (Fc gamma RI / RII) U937 cell bridging assay, and (2) CD16 (Fc gamma RIII) Jurkat Cell Bridging Assay. Both assays test the ability of AMEVIVE to bridge CHO cells expressing cell surface CD2 to cells expressing Fc-gamma receptors. The latter assay, assay (2), involves culturing adherent CHO-CD2 cells to form a monolayer in a 96-well plate; Adding AMEVIVE control and sample; Adding fluorescence labeled Jurkat-CD16 (+); And measuring fluorescence intensity.

Binding of the LFA-3-Ig fusion to CD2 immobilized on a substrate such as a chip can also be used to test the fusion protein.

CD2  Polypeptide

Soluble CD2 polypeptides can be derived from full-length CD2, in particular extracellular domains. The polypeptide may comprise all or part of the extracellular domain of CD2. Typical soluble CD2 polypeptides are described in PCT WO 90/08187, which is incorporated herein by reference.

Production of Soluble Polypeptides

Production of soluble polypeptides useful in the present invention can be accomplished by a variety of methods known in the art. For example, the polypeptide is derived from the native transmembrane LFA-3 or CD2 molecule or the original PI-linked LFA-3 molecule by exopeptidase, Edman degradation, or proteolysis with specific endopeptidase in combination with both. Can be. The original LFA-3 molecule or native CD2 molecule can in turn be purified from its natural source using conventional methods. Alternatively, the original LFA-3 or CD2 can be produced by known recombinant DNA techniques using cDNA (eg, US Pat. No. 4,956,281, Wallner et al .; Aruffo and Seed, Proc. Natl Acad. Sci., 84, pp. 2941-45 (1987); Sayre et al., Proc. Natl. Acad. Sci. USA, 84, pp. 2941-45 (1987).

Preferably, the soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for whole LFA-3 molecules or whole CD2 molecules as starting material. This can be accomplished by conventional chemical synthesis techniques or by well-known recombinant DNA techniques in which the DNA sequence encoding the desired peptide is expressed in a transformed host. For example, the gene encoding the desired soluble LFA-3 polypeptide or soluble CD2 polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. The oligonucleotides are designed based on the amino acid sequence of the desired soluble LFA-3 polypeptide or soluble CD2 polypeptide. Specific DNA sequences encoding the desired peptides can also be derived from full length DNA sequences by isolation of certain restriction enzyme fragments or PCR synthesis of specific regions.

Standard methods can be applied to synthesize genes encoding soluble LFA-3 polypeptides or soluble CD2 polypeptides useful in the present invention. For example, a complete amino acid sequence can be used to construct a back-translated gene. DNA oligomers comprising nucleotide sequences encoding soluble LFA-3 polypeptides or soluble CD2 polypeptides useful in the present invention can be synthesized in a single step. Alternatively, several smaller oligonucleotides encoding portions of the desired polypeptide can be linked after they have been synthesized. Preferably, the soluble LFA-3 polypeptide or soluble CD2 polypeptide useful in the present invention will be synthesized as several separate oligonucleotides that are linked together secondary. Individual oligonucleotides typically include 5 'or 3' overhangs for complementary assembly.

Once assembled, the preferred gene has a sequence that is recognized by the restriction enzyme (including the only restriction enzyme site for direct assembly into the cloning or expression vector), the preferred codon that takes into account the host expression system used, and stably efficient when transcribed. It is characterized by the sequence that produces the translated mRNA. Proper assembly can be confirmed by base sequencing, restriction enzyme mapping, and expression of the biologically active polypeptide in the appropriate host.

Due to the degeneracy of the genetic code, those skilled in the art will recognize that DNA molecules comprising many different nucleotide sequences can also encode soluble LFA-3 and CD2 polypeptides encoded by the specific DNA sequences described above. The degenerate sequence also encodes a polypeptide useful in the present invention.

The DNA sequence may be expressed in a single cell host, or preferably in an isolated mammalian host cell. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host. Preferably, the expression control sequence, and the gene of interest, will be included in an expression vector further comprising a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector may further comprise additional expression markers useful in the expression host.

The DNA sequence encoding the desired soluble polypeptide may or may not encode a signal sequence. If the expression host is a prokaryotic cell, it is generally preferred that the DNA sequence does not encode a signal sequence. If the expression host is a eukaryotic cell, it is generally preferred that the signal sequence is encoded.

Amino terminal methionine may or may not be present in the expressed product. If terminal methionine is not cleaved by the expression host, it can be removed chemically by standard techniques if desired.

Various expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences derived from SV40, soybean virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, eg, plasmids derived from E. coli, including colE1, pCRl, pBR322, pMB9 and derivatives thereof, broader host range plasmids such as RP4, phage DNA, eg For example, numerous derivatives of phage lambda, such as NM989, and other DNA phages, such as M13 and fibrous single stranded DNA phage. Useful expression vectors for yeast cells include 2μ plasmids and derivatives thereof. Vectors useful for insect cells include pVL 941.

In addition, any of a variety of expression control sequences can be used in the vector. Such useful expression control sequences include expression control sequences associated with structural genes of previous expression vectors. Examples of useful expression control sequences include early and late promoters of the SV40 or adenovirus, lac system, trp system, TAC or TRG system, major promoter and promoter sites of phage lambda, regulatory sites of fd coat protein, 3-phosphoglycer Promoters for late kinases or other glycolytic enzymes, promoters of acid phosphatase, such as Pho5, promoters of the yeast α-breeding system, and other sequences known to modulate the gene expression of prokaryotic or eukaryotic cells or viruses thereof , And various combinations thereof.

Various host cells are useful. The host cell may be a single cell organism or may be obtained from a cell isolated from a multicellular organism, eg, a multicellular host. Such hosts are well known eukaryotic and prokaryotic hosts, for example insect cells such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast strains, Spodoptera frugiperda (SF9), Animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells in tissue culture can do. For animal cell expression, CHO cells and COS 7 cells are preferred.

It should not be understood that all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Not all hosts will function equally well for the same expression system. However, one skilled in the art can choose between the vector, expression control sequences and host without undue experimentation. For example, when selecting a vector, the host must be considered because the vector must replicate in the host. The number of copies of the vector, the ability to control the copy number, the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.

When selecting expression control sequences, various factors must also be considered. This includes, for example, the relative strength of the sequence, its controllability, and its suitability with the DNA sequences discussed herein, particularly for potential secondary structures. Single-cell hosts are characterized by their suitability with the selected vector, the toxicity of the product encoded by the DNA sequence, its secretion properties, the ability to correctly fold soluble polypeptides, their fermentation or culture requirements, and the purification of the product encoded by the DNA sequence. They should be selected for ease of use.

Within these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations that will express the desired DNA sequence upon fermentation or large scale animal culture, for example using CHO cells or COS 7 cells.

The soluble LFA-3 and CD2 polypeptides can be isolated from fermentation or cell culture using any of a variety of conventional methods and can be purified. One skilled in the art can select the most appropriate separation and purification techniques.

While recombinant DNA technology is a preferred method of producing useful soluble CD2 polypeptides or soluble LFA-3 polypeptides having more than 20 amino acid sequences, shorter CD2 or LFA-3 polypeptides having less than about 20 amino acids are preferably conventional. Produced by phosphorus chemical synthesis technology. Synthetically produced polypeptides useful in the present invention can advantageously be produced in extremely high yields and can be readily purified.

Preferably, the soluble CD2 polypeptide or soluble LFA-3 polypeptide is synthesized by solution or solid phase polypeptide synthesis, optionally cleaved with carboxypeptidase (to remove C-terminal amino acids) or by manual Edman degradation ( To remove the N-terminal amino acid). The use of solution phase synthesis allows the direct addition of certain derived amino acids, such as O-sulfate esters of tyrosine, to advantageously growing polypeptide chains. This obviates the need for subsequent derivatization steps to modify any residue of a polypeptide useful in the present invention.

Correct folding of such polypeptides is described in Ken, “Chemical Synthesis of Polypeptides and Proteins”, Ann. Rev. Biochem., 57, pp. 957-89 (1988)] can be achieved under an oxidation state that favors disulfide bridge formation. Polypeptides produced in this manner can then be purified by separation techniques well known in the art.

term- LFA -3 and anti- CD2  Antibody homologues

As used herein, an “antibody homologue” is a protein comprising one or more polypeptides selected from immunoglobulin light chains, immunoglobulin heavy chains, and antigen-binding fragments thereof capable of binding an antigen. Polypeptide components of antibody homologues consisting of one or more polypeptides may optionally be disulfide bonds or otherwise covalently crosslinked.

Thus, antibody homologs include native immunoglobulins of type IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulins may be of kappa or lambda type. Antibody homologues also include portions of the original immunoglobulins that retain antigen-binding specificity, such as Fab fragments, Fab 'fragments, F (ab') ₂ fragments, F (v) fragments, heavy chain monomers or dimers, light chain monomers. Or dimers consisting of dimers, one heavy chain and one light chain, and the like. The term includes recombinant antibodies, chimeric, CDR-grafted and humanized antibodies, or other antibodies modified to less immunogenic in humans.

