HK1099638A - Methods of treating skin disorders - Google Patents

Description

Method for treating skin diseases

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application No.60/542,311, filed on 6/2/2004, the entire contents of which are hereby incorporated by reference herein.

Background

Psoriasis affects approximately four hundred and fifty thousand american adults. AMEVIVE^_(alefacept) is a biological agent approved for the treatment of psoriasis.

Summary of The Invention

The present invention provides methods of treating various diseases, including T cell mediated diseases, e.g., memory T cell mediated diseases, e.g., skin diseases, such as psoriasis, atopic dermatitis, cutaneous T cell lymphoma, allergic and irritant contact dermatitis, lichen planus (lichen planus), alopecia areataHead disorders, pyoderma gangrenosum, vitiligo, ocular scarring pemphigoid, UV damage, and urticaria. The methods described herein involve multi-cycle administration of an inhibitor of the LFA-3/CD2 interaction, e.g., a soluble LFA-3 polypeptide, e.g., a soluble LFA-3-immunoglobulin (Ig) fusion protein, such as AMEVIVE^_(alefacept) (hereinafter amevie). It has been found that multi-cycle therapy using the agents provides more significant results (e.g., a significantly longer remission period) than either single cycle therapy or dual cycle therapy, and surprisingly, does not have significant additional risk of side effects. In a preferred embodiment, the methods described herein relate to the treatment of psoriasis.

Accordingly, in one aspect, the invention features methods of treating a disease, such as a skin disease, psoriasis or other skin disease, as described herein. In one embodiment, the disease is mediated by memory effector T cells. The method comprises administering to the subject a multiple treatment regimen (preferably at least 3 treatment cycles) of soluble CD 2-binding LFA-3 polypeptide.

Preferably, the soluble CD 2-binding LFA-3 polypeptide is an LFA-3 fusion protein, e.g., an LFA-3/immunoglobulin (Ig) fusion protein. Exemplary LFA-3/Ig fusion proteins include soluble CD 2-binding LFA-3 polypeptides fused to all or part of the Fc region of an IgG, for example, to all or part of the IgG heavy chain hinge region and all or part of the heavy chain constant region. In a preferred embodiment, the Ig fusion protein consists of all or at least part of the N-terminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acids of the hinge region of human IgG1, the CH2 region of the human IgG1 heavy chain, and the CH3 region of the human IgG1 heavy chain. One such fusion protein is AMEVIVE. AMEVIVE is encoded by the insert contained in plasmid pSAB152, which is deposited with the American Type Culture Collection (American Type Culture Collection) under accession number ATCC 68720. AMEVIVE is described in more detail below.

The multiple course of treatment comprises at least 3 treatment cycles, each cycle comprising (a) a dosing period during which the therapeutic agent is administered at least2 times, followed by (b) a rest period during which no therapeutic agent is administered, wherein said rest period is substantially longer than the time interval between administrations in the cycle (IA,interval between administrations) and preferably at least as long as the administration period. In some embodiments, the multiple treatment sessions comprise at least 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 12 treatment cycles or more. The administration period for each cycle of the multi-course may be preselected or determined by, for example, the healthcare provider of the particular patient. Typically, the administration period is long enough to provoke a therapeutic response, e.g., a slowing of a selected level as measured by clinical measurements, e.g., PASI scoring. In some embodiments, the dosing period is at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, 20 weeks or more, but typically between 4-24 weeks. In a preferred embodiment, each cycle consists of a 12-week weekly polypeptide administration followed by a 12-week rest period during which the patient is evaluated at least once to determine the effect of the agent, e.g., a therapeutic effect or side effect. In a preferred embodiment, the rest period of each successive cycle of the multiple course is longer than the rest period of the preceding cycle in the multiple course. In some embodiments, the rest period of the last cycle of the multi-course may be at least 2 years, at least 18 months, at least 3 years, 4 years, 5 years, or more.

The soluble, CD 2-binding LFA-3 polypeptide may be administered at a dosage in the range of about 0.001 to about 50mg of binding agent per kg body weight. In one embodiment, the polypeptide is administered systemically, preferably by the Intramuscular (IM) or Intravenous (IV) route. The dosing period typically includes regular administration of the polypeptide, e.g., once a week, twice a week, half a week, or once a month. Typically, the polypeptide is administered in a unit dose in the range of 2-15mg when administered by the IV route (e.g., a 7.5mg IV bolus) and in a unit dose in the range of 2-30mg when administered by the IM route (e.g., a 10 or 15mg IM injection).

In one embodiment, the method comprises assessing the effect on a soluble CD 2-binding LFA-3 polypeptide during 1 or 2 dosing periods and rest periods of each cycle in a multiple course of therapy.

In one embodiment, the method comprises administering to the subject an additional therapeutic or prophylactic agent, e.g., UV radiation (such as UVB radiation), cyclosporin a, prednisone, FK506, methotrexate, steroids, retinoids, interferons, and nitrogen mustard, during the course of multiple course therapy. The additional agent may be administered during the dosing period, during the rest period, or both, during 1 or more cycles.

The subject is preferably a human. Preferred subjects include those patients with T cell mediated dermatological conditions such as psoriasis, for example, dermal cell proliferation, raised red spot formation (scaling), itch, cracking, stinging, burning, or bleeding spots, as well as those patients who have been diagnosed with psoriasis.

In another aspect, the invention features a method of treating a patient having psoriasis. The methods comprise (a) selecting a subject that has had at least 2 cycles of treatment with a soluble CD 2-binding LFA-3 polypeptide, e.g., based on having had at least 2 cycles, and (b) re-administering to the subject an additional cycle, e.g., 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th, 10 th or more cycles of treatment with a soluble CD 2-binding LFA-3 polypeptide.

In another aspect, the invention features methods of treating a subject, or suggesting or recommending a subject for treatment, with a soluble CD 2-binding LFA-3 polypeptide as described herein. The methods comprise teaching or providing guidance to a subject or other individual, e.g., a healthcare caregiver, such as a doctor, nurse, hospital employee, HMO, or other tissue providing healthcare, to administer a multi-course therapy described herein to the subject.

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

Brief description of the drawings

FIGS. 1A and 1B depict the 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; and the IgG1 region corresponds to amino acids 121-347 of FIG. 1A.

Figure 2 is a bar graph of the percentage of psoriasis patients who obtained PASI50 at 2 or 12 weeks following a-D cycle treatment using AMEVIVE multi-course therapy.

Figure 3 is a graph of the benefit and repeat response in a multi-course treatment of psoriasis using AMEVIVE.

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

Fig. 5 is a graph of mean CD4+ T cell counts for patients with multiple courses of treatment using AMEVIVE.

Fig. 6A is a graph of the percentage of patients who obtained PASI 75 at any time during multiple course Intravenous (IV) treatment using AMEVIVE. Fig. 6B is a graph of the percentage of patients who obtained PASI50 at any time during multiple courses of IV therapy using AMEVIVE.

Fig. 7A is a graph of the percentage of patients who obtained a "clear" or "nearly clear" response to PGA at any time during multiple courses of IV therapy using AMEVIVE. Fig. 7B is a graph of the percentage of patients who obtained a "clear" or "nearly clear" response to PGA at any time during multiple courses of IM therapy using AMEVIVE.

Detailed description of the invention

The methods described herein generally relate to multi-course therapy using soluble CD2 binding LFA-3 polypeptides for the treatment of T cell mediated diseases (e.g., psoriasis). It has been found that multi-course therapy provides significantly longer periods of remission than either single or double cycle therapy, and surprisingly, without significant additional risk of side effects.

Multiple course of treatment

As used herein, a "cycle" of treatment includes (a) a dosing period during which the therapeutic agent is administered at least 2 times (the time interval between administrations is referred to as IA), followed by (b) a rest period during which the therapeutic agent is not administered. The rest period is substantially longer than the longest IA, e.g. at least 4-5 times longer, and preferably at least as long as the administration period. The agent may be administered at least 2,4, 6, 8, 10, 12, 14, 16, 18 or 20 times at intervals (preferably regularly) during the dosing period of the cycle. Typically, the dosing period is sufficiently long for the patient to exhibit a preselected level of disease improvement, e.g., a preselected PASI score, e.g., PASI50 or PASI 75. The rest period can include monitoring the patient's response (e.g., efficacy or side effects) to the therapeutic agent. In a preferred cycle, the medicament is administered once per week during a dosing period of 12 weeks, followed by a rest period of 12 weeks during which the patient is evaluated at least once by a healthcare provider.