As used herein, “humanized recombinant or humanized antibody homolog” is an antibody homolog produced by recombinant DNA technology, wherein all or part of the amino acids of the human immunoglobulin light chain or heavy chain required for antigen binding are non-human mammalian immunoglobulin light chains or Substituted for the corresponding amino acid from the heavy chain.

As used herein, a “chimeric recombinant antibody analog” is an antibody homologue produced by recombinant DNA technology, wherein all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both are derived from another immunoglobulin light chain or heavy chain. It was substituted for the corresponding site of.

Many types of anti-LFA-3 or anti-CD2 antibody analogs are useful in the methods of the invention. This includes monoclonal antibodies, recombinant antibodies, chimeric recombinant antibodies, humanized recombinant antibodies as well as previous antigen-binding portions.

It is preferable to use a monoclonal anti-LFA-3 antibody among the said anti-LFA-3 antibody homologs. Accession Nos. Monoclonal anti-LFA- produced by hybridomas selected from the hybridoma group with ATCC HB 10693 (1E6), ATCC HB 10694 (HC-1B11), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8) More preferred is the use of 3 antibodies or monoclonal antibodies known as TS2 / 9 (Sanchez-Madrid et al., "Three Distinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3"). Proc. Natl. Acad. Sci. USA, 79, pp.7489-93 (1982). Most preferably, said monoclonal anti-LFA-3 antibody comprises Accession Nos. Produced by hybridomas selected from the group of hybridomas having ATCC HB 10695 (7A6) and ATCC HB 10693 (1E6).

Among the anti-CD2 antibody homologues, it is preferable to use monoclonal anti-CD2 antibodies, such as anti-CD2 monoclonal antibodies known as T11 ₁ epitope antibodies comprising TS2 / 18 (Sanchez-Madrid et al., "Three Distinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3 ", Proc. Natl. Acad. Sci. USA, 79, pp.7489-93 (1982)).

Techniques for producing monoclonal antibodies are well known. See Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Kohler et al., Nature, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", 256, pp. 495-97 (1975). Useful immunogens for the purposes of the present invention include cell-free preparations comprising cells with CD2- or LFA-3 as well as LFA-3, CD2 or counter receptor-binding fragments thereof (eg, in LFA-3 CD2 fragment binding or LFA-3 fragment binding to CD2).

Immunization can be performed using standard procedures. Unit dosage and immunization management depend on the species of the immunized mammal, its immune status, the body weight of the mammal, and the like. Typically, the immunized mammals are drawn and serum from each blood sample is analyzed for specific antibodies using appropriate screening assays. For example, useful anti-LFA-3 or anti-CD2 antibodies are immune that prevent the sheep red blood cell binding of Jurkat cells due to the presence of LFA-3 and CD2 on each surface of Jurkat cells. This can be confirmed by testing the ability of the serum. Lymphocytes used in the production of hybridoma cells are typically isolated from immunized mammals whose serum has already been tested positive for the presence of the desired antibody using this screening assay.

Anti-CD2 and anti-LFA-3 antibody analogs useful in the present invention may also be recombinant antibodies produced by host cells transformed with DNA encoding the immunoglobulin light and heavy chains of the desired antibody. Recombinant antibodies can be produced by known genetic engineering techniques. See, eg, US Pat. No. 4,816,397, which is incorporated herein by reference. For example, recombinant antibodies can be produced by cloning cDNA or genomic DNA encoding the immunoglobulin light and heavy chains of the desired antibody from hybridoma cells that produce antibody homologues useful in the present invention. The cDNA or genomic DNA encoding the polypeptide is then inserted into an expression vector such that both genes are operably linked to its own transcriptional and translational expression control sequences. The expression vector and expression control sequences are chosen to be suitable for the expression host cell used. Typically, both genes are inserted into the same expression vector.

Prokaryotic or eukaryotic host cells can be used. Expression in eukaryotic host cells is preferred because they are likely to assemble and secrete more correctly folded and immunologically active antibodies than prokaryotic cells. It is possible for the host cell to produce a portion of the original antibody, eg a light chain dimer or a heavy chain dimer which is the antibody homologue according to the invention.

It will be appreciated that changes to the above procedures are useful in the present invention. For example, it may be desirable to transform host cells with DNA encoding the light chain or heavy chain (but not both) of the antibody homologues. Recombinant DNA technology can also be used to remove all or part of the DNA encoding both or one of the light and heavy chains that are not required for CD2 or LFA-3 counter receptor binding. Molecules expressed from the cleaved DNA molecules are useful in the methods of the present invention. In addition, one heavy and one light chain is an anti-CD2 or anti-LFA-3 antibody homologue and the other heavy and light chains are specific for other antigens other than CD2 or LFA-3 or another epitope of CD2 or LFA-3. Bifunctional antibodies can be produced.

Chimeric recombinant anti-LFA-3 or anti-CD2 antibody homologues are DNA from the corresponding site of an immunoglobulin light or heavy chain of a species of which all or part of the DNA encoding the hinge and constant regions of the heavy and / or light chain is different. It can be produced by transforming a host cell with a suitable expression vector comprising DNA encoding the desired and desired immunoglobulin light and heavy chains. When the original recombinant antibody is non-human and the inhibitor is administered to a human, substitution of the corresponding human sequence is preferred. Typical chimeric recombinant antibodies have mouse variable regions and human hinges and constant regions. See, generally, US Pat. No. 4,816,397; Morrison et al., “Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains with Human Constant Region Domains”, Proc. Natl. Acad. Sci. USA, 81, pp. 6851-55 (1984); Robinson et al., International Patent Publication PCT / US86 / 02269; Akia, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Neuberger et al., International Application WO 86/01533; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al., J. Immunol. 139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al., 1987, Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559.

Humanized recombinant anti-LFA-3 or anti-CD2 antibodies can be generated by replacing sequences of Fv variable regions not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are described in Morrison, S. L., 1985, Science 229: 1202-1207, Oi et al., 1986, BioTechniques 4: 214, and Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762. The method includes separating, manipulating and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable region from one or more heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from hybridomas producing anti-LFA-3 or anti-CD2 antibodies. The nucleic acid encoding the humanized antibody, fragment thereof, may then be cloned into a suitable expression vector.

Humanized or CDR-grafted antibody molecules or immunoglobulins can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be substituted. For example, US Pat. No. 5,225,539; all of which are hereby expressly incorporated by reference; Jones et al. 1986 Nature 321: 552-525; Verhoeyan et al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141: 4053-4060; Winter US 5,225,539. Winter describes a CDR-grafting method that can be used to prepare human cyclized antibodies of the invention, the contents of which are hereby expressly incorporated by reference (UK Patent Application GB 2188638A, filed March 26, 1987; Winter US 5,225,539). ). All CDRs of a particular human antibody may be substituted with at least part of a non-human CDR or only a portion of the CDR may be substituted with a non-human CDR. It is only necessary to substitute the number of CDRs required for the binding of a humanized antibody to a predetermined antigen, eg, LFA-3 or CD2.

Also within the scope of the present invention are humanized antibodies comprising immunoglobulins, wherein certain amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions at the framework sites, for example, to improve binding to the antigen. For example, a selected small number of acceptor backbone residues of the humanized immunoglobulin chain may be substituted by the corresponding donor amino acid.

Preferred positions of substitution include amino acid residues that are adjacent to or can interact with the CDRs (see, eg, US 5,585,089). Criteria for selecting amino acids from donors are described in US Pat. No. 5,585,089, eg, column 12-16 of US Pat. No. 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing immunoglobulin chains, including antibodies, are disclosed in Padlan et al. EP 519596 Al.

Human monoclonal antibodies (mAb) against human LFA-3 or CD2 can be generated using transgenic mice with a complete human immune system rather than a mouse system. Splenocytes from the transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinity for epitopes derived from human proteins (eg, Wood et al. International Application WO 91/00906; Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368: 856-859; Green, LL et al. 1994 Nature Genet. 7: 13-21; Morrison, SL et al. 1994 Proc. Natl. Acad. Sci. USA 81: 6851-6855; Bruggeman et al. 1993 Year Immunol. 7: 33-40; Tuaillon et al. 1993 PNAS 90: 3720-3724; Bruggeman et al. 1991 Eur J Immumnol 21: 1323-1326).

Monoclonal antibodies can also be produced by other methods known to those skilled in the art of recombinant DNA technology. Alternative methods referred to as "combined antibody manifestations" have been developed to identify and isolate antibody fragments with specific antigen specificities and can be used to produce monoclonal antibodies (for description of combinatorial antibody manifestations, for example, , Sastry et al. 1989 PNAS 86: 5728; Huse et al. 1989 Science 246: 1275; and Orlandi et al. 1989 PNAS 86: 3833). After immunizing the animal with the immunogen described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods of obtaining DNA sequences of variable regions of various populations of immunoglobulin molecules by using a mixture of oligomeric primers and PCR are generally known (Larrick et al., 1991, Biotechniques 11: 152-156; Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2: 106-110).

Examples of methods and reagents that are particularly familiar for use in generating altered antibody display libraries can be found, for example, in Ladner et al. US Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; And Barbas et al. (1991) PNAS 88: 7978-7982. Kits for generating phage manifest libraries are commercially available (eg, the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP ™ phage display kit, catalog no. 240612).

In certain embodiments, the V region domains of the heavy and light chains can be expressed on the same polypeptide linked by a flexible linker to form a single chain Fv fragment, wherein the scFV gene is then cloned into the desired expression vector or phage genome. As generally described in McCafferty et al., Nature (1990) 348: 552-554, the complete VH and VL domains of antibodies linked by a flexible (Gly4-Ser) 3 linker are present in a manifestation package based on antigen affinity. It can be used to produce single chain antibodies that can be separable. Isolated scFV antibodies that are immunoreactive to the antigen can then be formulated into pharmaceutical formulations for use in the subject methods.

Certain antibodies with high affinity for surface proteins can be prepared according to methods known to those skilled in the art, for example, including screening of libraries (Ladner, RC, et al., US Pat. No. 5,233,409; Ladner, RC, et al., US Pat. No. 5,403,484). In addition, the methods of the library can be used in screens to obtain binding determinants that are mimics of structural determinants of antibodies. For example, Bajorath, J. and S. Sheriff, 1996, Proteins: Struct., Funct., And Genet. 24 (2), 152-157; Webster, D. M. and A. R. Rees, 1995, "Molecular modeling of antibody-combining sites," in S. Paul, Ed., Methods in Molecular Biol. 51, Antibody Engineering Protocols, Humana Press, Totowa, NJ, pp 17-49; And Johnson, G., Wu, T. T. and E. A. Kabat, 1995, "Seqhunt: A program to screen aligned nucleotide and amino acid sequences,", Methods in Molecular Biol. 51, op. See cit., pp 1-15.

Anti-CD2 and anti-LFA-3 antibody analogs which are not native antibodies are also useful in the present invention. The homologue may be derived from any of the antibody homologs described above. For example, antigen-binding fragments as well as full-length monomers, dimers or trimer polypeptides derived from the aforementioned antibodies are useful in themselves. Useful antibody homologues of this type include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) a F (ab ') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge site; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); And (vi) isolated complementarity determining sites (CDRs).

Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, this is a synthesis that allows recombinant VL and VH regions to be prepared as paired single protein chains to form monovalent molecules using recombinant methods. Linker (known as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be included within the term “antigen-binding fragment” of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for availability in the same manner as the original antibodies. Anti-LFA-3 heavy chains are preferred anti-LFA-3 antibody fragments.

Antibody fragments can also be produced by chemical methods, for example, cleaving the original antibody with a protease such as pepsin or papain and optionally treating the cleaved product with a reducing agent. Alternatively, useful fragments may be produced by using host cells transformed with truncated heavy and / or light chain genes. Heavy and light chain monomers can be produced by treating the original antibody with a reducing agent such as dithiothreitol and then purifying to separate the chains. Heavy and light chain monomers can also be produced by host cells transformed with, but not both, DNA encoding the desired heavy or light chain. See, eg, Ward et al., "Binding Activities of a Repertoire of Single Imminoglobulin Variable Domains Secreted from Escherichia coli", Nature, 341, pp. 544-46 (1989); Sastry et al., "Cloning of the Immunological Repertoire in Eschrichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library", Proc. Natl. Acad. Sci. USA, 86, pp. See 5728-32 (1989).

LFA -3 and CD-2 Mimic  or Small molecule  matter

Also useful in the method of the invention are LFA-3 and CD2 mimetic materials. Such materials, which may be peptides, semi-peptide compounds or non-peptide compounds (eg organic small molecules), are inhibitors of the CD2: LFA-3 interaction. Preferred CD2 and LFA-3 mimetic materials will inhibit at least CD2: LFA-3 interactions as well as anti-LFA-3 monoclonal antibody 7A6 or anti-CD2 monoclonal antibody TS2 / 18 (described above).

In a preferred embodiment, the test substance is a member of a combinatorial library, eg, a peptide or organic combinatorial library, or a natural product library. In a preferred embodiment, the plurality of test compounds, eg, library members, comprises at least 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ compounds. In a preferred embodiment, a plurality of test compounds, eg, library members, share structural or functional properties.

In one embodiment, the present invention provides a library of LFA-3 and / or CD2 inhibitors. Synthesis of combinatorial libraries is well known in the art and reviewed (eg, EM Gordon et al., J. Med. Chem. (1994) 37: 1385-1401; DeWitt, SH; Czarnik, AW Acc. Chem) Res. (1996) 29: 114; Armstrong, RW; Combs, AP; Tempest, PA; Brown, SD; Keating, TA Acc. Chem. Res. (1996) 29: 123; Ellman, JA Acc. Chem. Res. (1996) 29: 132; Gordon, EM; Gallop, MA; Patel, DV Acc. Chem. Res. (1996) 29: 144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelle et al. Trends Anal.Chem. (1995) 14: 83; Chen et al. J. Am. Chem. Soc. (1994) 116: 2661; US Patents 5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. WO 92/10092, WO 93/09668, WO 91/07087, WO 93/20242, WO 94/08051).

Libraries of the compounds of the invention can be prepared using various methods, some of which are known in the art. For example, a "split-pool" strategy can be performed in the following way: The beads of the functionalized polymeric support are located in a plurality of reaction vessels; Various polymeric supports suitable for solid phase peptide synthesis are known and some are commercially available (see, eg, M. Bondansky "Principles of Peptide Syntehsis", 2nd edition, Springer-Verlag, Berlin (1993)). A different activated amino acid solution is added to each aliquot of the beads and the reaction proceeds to produce a plurality of immobilized amino acids, one in each reaction vessel. An aliquot of the induced beads is then washed, "combined" (ie, recombined) and the pool of beads is subdivided again so that each aliquot is placed in a separate reaction vessel. Another activated amino acid is then added to each aliquot of the beads. The synthesis cycle is repeated until the desired peptide length is obtained. The amino acid residues added in each synthesis cycle can be chosen randomly; Alternatively, the amino acids may be combined with known peptides, such as anti-idiotype antibody antigen binding sites, to which certain portions of the "biased" library, eg, inhibitors, may interact, for example, with the antibody. It can be chosen to provide a library that is randomly selected to provide an inhibitor with known structural similarity or homology. It will be appreciated that various peptides, peptidomimetics, or non-peptide compounds can be readily produced in this manner.

This "split-pool" strategy results in a library of peptides such as inhibitors that can be used to prepare libraries of test compounds of the invention. In another exemplary synthesis, the "diversomer library" is generated by the method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U. S. A. 90: 6909 (1993)). Other synthetic methods, including the "tea-bag" technique of Houghten (see, for example, Houghten et al., Nature 354: 84-86 (1991)), also synthesize a library of compounds according to the present invention. It can be used to.

Libraries of compounds can be screened to determine if any member of the library has the desired activity, and to identify the active species if it has the desired activity. A method for screening combinatorial libraries has been described (eg, Gordon et al., J Med. Chem., Below). Soluble compound libraries can be screened by affinity chromatography using appropriate receptors to separate ligands for receptors, and the isolated ligands can then be identified by conventional techniques (eg, mass spectrometry, NMR, etc.). . Immobilized compounds can be screened by contacting the compounds with soluble receptors; Preferably, the soluble receptor is conjugated to a label (eg, fluorophore, colorimetric enzyme, radioisotope, luminescent compound, etc.) that can be detected to exhibit ligand binding. Alternatively, the immobilized compound can be selectively released and can diffuse through the membrane to interact with the receptor. Typical assays useful for screening libraries of the present invention are described below.

In one embodiment, the compounds of the present invention can be used, for example, by assaying for activities that directly bind to the polypeptides of each compound or inhibit the CD2: LFA-3 interaction, such as, for example, test compounds for CD2 or LFA-3 polypeptides and The ability to interact with CD2 or LFA-3 polypeptides by incubating lysates, such as T or APC cell lysates, in one well of a multiwell plate, eg, a standard 96-well microtiter plate. Can be screened for. In such embodiments, the activity of each individual compound can be measured. Well (s) without test compound can be used as a control. After incubation, the activity of each test compound can be measured by analyzing each well. Thus, the activity of a plurality of test compounds can be measured in parallel.

In yet another embodiment, a greater number of test compounds can be tested simultaneously for binding activity. For example, test compounds can be synthesized on solid resin beads in a "one bead-1 compound" synthesis; The compound can be immobilized on the resin support via a photolabile linker. The plurality of beads (e.g., as many as 100,000 beads or more) are then combined with the yeast cells and can be sprayed into a plurality of "nano-droplets", each droplet containing a single bead (and therefore a dinyl test compound). . Exposure of the nano-droplets to UV light then results in cleavage of the compound from the beads. It will be appreciated that the assay format allows for the screening of large libraries of test compounds in a rapid format.