In preferred embodiments, there will be less than 50, 40, 30, 20 or 15 administrations during the dosing period. In preferred embodiments, the maximum time Interval (IA) between any two adjacent administrations within 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 time interval between administrations is about 1 week. The administration period of the cycle may vary taking into account the dosage strategy. For example, if the dosing period is measured in weeks or months, the dosing period may include monthly, weekly, bi-weekly, semi-weekly, or daily administration of the medicament for a specified number of weeks, as determined by the health care professional for the particular patient. A preferred dosing period of one cycle comprises about 6-24 administrations with an IA of 3-15 days. More preferably, the dosing period of one cycle comprises about 10-14 administrations with 5-9 days of IA.

In some cases, the rest period is equal to or longer than the period during which the agent has a substantially reduced effect on the patient, as measured by standard clinical measurements. For example, a rest period for a psoriatic patient during a treatment cycle may be a period during which a particular Psoriasis Area and Severity Index (PASI) score (e.g., PASI50 or PASI 75) or a specific physician integrated assessment (PGA) score (e.g., PGA "clear" or "almost clear") is maintained, or longer. However, the rest period, which is at least equal to the remission period, is typically between 1 year and 10 years, e.g. between 2 years and 5 years. In some 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.

By "multiple course of treatment" is meant at least 3 treatment cycles. The cycles in a multi-course treatment may be the same but they need not be, e.g., they may differ in dosage strategy during the administration period; or in IA, the length of the administration period, rest period, or both. For example, a multi-course treatment may include (a) a first cycle consisting of a 12-week weekly administration of AMEVIVE followed by a 12-week rest, followed by (b) a second cycle consisting of a 12-week weekly administration of AMEVIVE followed by a 1-year rest period during which the agent has a substantially reduced effect on the patient, followed by (c) a third cycle consisting of a 10-week half-weekly administration of therapeutic agent followed by a 2-year rest, optionally followed by (d) subsequent cycles, e.g., additional 4 th, 5 th, 6 th, 7 th, 8 th cycles or more.

In a preferred embodiment, the rest period and the slowing effect increase with an increasing number of cycles during the course of multi-course treatment. The increase with each increased treatment cycle in the rest period or reduced duration of action is preferably at least 10%, more preferably at least 15% or 20%, more preferably at least 25%, 30%, 40%, 50% or more. In some embodiments, the rest period and the reduction effect for the third cycle (and/or subsequent cycles) of the multi-course treatment is at least 18 months, preferably at least 2 years, more preferably at least 30 months, 36 months, 42 months, 48 months or more.

CD 2: inhibitors of LFA-3 interaction

CD 2: any inhibitor of LFA-3 interaction is useful in the methods of the invention. Such inhibitors include soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD 2 antibody homologs, soluble CD2 polypeptides, small molecules (e.g., chemical agents having a molecular weight of less than 2500Da, preferably less than 1500Da, chemical agents, such as small organic molecules, e.g., products of combinatorial libraries), LFA-3 and CD2 mimetics, and derivatives thereof.

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

Soluble CD2 and LFA-3 polypeptides

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. Soluble LFA-3 polypeptides are preferred, particularly soluble LFA-3/Ig fusions.

As used herein, a "soluble CD 2-binding LFA-3 polypeptide" is a polypeptide that includes at least the CD 2-binding domain of LFA-3(SEQ ID NO: 2) and is incapable of anchoring itself in a membrane. Such soluble polypeptides include, for example, LFA-3 polypeptides that lack a sufficient portion of their transmembrane domain to anchor the polypeptide or that are modified such that the transmembrane domain is nonfunctional. Soluble CD 2-binding LFA-3 polypeptides include soluble fusion proteins comprising at least the CD 2-binding domain of LFA-3 fused to a heterologous polypeptide. In one embodiment, the heterologous polypeptide is an Fc region of an immunoglobulin (e.g., an IgG1 hinge region and CH2-CH3 domains) or a substantial portion thereof.

Soluble LFA-3 polypeptides may be derived from the transmembrane form of LFA-3, particularly the extracellular domain (e.g., amino acids 1-187 of SEQ ID NO: 2 of US6,162,432, which is incorporated herein by reference). Such polypeptides are described in U.S. patent No.4,956,281 and U.S. patent No.6,162,432, which are incorporated herein by reference. Preferred soluble LFA-3 polypeptides include those comprising seq id NO: 2, residues 1-92 of SEQ ID NO: 2, residues 1-80 of SEQ ID NO: 2 and residues 50-65 of SEQ ID NO: 2, wherein the amino acid sequence of SEQ ID NO: 2 is shown in us patent No.6,162,432. Will comprise a nucleic acid sequence encoding SEQ ID NO: 2 (SEQ ID NO: 1) with deposit No.75107 at the American Type Culture Collection (Rockville, Maryland) of Rockville American Type Culture Collection, Maryland, wherein the amino acid sequence of SEQ ID NO: 1 and 2 are shown in US6,162,432.

Soluble LFA-3 polypeptides may also be derived from PI-linked forms of LFA-3, such as those described in PCT patent application Ser. No. WO 90/02181. A vector comprising a DNA sequence encoding PI-linked LFA-3(SEQ ID NO: 3) is deposited with the American type culture Collection of Rockville, Md., accession No. 68788. It will be appreciated 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 cross-modal form of LFA-3.

The most preferred soluble CD2 binding for use in the present inventionThe LFA-3 polypeptide is LFA-3/Ig fusion protein. An example of such a fusion protein is AMEVIVE^_(alefacept)。

AMEVIVE^_(alefacept)

AMEVIVE is a fusion protein that includes the first extracellular domain of human LFA-3(CD58) fused to the Fc portion of human IgG 1. In particular, AMEVIVE comprises the N-terminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acids of the human IgG1 hinge region (which contains 2 cysteine residues believed to be involved in interchain disulfide bonds), and a substantial portion of the CH2 and CH3 regions of the human IgG1 heavy chain constant region (e.g., SEQ ID NO: 3). The protein is a glycosylated, disulfide-linked dimer having a molecular weight of about 112kD under PAGE non-reducing conditions. The constant region of AMEVIVE has C-terminal variability, which corresponds to the splice variant form of the full-length fusion polypeptide.

Plasmid pSAB152, which encodes AMEVIVE, is deposited with the American type culture Collection of Rockville, Md. under accession number ATCC 68720.

pMDR (92) Ig-3 is an example of an expression vector that can be used to produce AMEVIVEE. pMDR (92) Ig-3 includes the following elements: (a) a pBR322 fragment containing the ColE1 origin and the beta lactamase expression cassette (GenBank accession No. j01749); (b) a DHFR expression cassette consisting of the following elements: the SV40 early promoter (part of GenBank accession No. J02400) with enhancer deleted, murine DHFR cDNA (GenBank accession No. L26316), the SV40 polyadenylation (SV40 poly A) site and the small t intron (part of GenBank accession No. J02400), and the human gastrin transcription terminator sequence, 3' UTR (Sato et al (1986) Mol CellBiol 6: 1032-1043); (c) an AMEVIVE expression cassette comprising, preferably in the following order: SV40 early Promoter/enhancer (GenBank accession No. j02400), Adenovirus major terminal Promoter including splice donor and intron sequences (Adenovirus major Late Promoter) and tripartite leader sequence (part of GenBank accession No. j01917), murine Ig heavy chain variable region intron sequences and splice acceptor (Kaufman and Sharp (1982) Mol Cell biol.2: 1304-1319), (optionally) cloning junctions, the first 92 amino acids of LFA-3 gene isolated from human tonsil cDNA library fused in-frame to nucleic acids encoding the hinge CH2 and CH3 regions of the human IgG1 gene isolated from human fibula-like genomic DNA library, cloning junctions (optionally), MIS 3' UT region containing a polyadenylation site (GenBank accession No. 03474), and SV40 polyadenylation site and small-t intron (GenBank accession No. 02400); and a fragment of pBR327 (GenBank accession No. l 08856).