Combinatorial libraries of compounds can be synthesized using “tags” to encode the identity of each member of the library (eg, WC Still et al., US Pat. No. 5,565,324 and PCT Publication Nos. WO 94/08051). And WO95 / 28640). In general, the method is characterized by the use of inert but easily detectable tags attached to a solid support or compound. When the active compound is detected (eg, by one of the techniques described above), the identity of the compound is determined by the identification of the unique attached tag. The tagging method allows the synthesis of large libraries of compounds that can be identified at very low levels. The tagging scheme can be useful, for example, in the "nano-droplet" screening assay described above to identify compounds released from the beads.

In a preferred embodiment, the library of compounds of the invention comprises at least 30 compounds, more preferably at least 100 compounds, and still more preferably at least 500 compounds. In a preferred embodiment, the library of compounds of the invention comprises less than 10 ⁹ compounds, more preferably less than 10 ⁸ compounds, and still more preferably less than 10 ⁷ compounds.

Induced  Inhibitor

Also useful in the methods of the invention are the induced inhibitors of the CD2: LFA-3 interaction, for example, any of the antibody homologues, soluble CD2 and LFA-3 polypeptides described herein, or CD2 and LFA-3 mimetics Functionally (chemically coupled to one or more members independently selected from the group consisting of anti-LFA-3 and anti-CD2 antibody analogs, soluble LFA-3 and CD2 polypeptides, CD2 and LFA-3 mimetics, cytotoxic agents and drugs , By genetic fusion or other methods.

One type of induced inhibitor is produced by crosslinking two or more inhibitors (same or different types). Suitable crosslinkers are heterodifunctional (e.g. m-maleimidobenzoyl-N-hydroxysuccinimide esters) having two uniquely reactive functional groups separated by suitable spacers or homodifunctional (e.g. Disuccinimidyl suverate). The linker is commercially available from Pierce Chemical Company (Rockford, Illinois).

Another possibility for crosslinking utilizes PI linkage signal sequences in PI-linked LFA-3, or fragments thereof. Specifically, the DNA encoding the PI-linked signal sequence (eg, amino acids 162-212 of SEQ ID NO: 4) is linked downstream of the DNA encoding the desired polypeptide, preferably soluble LFA-3 polypeptide. If the construct is expressed in a suitable eukaryotic cell, the cell will recognize a PI linkage signal sequence and will covalently link PI to the polypeptide. The hydrophobic nature of the PI can then be used to form micelle aggregates of the polypeptide.

In addition, inhibitors linked to one or more cytotoxic agents or agents are useful. Useful agents include antibody analogs specific for human polypeptides other than biologically active peptides, polypeptides and proteins such as CD2 or LFA-3, or portions thereof. Useful agents and cytotoxic agents also include UV radiation (eg, UVB), cyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids, interferons, and nitrogen mustards.

Preferred inhibitors derived from the medicament include recombinantly produced polypeptides, wherein the soluble LFA-3 polypeptide, soluble CD2 polypeptide, or peptidyl CD2 or peptidyl LFA-3 mimetics are all or at the immunoglobulin heavy chain hinge sites. Fused to all or part of the portion and heavy chain constant region. Preferred polypeptides for preparing such fusion proteins are soluble LFA-3 polypeptides. Amino acid 1-92 (two cysteine residues believed to participate in interchain disulfide bonds) of mature LFA-3 fused to the C _H 2 and C _H 3 regions of the IgG ₁ heavy chain constant domain and to portions of the human IgG _l hinge region. Most preferred is a fusion protein comprising the C-terminal ten amino acids of the containing hinge region). The fusion proteins are expected to exhibit long-term serum half-lives and enable inhibitor dimerization.

The utility of certain soluble CD2 polypeptides, soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD2 antibody homologs or CD2 and LFA-3 mimetics in the methods of the invention inhibits LFA-3 / CD2 interactions. It can be easily measured by analyzing its ability. This ability can be analyzed using a simple cell binding assay that allows, for example, a visual (under magnification) assessment of the ability of a hypothetical inhibitor to inhibit the interaction between LFA-3 and CD2 on cells with the molecule. Can be. Jurkat cells are preferred as CD2 ⁺ substrates, and both red blood cells or human JY cells are preferred as LFA-3 ⁺ substrates. The binding properties of soluble polypeptides, antibody homologues and mimetics useful in the present invention can be determined by radiolabeling (eg, ³⁵ S or ¹²⁵ I) in some known manner, for example, antibody homologues, polypeptides or substances. Labeled polypeptides, mimetics or antibody homologues can be analyzed by contacting appropriately with CD2 ⁺ of LFA-3 ⁺ cells. Binding properties can also be analyzed by using appropriate enzymatically labeled secondary antibodies. Seed et al. Rosetting competition assays such as those described in (Proc. Natl. Acad. Sci. USA, 84, pp. 3365-69 (1987)) can also be used.

Combination Therapy

Such materials, such as soluble CD2 binding LFA-3 polypeptides, can be used in combination with other therapies, such as other materials. Other material (s) are referred to herein as "second material (s)" or "additional materials" and include one or more of the following: immunosuppressive agents (eg, methotrexate, cyclosporine, or chlorambucil) , Cyclophosphamide, prednisone, FK506, steroid, retinoid, interferon, nitrogen mustard, cytokine binding material (e.g., type 2 cytokine binding material, e.g. IL-2- or IL-8-binding material, e.g. For example, anti-IL-2 or IL-8 monoclonal antibody (Abgenix), inhibitors of ICAM / LFA-1 interaction, eg, ICAM-1 (eg, humanized, chimeric, or human anti ICAM-binding agents (eg, antibodies, eg, monoclonal antibodies) against -ICAM-1 antibodies; Or LFA-1 (also known as CD1la) binding to LFA-1 (eg, humanized, chimeric, or human anti-LFA-1 antibody, eg, Raptiva (GenentechlXoma)) (Eg, antibodies, eg monoclonal antibodies); Co-promoter molecular binding agents, eg, B7-1 (CD8O) binding agents (eg, anti-B7-1 monoclonal antibody (IDEC); vasodilators (eg, ACE inhibitors or minoxidil); corticosteroids or Penicillamine In one embodiment, the agent, such as an inhibitor of CD2: LFA-3 interaction, is interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL- 8, in combination with one or more inhibitors of TGF-β, PDGF, granzyme A or leukotrien B4 The combination therapy advantageously utilizes lower doses of therapeutic or prophylactic agents.

As used herein, "administered in combination" means that two (or more) different therapies are delivered to a subject during the pain course of a subject with a disease, eg, two or more treatments cause the subject to It is meant to be delivered after diagnosis and before the disease is treated or eliminated. In certain embodiments, delivery of one treatment is still occurring when the second delivery is initiated and there is overlap. This is often referred to herein as "simultaneous" or "simultaneous delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of the above cases, the treatment is more effective because of the combined administration. For example, the second treatment is more effective, for example, an equivalent effect is observed for fewer second treatments, or the second treatment is observed when the second treatment is administered in the absence of the first treatment, Or a similar situation reduces the symptoms to a greater extent than that observed for the first treatment.

In certain embodiments, delivery is that the reduction in symptoms, or other variables associated with the disease, eg, a decrease in T cell levels or activity, is greater than that observed for treatment delivered in the absence of other. The effects of the two treatments are partially additive, totally additive, or additive. Delivery may still be detectable when the effect of the delivered first treatment is delivered, for example, when a CD2- or LFA-3 binding agent is delivered first, a decrease in T cell levels or activity is achieved. It is still detectable when the second substance is delivered. In a preferred embodiment, delivery of the first treatment and delivery of the second treatment occur within 1, 2, 5, 10, 15, or 30 days of each other.

In a preferred embodiment, the CD2-binding substance (eg LFA-3 / Ig fusion), the second substance (or both) or the pharmaceutical composition comprising the above is systemic, for example intravenously, intramuscularly, It is administered subcutaneously, intraarticularly, transdermally, intradurally, into the periosteum, into a tumor, into a lesion, infusion (eg, using an infusion device), around a lesion, orally, locally or by inhalation. Preferably, the CD2-binding substance is administered intramuscularly or intravenously. In other embodiments, the CD2-binding agent is administered locally, eg, locally or by needleless injection to the affected site.

Parenteral administration of a CD2-binding substance (e.g., LFA-3 / Ig fusion), a second substance (or both), or a pharmaceutical composition comprising the above, may be carried out by a needle or needleless syringe by procedures known in the art. It can be performed using. Examples of needleless syringe systems and modes of administration are described in US 6,132,395, US 6,096,002, US 5,993,412, US 5,893,397, US 5,520,639, US 5,503,627, US 5,399,163, US 5,383,851, US 5,312,577, US 5,312,577, US 5,312,577, the contents of which are incorporated herein by reference. Described.

Pharmaceutical composition

Preferably, an effective amount of a CD2: LFA3 inhibitor (eg, soluble CD2-binding LFA-3 polypeptides described herein) is administered. "Effective amount" means an amount that can reduce the metastases or severity of the conditions described herein. In a therapeutic embodiment, an effective amount of a substance inhibits, decreases, or ameliorates a disease (eg, improves a PASI score or PGA score for a psoriasis patient) or has the disease beyond what is expected in the absence of the treatment. When prolonging the survival of a patient, it refers to the amount of substance effective at a single or multiple doses to the subject. Improvements in psoriasis are expected to result in improved quality of life, for example, as assessed by the SF-36 Mental Health Index developed by RAND Health, a division of RAND Corporation (Santa Monica, CA). An effective amount does not necessarily indicate complete elimination of the disease. In prophylactic embodiments, an effective amount of a CD2- or LFA-3 binding agent described herein refers to an amount of the substance effective at single or multiple administrations to a subject when preventing or delaying the frequency of onset or recurrence of the disease.

An effective amount of a substance can be determined in particular by the disease being treated (e.g., T cell mediated disease of the skin for T cell mediated diseases of organs other than the skin), dosing schedule, unit dosage administered, and the substance used in combination with other therapeutic agents It will be apparent to those skilled in the art that the dosage will depend on the patient's immune status and health, the therapeutic or prophylactic activity of the particular substance administered and the serum half-life. Depending on the disease to be treated, the materials may be packaged differently.

Preferably, the soluble CD2-binding LFA-3 polypeptide (eg, LFA3TIP) is from about 0.001 to about 50 mg of substance per kg of body weight, more preferably from about 0.01 to about 10 mg of substance per kg of body weight, most Preferably at a dose of about 0.1 to about 4 mg of substance per kg of body weight. In a preferred embodiment, the soluble CD2-binding LFA-3 polypeptide is administered in a unit dose of 2-15 mg (eg 7.5 mg IV bolus) when administered by the IV route and 2 when administered by the IM route. To a unit dosage of from 30 mg (eg, 10 mg or 15 mg IM injection). IM and IV administration is preferred.

Unit doses are typically administered until an effect is observed. The effect can be measured by a variety of methods, including in vitro T cell activity analysis and removal or amelioration of diseased skin areas, or other diseased body parts, as may be associated with a particular disease. . Preferably, the unit dose is administered at regular intervals, such as once a week, during the treatment cycle. More preferably, they are administered at regular intervals, for example at weekly intervals of several weeks, for example 12 weeks. More frequent dosing, for example twice or three times a week, may also be considered and may be adapted if the disease in the subject is severe or indicates an urgent interference. Less frequent administration, for example once or twice a month, may also be contemplated, and may be adopted if the subject responds well to treatment so that maintenance administration is appropriate. However, it will be appreciated that lower or higher dosages and other dosing regimens may be used for any particular dosing cycle.

The agent, eg, CD2-binding LFA-3 polypeptide (eg AMEVIVE), is also preferably administered in a composition comprising a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a carrier that does not cause an allergic reaction or other adverse effect in the administered patient.

Suitable pharmaceutically acceptable carriers include, for example, one or more water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the materials.

Formulations of CD2-binding material (s), such as pharmaceutical formulations, may be prepared in aqueous or non-aqueous, eg, lyophilized form. Preferred pharmaceutical formulations are suitable for injection. Examples of aqueous formulations encompassed by the present invention include phosphate buffered saline (PBS) frozen liquid formulations. Examples of lyophilized formulations include one or more of the following: citrate, glycine and sucrose. For example, preferred lyophilized formulations have sodium citrate-citric acid buffer (at least 10 mM, buffered at a pH of at least about 4, preferably 5, more preferably 6 (or even more preferably 6.8)). Preferably 25 mM), 1-5% sucrose, preferably 2.5% sucrose, and 0.5% -2% glycine, preferably 1% glycine.

The second agent may be a single dosage form (ie, as part of the same pharmaceutical composition) with CD2-binding material (s), multiple dosage forms separately or simultaneously with the CD2-binding material (s), or two components. It may be administered in a plurality of dosage forms administered separately and continuously.

Alternatively, the CD2-binding material and other active materials may be in the form of a single conjugated molecule. Conjugation of the two components can be accomplished by standard crosslinking techniques well known in the art. Single molecules can also take the form of recombinant fusion proteins. In addition, the pharmaceutical compositions useful in the present invention can be used in combination with other therapies such as PUVA, chemotherapy and UV light. The combination therapy may advantageously utilize low doses of the therapeutic or prophylactic agent.

The CD2-binding substance, or pharmaceutical composition may be in various forms. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectables, injectables, and topical preparations. Preferred forms depend on the intended mode of administration and therapeutic application. Preferred forms are solutions for injection or infusion.

The present invention includes formulations suitable for use as topically applied sunscreens or UV-protectors. Preferred embodiments include AMEVIVE formulations. The active ingredient may be formulated into liposomes. The product may be applied before, during, or after UV exposure, or may be applied before, during, or after the occurrence of redness.

order

The following is a summary of the sequences described in US Pat. No. 6,162,432 and the sequences mentioned throughout this application:

SEQ ID NO: 1 DNA sequence of transmembrane LFA-3

SEQ ID NO: 2 amino acid sequence of transmembrane LFA-3

SEQ ID 3: DNA sequence of PI-linked LFA-3

SEQ ID NO 4: Amino acid sequence of PI-linked LFA-3

SEQ ID NO: 5 DNA sequence of CD2

SEQ ID NO: 6 amino acid sequence of CD2

SEQ ID NO: 7: DNA sequence of AMEVIVE

Amino acid sequence of SEQ ID NO: AMEVIVE

1A and 1B show amino acid and nucleotide sequences of LFA-3 / IgG fusion proteins. The signal peptide corresponds to amino acids 1-28 of FIG. 1A; The mature LFA-3 region corresponds to amino acids 29-120 of FIG. 1A; IgG1 sites correspond to amino acids 121-347 of FIG. 1A.

FIG. 2 is a bar graph of the percentage of psoriasis patients who achieved PASI 50 at 2 or 12 weeks after treatment for Cycle A-D of multiple courses of treatment with AMEVIVE.

3 is a graph of the benefits and repeat response in the treatment of multiple processes with AMEVIVE for psoriasis.

4 is a bar graph of the maximum length of response time in four psoriasis patients receiving multiple courses of treatment with AMEVIVE.

5 is a graph of average CD4 + T-cell numbers for patients with multiple courses of treatment with AMEVIVE.

6A is a graph of the percentage of patients who achieved PASI 75 at any time during multiple course intravenous (IV) treatment with AMEVIVE. 6B is a graph of the percentage of patients who achieved PASI 50 at any time during multiple course IV treatment with AMEVIVE.

FIG. 7A is a graph of the percentage of patients who achieved a PGA of “apparent” or “nearly apparent” response at any time during multiple course IV treatment with AMEVIVE. FIG. 7B is a graph of the percentage of patients who achieved a PGA of “obvious” or “nearly apparent” response at any time during multiple courses of IM treatment with AMEVIVE.

( alefacept )를 이용한 건선의 복수 과정 치료 Example 1: AMEVIVE

Multi-course treatment of psoriasis with (alefacept)

를 이용한 9 사이클 이하의 치료의 복수 의 과정의 치료를 받은 환자에서 효능 및 안전성을 조사하였다.This example is AMEVIVE over 4.5 years.

Efficacy and safety were investigated in patients who received multiple courses of treatment with up to 9 cycles of therapy.

cure

In the open-label study the initial treatment cycle is referred to as Cycle A. This cycle is labeled cycles B, C, D, and the like. Patients were treated once per week for 12 weeks (dose period) followed by 12 weeks (rest period) in each cycle.

AMEVIVE 7.5 mg was administered by intravenous (IV) bolus injection.

Prior to initiation of treatment in the extended study, the need for systemic treatment was established based on the physician's assessment of disease severity, and the CD4 + T-cell number was at or below the lower limit of normal (LLN; 404 cells / mm ³ ).

Eligibility for subsequent cycles was based on the aforementioned criteria, and furthermore: the patient must have received at least eight doses of AMEVIVE during the 12 week treatment period of the previous cycle and during Cycle C and subsequent cycles, and the lymphocyte count was the screening visit of the study. It was required to be at least 75% of the number recorded in the visit.

Within any given cycle, if the patient had a CD4 + T-cell number of 200-300 cells / mm ³ , the dose of AMEVIVE was reduced by 50% (3.75 mg). If the CD4 + T-cell count was less than 200 cells / mm ³ , the scheduled dosing was withheld. If the CD4 + T-cell number was less than 200 cells / mm ³ for 4 consecutive visits, AMEVIVE was forever suspended. AMEVIVE administration was withheld for two weeks when evidence of clinically significant infection was observed.

evaluation

Efficacy was assessed by psoriasis site severity index (PASI) and physician global assessment (PGA). For Cycle A, assessments were made at 1, 3, 5, 7, 9, 11, and 12 weeks during treatment and at 2, 4, 6, 8, and 12 weeks after treatment. This was assessed for cycles at 1 and 7 weeks during treatment and at 2 and 12 weeks after treatment. From baseline, the proportion of patients who achieved at least 50% and at least 75% improvement in PASI (PASI 50 and PASI 75, respectively) and patients who achieved “obvious” or “nearly apparent” PGA were reported.

Total lymphocyte and lymphocyte subset analysis was performed at each study visit, except 4 weeks after treatment in Cycle A and 4 and 8 weeks after treatment in all subsequent cycles. Patients with new or ongoing viral, bacterial, or fungal infections were monitored at all visits. Adverse events were monitored throughout the study.

result

In the analysis, the patient received a repeated cycle of treatment with AMEVIVE over 4.5 years. In this study, 175 patients had at least 1 cycle; 126 more than 2 cycles; 96 more than 3 cycles; And 71 received at least 4 cycles of AMEVIVE. Some patients received less than 9 cycles of AMEVIVE as a result of exposure to AMEVIVE in a previous phase 2 study.

efficacy

The proportion of patients achieving PASI 50 at 2 or 12 weeks after treatment for cycle A-D is shown in FIG. 2. The reaction rate of cycles C and D is significantly increased compared to cycles A and B. The proportion of patients achieving PASI 75 was 29%, 33%, 34%, and 52% in cycles A, B, C, and D, respectively. Corresponding reaction rates for “obvious” or “nearly apparent” PGAs were 24%, 29%, 33%, and 34%, respectively.

For cycles A-D, the increased benefit and repeat response of treatment of additional cycles with AMEVIVE are shown in FIG. 3. For Cycle A responders who received an additional cycle of AMEVIVE (ie, patients who achieved PASI 50 in Cycle A), the proportion of patients who achieved PASI 50 increased with each secondary cycle. In general, patients began to respond to repeated treatment with AMEVIVE without evidence of tolerability. Among patients who achieved PASI 50 in a given cycle, 75% to 90% of patients achieved PASI 50 in subsequent cycles (ie, repeated responses).

Adverse events

The occurrence of adverse events generally did not change significantly over the cycle. The overall safety profile of AMEVIVE following multiple cycles of administration (treatment of multiple processes) is similar to that reported in the Phase 3 study. The incidence of severe adverse events was less than 7% in any cycle, and the spectrum of severe adverse events was similar to previous phase 2 and 3 studies.

Two patients each in Cycles A and B and one patient in Cycle E discontinued treatment because of adverse events. The incidence of malignancy was low: no more than 3% in any cycle; Most of them were skin cancer.

No treatment  Duration of reaction

In phase 3 studies, the palliative action of AMEVIVE was demonstrated, while patients maintained a PASI 50 response for the middle of 7 months. In this example, some patients were followed for a long time after a successful treatment cycle. 4 shows the maximum length of response time in four of these patients. Response to treatment with AMEVIVE was maintained for 18-24 months in some patients.

Lymphocyte count

Over several cycles of treatment with AMEVIVE, the decrease in lymphocyte count was consistent. The decrease in the number of lymphocytes observed for each cycle was not cumulative.

Average CD4 + T-cell numbers were above LLN for all cycles and did not decrease following multiple course exposure to AMEVIVE (FIG. 5).

In summary, the present study shows that multiple courses of treatment (three cycles of treatment or more) provide more pronounced results than a single course of treatment without the apparent risk of additional side effects. The multiple cycles of AMEVIVE were well tolerated by the patient, and the incidence of adverse events did not change significantly over the cycle.

Example  2: AMEVIVE Using Psoriasis  Multiple course therapy

This example shows the treatment of a second cycle with AMEVIVE in a psoriasis patient who did not achieve a 50% or more reduction (PASI 50) or a 25% or more reduction (PASI 25) in the psoriasis area severity index during the first cycle of treatment with AMEVIVE. The efficacy of treatment of multiple cycles as well as clinical response to was investigated.

patient

The patient was 16 years old or older and had chronic plaque psoriasis for at least 12 months, including at least 10% of the body surface area. CD4 + T-cell numbers required more than the lower limit of normal (LLN). Treatment with phototherapy, systemic retinoids, systemic corticosteroids, systemic fumarates, immunosuppressive agents (methotrexate, cyclosporin, azathioprine, and thioguanine), and high potency localized corticosteroids, is within 4 weeks prior to treatment with AMEVIVE and It was banned throughout the study. The use of moderately potent topical corticosteroids, topical retinoids, coal tar, keratinants, and vitamin D analogs were prohibited within two weeks of treatment with AMEVIVE and throughout the study, except for the scalp, palm, groin, and sole. To be eligible for enrollment in the extended study, patients were required to receive AMEVIVE of at least 8 doses and complete the final follow-up visit of the previous cycle of treatment. Patients were excluded from the extension study if they were enrolled in drug or non-drug treatment or any other investigational study of the disclosed alternative systemic psoriasis treatment, phototherapy, or other disallowed treatment 8 weeks prior to the previous cycle.

cure

Phase III studies were multicenter, randomized, double-blind and placebo controlled (Krueger et al., J. Am. Acad. Dermatol. 47: 821-833, 2002; Lebwohl et al., Arch. Dermatol. 139: 719-727, 2003). In a phase 3 study of intravenous (IV) treatment with AMEVIVE, the patient received two cycles of treatment, each cycle consisting of (i) a weekly dosing period of AMEVIVE (7.5 mg) or placebo once per week, and (ii) Twelve weeks follow-up (rest period) was made. In a phase 3 study of intramuscular (IM) treatment with AMEVIVE, the patient was treated with a single cycle of 12 weeks of dosing (EV or rest) followed by 12 weeks of AMEVIVE (10 mg or 15 mg) or placebo per week. I was treated.

In multiple course therapy studies, patients who received additional cycles of treatment with AMEVIVE (same dosing regimen) were patients whose disease had advanced to require systemic therapy or phototherapy as measured by the investigator, with LLN or higher circulating Had a CD4 + T-cell number (Gordon et al., J. Drugs Dermatol. 2: 624-628, 2003).

evaluation

During the Phase 3 study, PASI and PGA were assessed at baseline every two weeks during treatment and every two to four weeks during follow-up. PASI and PGA were also evaluated at several time points during the IV multi-course study, whereas only PGA was evaluated during the IM multi-course study.

analysis

Data from a 2-cycle phase 3 study of IV treatment with AMEVIVE was used to determine the efficacy of a second cycle of treatment with AMEVIVE in two groups of patients: (a) failure to achieve PASI 50 in the first cycle. Patients and (b) patients who did not achieve PASI 25 in the first cycle. Determining the proportion of patients who did not achieve PASI 50 and PASI 25 during cycle 1 treatment with AMEVIVE for patients who achieved PASI 50 or 75% or more PASI reduction (PASI 75) at any time during the second cycle of treatment. It was. Odds ratios and corresponding 95% confidence intervals (CI) were calculated to compare response rates between patients receiving a second cycle of treatment with AMEVIVE and patients receiving placebo.

Percentage of patients who achieved PASI 50, PASI 75, and “obvious” or “nearly apparent” PGA at any point during each IV treatment cycle, and “obvious” or “at any point during each IM treatment cycle. The proportion of patients who achieved a nearly evident PGA was also measured.

result

The patient had an average age of about 45 years and about 70% were male. The median duration of psoriasis was about 19 years and median body surface involvement was about 22%. The number of patients receiving IV and IM treatment for each cycle with AMEVIVE is summarized in Table 1.

Number of patients who received multiple cycles of AMEVIVE Treatment cycle One 2 3 4 5 IV AMEVIVE 521 327 217 158 39 IM AMEVIVE 457 320 156 100 50

Before PASI  50 or PASI  Failed to achieve 75 In the patient  Efficacy of Treatment in the Second Cycle

PASI improvement was observed during IV treatment of the second cycle with AMEVIVE in 93% of patients who did not achieve PASI 50 during the first cycle and 89% of patients who did not achieve PASI 25 during the first cycle. Treatment with AMEVIVE in the second cycle resulted in a significantly greater proportion of patients who achieved PASI 50 and PASI 75 than placebo treatment (Table 2). 19% of patients who did not achieve PASI 50 during the initial cycle of treatment with AMEVIVE achieved PASI 75 during the second cycle and 53% achieved PASI 50. 14% of patients who did not achieve PASI 25 during the initial cycle of treatment with AMEVIVE achieved PASI 75 during the second cycle and 47% achieved PASI 50 (Table 2). Crossover ratios showed that patients who did not achieve ASI 50 or PASI 25 in Cycle 1 were likely to achieve 2-3 times more PASI 50 or PASI 75 in Cycle 2 compared to patients who did not receive another cycle (Table 2 ).

PASI improvement at any point during AMEVIVE of the second cycle in patients who did not achieve PASI 50 or PASI 25 during the first cycle cure AMEVIVE- AMEVIVE AMEVIVE- placebo Cross Ratio (95% CI) Patients failing to achieve PASI 50 in the first AMEVIVE cycle N 97 86 PASI 75, n (%) 18 (18.6) 7 (8.1) 2.57 (1.02-6.50) PASI 50, n (%) 51 (52.6) 28 (32.6) 2.30 (1.26-4.19) Any PASI improvement, n (%) q 90 (92.8) 74 (86.0) 2.08 (0.78-5.56) Patients failing to achieve PASI 25 in the first AMEVIVE cycle N 66 46 PASI 75, n (%) 9 (13.6) 3 (6.5) 2.26 (0.58-8.86) PASI 50, n (%) 31 (47.0) 11 (23.9) 2.82 (1.23-6.48) Any PASI improvement, n (%) q 59 (89.4) 38 (82.6) 1.77 (0.59-5.29)

Benefits of Multiple Course Therapy

Patients receiving multiple cycles of IV treatment with AMEVIVE showed increasing improvement in PASI during each subsequent treatment cycle. The proportion of patients who achieved PASI 75 increased from 29% during Cycle 1 up to 54% during Cycle 5 (FIG. 6A). Similarly, the proportion of patients who achieved PASI 50 increased from 56% during Cycle 1 up to 74% during Cycle 5 (FIG. 6B).

PGA results also support increasing clinical improvement in observed psoriasis for the treatment of multiple cycles with AMEVIVE. 23% of patients had "clear" or "nearly clear" PGA during IV treatment of Cycle 1 with AMEVIVE. During cycle 5, 44% of patients achieved this level of response (FIG. 7A). For IM treatment with AMEVIVE, the PGA “obvious” / “nearly apparent” response rate increased from 21% for Cycle 1 to up to 41% during Cycle 4 (FIG. 7B).

In summary, increasing clinical improvement was observed following continuous cycle of treatment with AMEVIVE, indicating its efficacy for long-term treatment of patients with chronic plaque psoriasis. Multiple course treatment data show that patient response to additional treatment with AMEVIVE is advantageously increased, regardless of response to initial treatment.

                         SEQUENCE LISTING <110> Astellas US LLC        MAGILAVY, Daniel   <120> METHODS OF TREATING SKIN DISORDERS <130> 253762 <150> 60 / 542,311 <151> 2004-02-06 <150> PCT / US2005 / 003907 <151> 2005-02-07 <160> 8 <170> PatentIn version 3.3 <210> 1 <211> 753 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (750) <220> <221> sig_peptide (222) (1) .. (84) <220> <221> misc_feature (222) (1) .. (750) <223> "Human transmembrane LFA-3" <220> <221> mat_peptide (222) (85) .. (750) <220> <221> misc_feature 646 (646) .. (646) <223> "Transmembrane domain" <400> 1 atg gtt gct ggg agc gac gcg ggg cgg gcc ctg ggg gtc ctc agc gtg 48 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 gtc tgc ctg ctg cac tgc ttt ggt ttc atc agc tgt ttt tcc caa caa 96 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 ata tat ggt gtt gtg tat ggg aat gta act ttc cat gta cca agc aat 144 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 gtg cct tta aaa gag gtc cta tgg aaa aaa caa aag gat aaa gtt gca 192 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 gaa ctg gaa aat tct gaa ttc aga gct ttc tca tct ttt aaa aat agg 240 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 gtt tat tta gac act gtg tca ggt agc ctc act atc tac aac tta aca 288 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 tca tca gat gaa gat gag tat gaa atg gaa tcg cca aat att act gat 336 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 acc atg aag ttc ttt ctt tat gtg ctt gag tct ctt cca tct ccc aca 384 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100 cta act tgt gca ttg act aat gga agc att gaa gtc caa tgc atg ata 432 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile                 105 110 115 cca gag cat tac aac agc cat cga gga ctt ata atg tac tca tgg gat 480 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp             120 125 130 tgt cct atg gag caa tgt aaa cgt aac tca acc agt ata tat ttt aag 528 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys         135 140 145 atg gaa aat gat ctt cca caa aaa ata cag tgt act ctt agc aat cca 576 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro     150 155 160 tta ttt aat aca aca tca tca atc att ttg aca acc tgt atc cca agc 624 Leu Phe Asn Thr Thr Ser Ser Ser Ile Leu Thr Thr Cys Ile Pro Ser 165 170 175 180 agc ggt cat tca aga cac aga tat gca ctt ata ccc ata cca tta gca 672 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala                 185 190 195 gta att aca aca tgt att gtg ctg tat atg aat ggt att ctg aaa tgt 720 Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Ile Leu Lys Cys             200 205 210 gac aga aaa cca gac aga acc aac tcc aat tga 753 Asp Arg Lys Pro Asp Arg Thr Asn Ser Asn         215 220 <210> 2 <211> 250 <212> PRT <213> Homo sapiens <400> 2 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile                 105 110 115 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp             120 125 130 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys         135 140 145 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro     150 155 160 Leu Phe Asn Thr Thr Ser Ser Ser Ile Leu Thr Thr Cys Ile Pro Ser 165 170 175 180 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala                 185 190 195 Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Ile Leu Lys Cys             200 205 210 Asp Arg Lys Pro Asp Arg Thr Asn Ser Asn         215 220 <210> 3 <211> 723 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (720) <220> <221> sig_peptide (222) (1) .. (84) <220> <221> misc_feature (222) (1) .. (720) <223> "Human PI-linked LFA-3" <220> <221> mat_peptide (222) (85) .. (720) <220> <221> misc_feature <222> (568) .. (720) <223> "signal sequence for PI-linkage" <400> 3 atg gtt gct ggg agc gac gcg ggg cgg gcc ctg ggg gtc ctc agc gtg 48 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 gtc tgc ctg ctg cac tgc ttt ggt ttc atc agc tgt ttt tcc caa caa 96 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 ata tat ggt gtt gtg tat ggg aat gta act ttc cat gta cca agc aat 144 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 gtg cct tta aaa gag gtc cta tgg aaa aaa caa aag gat aaa gtt gca 192 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 gaa ctg gaa aat tct gaa ttc aga gct ttc tca tct ttt aaa aat agg 240 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 gtt tat tta gac act gtg tca ggt agc ctc act atc tac aac tta aca 288 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 tca tca gat gaa gat gag tat gaa atg gaa tcg cca aat att act gat 336 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 acc atg aag ttc ttt ctt tat gtg ctt gag tct ctt cca tct ccc aca 384 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100 cta act tgt gca ttg act aat gga agc att gaa gtc caa tgc atg ata 432 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile                 105 110 115 cca gag cat tac aac agc cat cga gga ctt ata atg tac tca tgg gat 480 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp             120 125 130 tgt cct atg gag caa tgt aaa cgt aac tca acc agt ata tat ttt aag 528 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys         135 140 145 atg gaa aat gat ctt cca caa aaa ata cag tgt act ctt agc aat cca 576 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro     150 155 160 tta ttt aat aca aca tca tca atc att ttg aca acc tgt atc cca agc 624 Leu Phe Asn Thr Thr Ser Ser Ser Ile Leu Thr Thr Cys Ile Pro Ser 165 170 175 180 agc ggt cat tca aga cac aga tat gca ctt ata ccc ata cca tta gca 672 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala                 185 190 195 gta att aca aca tgt att gtg ctg tat atg aat ggt atg tat gct ttt 720 Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Met Tyr Ala Phe             200 205 210 taa 723 <210> 4 <211> 240 <212> PRT <213> Homo sapiens <400> 4 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr 85 90 95 100 Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile                 105 110 115 Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp             120 125 130 Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys         135 140 145 Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro     150 155 160 Leu Phe Asn Thr Thr Ser Ser Ser Ile Leu Thr Thr Cys Ile Pro Ser 165 170 175 180 Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala                 185 190 195 Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Met Tyr Ala Phe             200 205 210 <210> 5 <211> 1056 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (1053) <220> <221> sig_peptide (222) (1) .. (72) <220> <221> misc_feature (222) (1) .. (1053) <223> "Human CD2" <220> <221> mat_peptide (222) (73) .. (1053) <220> <221> misc_feature (628) .. (702) <223> "Transmembrane domain" <400> 5 atg agc ttt cca tgt aaa ttt gta gcc agc ttc ctt ctg att ttc aat 48 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn                 -20 -15 -10 gtt tct tcc aaa ggt gca gtc tcc aaa gag att acg aat gcc ttg gaa 96 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu             -5 -1 1 5 acc tgg ggt gcc ttg ggt cag gac atc aac ttg gac att cct agt ttt 144 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe     10 15 20 caa atg agt gat gat att gac gat ata aaa tgg gaa aaa act tca gac 192 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 25 30 35 40 aag aaa aag att gca caa ttc aga aaa gag aaa gag act ttc aag gaa 240 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu                 45 50 55 aaa gat aca tat aag cta ttt aaa aat gga act ctg aaa att aag cat 288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His             60 65 70 ctg aag acc gat gat cag gat atc tac aag gta tca ata tat gat aca 336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr         75 80 85 aaa gga aaa aat gtg ttg gaa aaa ata ttt gat ttg aag att caa gag 384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu     90 95 100 agg gtc tca aaa cca aag atc tcc tgg act tgt atc aac aca acc ctg 432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 105 110 115 120 acc tgt gag gta atg aat gga act gac ccc gaa tta aac ctg tat caa 480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln                 125 130 135 gat ggg aaa cat cta aaa ctt tct cag agg gtc atc aca cac aag tgg 528 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp             140 145 150 acc acc agc ctg agt gca aaa ttc aag tgc aca gca ggg aac aaa gtc 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val         155 160 165 agc aag gaa tcc agt gtc gag cct gtc agc tgt cca gag aaa ggt ctg 624 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu     170 175 180 gac atc tat ctc atc att ggc ata tgt gga gga ggc agc ctc ttg atg 672 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met 185 190 195 200 gtc ttt gtg gca ctg ctc gtt ttc tat atc acc aaa agg aaa aaa cag 720 Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln                 205 210 215 agg agt cgg aga aat gat gag gag ctg gag aca aga gcc cac aga gta 768 Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val             220 225 230 gct act gaa gaa agg ggc cgg aag ccc cac caa att cca gct tca acc 816 Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala Ser Thr         235 240 245 cct cag aat cca gca act tcc caa cat cct cct cca cca cct ggt cat 864 Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His     250 255 260 cgt tcc cag gca cct agt cat cgt ccc ccg cct cct gga cac cgt gtt 912 Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Gly His Arg Val 265 270 275 280 cag cac cag cct cag aag agg cct cct gct ccg tcg ggc aca caa gtt 960 Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val                 285 290 295 cac cag cag aaa ggc ccg ccc ctc ccc aga cct cga gtt cag cca aaa 1008 His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys             300 305 310 cct ccc cat ggg gca gca gaa aac tca ttg tcc cct tcc tct aat taa 1056 Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn         315 320 325 <210> 6 <211> 351 <212> PRT <213> Homo sapiens <400> 6 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn                 -20 -15 -10 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu             -5 -1 1 5 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe     10 15 20 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 25 30 35 40 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu                 45 50 55 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His             60 65 70 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr         75 80 85 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu     90 95 100 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 105 110 115 120 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln                 125 130 135 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp             140 145 150 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val         155 160 165 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu     170 175 180 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met 185 190 195 200 Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln                 205 210 215 Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val             220 225 230 Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala Ser Thr         235 240 245 Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His     250 255 260 Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Gly His Arg Val 265 270 275 280 Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val                 285 290 295 His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys             300 305 310 Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn         315 320 325 <210> 7 <211> 1050 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (1041) <220> <221> sig_peptide (222) (1) .. (84) <220> <221> mat_peptide (222) (85) .. (1041) <220> <221> misc_feature (222) (85) .. (1041) <223> "LFA3TIP" <220> <221> misc_feature (222) (360) .. (361) <223> "LFA-3 / IgG fusion point" <400> 7 atg gtt gct ggg agc gac gcg ggg cgg gcc ctg ggg gtc ctc agc gtg 48 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 gtc tgc ctg ctg cac tgc ttt ggt ttc atc agc tgt ttt tcc caa caa 96 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 ata tat ggt gtt gtg tat ggg aat gta act ttc cat gta cca agc aat 144 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 gtg cct tta aaa gag gtc cta tgg aaa aaa caa aag gat aaa gtt gca 192 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 gaa ctg gaa aat tct gaa ttc aga gct ttc tca tct ttt aaa aat agg 240 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 gtt tat tta gac act gtg tca ggt agc ctc act atc tac aac tta aca 288 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 tca tca gat gaa gat gag tat gaa atg gaa tcg cca aat att act gat 336 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 acc atg aag ttc ttt ctt tat gtc gac aaa act cac aca tgc cca ccg 384 Thr Met Lys Phe Phe Leu Tyr Val Asp Lys Thr His Thr Cys Pro Pro 85 90 95 100 tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 432 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 105 110 115 cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 480 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             120 125 130 tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 528 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         135 140 145 tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 576 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     150 155 160 gag gag cag tac aac agc acg tac cgg gtg gtc agc gtc ctc acc gtc 624 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 165 170 175 180 ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 672 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 185 190 195 aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 720 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             200 205 210 ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat 768 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         215 220 225 gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 816 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     230 235 240 tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 864 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 245 250 255 260 aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 912 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 265 270 275 ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 960 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             280 285 290 aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1008 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         295 300 305 acg cag aag agc ctc tcc ctg tct ccg ggt aaa tgagtgcgg 1050 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     310 315 <210> 8 <211> 347 <212> PRT <213> Homo sapiens <400> 8 Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val             -25 -20 -15 Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln         -10 -5 -1 1 Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn 5 10 15 20 Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala                 25 30 35 Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg             40 45 50 Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr         55 60 65 Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp     70 75 80 Thr Met Lys Phe Phe Leu Tyr Val Asp Lys Thr His Thr Cys Pro Pro 85 90 95 100 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 105 110 115 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             120 125 130 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         135 140 145 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     150 155 160 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 165 170 175 180 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 185 190 195 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             200 205 210 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         215 220 225 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     230 235 240 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 245 250 255 260 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 265 270 275 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             280 285 290 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         295 300 305 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     310 315  

Claims (30)

A method of treating a subject with psoriasis comprising administering to the subject a treatment of a plurality of processes of soluble CD2-binding LFA-3 polypeptide, the plurality of processes comprising a plurality of cycles of treatment, each cycle of administration And a rest period. The method of claim 1, The soluble CD2-binding LFA-3 polypeptide is an LFA-3 fusion protein. The method of claim 1, The soluble CD2-binding LFA-3 polypeptide is an LFA-3 / immunoglobulin (Ig) fusion protein. The method of claim 1, Wherein said soluble CD2-binding LFA-3 polypeptide comprises a soluble LFA-3 polypeptide fused to all or a portion of an IgG heavy chain hinge region and to all or a portion of a heavy chain constant region. The method of claim 1, The soluble CD2-binding LFA-3 polypeptide comprises 92 amino acids of the amino terminus of mature LFA-3, 10 amino acids of C-terminus of the human IgGl hinge region, a CH2 region of human IgGI heavy chain, and at least a portion of a CH3 region of human IgGl heavy chain. Method comprising a fusion protein consisting of. The method of claim 1, The soluble CD2-binding LFA-3 polypeptide is AMEVIVE (FIG. 1). The method of claim 1, Wherein said soluble CD2-binding LFA-3 polypeptide is encoded by an insert contained in plasmid pSAB152 deposited in the American Type Culture Collection under Accession Number ATCC 68720. The method according to any one of claims 1 to 7, Wherein said plurality of processes comprises at least 4 cycles of treatment. The method according to any one of claims 1 to 7, Wherein the plurality of processes comprise at least 5 cycles of treatment. The method according to any one of claims 1 to 7, Wherein said plurality of processes comprises at least six cycles of treatment. The method according to any one of claims 1 to 7, Wherein said plurality of procedures comprises at least seven cycles of treatment. The method according to any one of claims 1 to 7, Wherein said plurality of processes comprises at least eight cycles of treatment. The method according to any one of claims 1 to 7, And the rest period of each successive cycle of the plurality of processes is longer than the rest period of a previous cycle in the plurality of processes. The method according to any one of claims 1 to 7, And the rest period of the last cycle of the plurality of processes is at least two years. The method according to any one of claims 1 to 7, The rest period of the last cycle of the plurality of processes is at least three years. The method according to any one of claims 1 to 7, And the administration period of each cycle of the plurality of procedures is at least 8 weeks. The method according to any one of claims 1 to 7, And the administration period of each cycle of the plurality of procedures is at least 10 weeks. The method according to any one of claims 1 to 7, And the administration period of each cycle of the plurality of procedures is at least 12 weeks. The method according to any one of claims 1 to 7, And said polypeptide is administered intramuscularly. The method according to any one of claims 1 to 7, And said polypeptide is administered intravenously. The method according to any one of claims 1 to 7, Wherein said polypeptide is administered in a unit dose of 2 to 30 mg. The method according to any one of claims 1 to 7, The method further comprises administering to the subject an additional therapeutic or prophylactic agent during the treatment of the plurality of processes. A method of treating a subject in need of treatment for psoriasis, comprising administering to the subject a treatment of a plurality of processes of AMEVIVE (FIG. 1), wherein the treatment of the plurality of processes comprises at least three cycles of treatment, The treatment of each cycle includes a dosing period of at least 12 weeks, followed by a dosing period of weekly administration of AMEVIVE (FIG. 1) for 12 weeks. The method of claim 23, And the treatment of the plurality of processes comprises at least four cycles of treatment. The method of claim 23, Wherein the treatment of the plurality of processes comprises at least 5 cycles of treatment. The method of claim 23, The method comprising evaluating the subject for the effect of AMEVIVE (FIG. 1) during one or both of the administration period and the resting period of each cycle in a plurality of procedures. The method of claim 23, The method further comprises administering to the subject an additional therapeutic or prophylactic agent during the treatment of the plurality of processes. (a) selecting a subject based on having at least two cycles of treatment with a soluble CD2-binding LFA-3 polypeptide, and (b) subjecting the subject to a third cycle of treatment of the soluble CD2-binding LFA-3 polypeptide. A method of treating a subject with psoriasis, comprising administering. The method of claim 28, The soluble CD2-binding LFA-3 polypeptide is AMEVIVE (FIG. 1). A pharmaceutical composition comprising AMEVIVE and instructions for administering the pharmaceutical composition to a patient who previously had two cycles of treatment with AMEVIVE (FIG. 1).

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