The host cell line that can be used to produce AMEVIVE can be derived from CHO-DUkX-B1 cells. In one embodiment, DHFR (-) mutants of this cell line can be transfected with the vector pMDR (92) Ig-3, and DHFR (+) transformants can be cultured in selection medium (e.g., containing 25nM Methotrexate (MTX)). Positive transformants can be subjected to increased concentrations of MTX (e.g., 50nM) and colonies that produce high levels of AMEVIVE can then be selected.

The production of AMEVIVE can be carried out as follows: CHO host cells were thawed, scaled up to 2000L of culture, maintained in culture for 6-7 days under pH control and nutrient feed (at 48hrs., 96hrs., and 120hrs.), after which conditioned media was harvested by microfiltration. Preferably MTX is present in the culture medium. AMEVIVE can be recovered from conditioned medium by performing the following steps: (i) protein a chromatography, (ii) ceramic hydroxyapatite (chromatography), (iii) viral inactivation at low pH, (iv) hydrophobic interaction chromatography, (v) followed by concentration, diafiltration, viral filtration, and a second concentration step, which will yield the fusion product.

Another method of producing AMEVIVE for use in the method of the present invention is described in co-pending, commonly assigned U.S. patent application serial No.07/770,967. Typically, conditioned media of COS7 or CHO cells transfected with pSAB152 were concentrated using an AMICON S1Y30 spiral cartridge system (AMICON, Danvers, massachusetts) and subjected to protein a-sepharose 4B (Sigma, st. The bound protein was eluted and subjected to Superose-12(Pharmacia/LKB, Piscataway, N.J.) gel filtration chromatography.

Superose-12 fractions containing AMEVIVE with minimal contaminating proteins as determined on SDS-PAGE gels and by Western blot analysis (see, e.g., Towbin et al, Proc. Natl. Acad. Sci. USA, 74, pp 4350-54 (1979); Antibodies: A Laboratory Manual, pp 474-510 (Cold Spring Harbor Laboratory (1988)), pooled and concentrated in YM30 concentration tubes (AMICON). Rabbit anti-LFA-3 polyclonal antiserum was used followed by detection of AMEVEVE on Western blots by detectably labeled goat anti-rabbit IgG. purified COS7 or the AMEVE of CHO cells was a dimer of two monomeric A-3-Ig fusion proteins linked by disulfide bonds.

LFA-3-Ig fusion activity can be tested using the following bioassays: (1) CD32/64(Fc γ RI/RII) U937 cell bridge assay, and (2) CD16(Fc γ RIII) Jurkat cell bridge assay. Both assays tested the ability of AMEVIVE to bridge CHO cells displaying cell surface CD2 with Fc-gamma receptor expressing cells. The latter assay, assay (2), comprising culturing adherent CHO-CD2 cells in a 96-well plate to form a monolayer; adding amevite control and sample; adding fluorescently labeled Jurkat-CD16 (+); and measuring the fluorescence intensity.

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

CD2 polypeptide

Soluble CD2 polypeptides may be derived from full-length CD2, particularly the extracellular domain. Such a polypeptide may comprise all or part of the extracellular domain of CD 2. Exemplary 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 invention can be accomplished by a variety of methods known in the art. For example, the polypeptide may be derived from an intact transmembrane LFA-3 or CD2 molecule or an intact PI-bound LFA-3 molecule by proteolysis, which uses a specific endopeptidase to bind to an exopeptidase, Edman degradation, or a combination of both. The intact LFA-3 molecule or intact CD2 molecule may in turn be purified from its natural source using conventional methods. Alternatively, the entire LFA-3 or CD2 molecule can be produced by known recombinant DNA techniques using cDNAs (see, e.g., U.S. Pat. No.4,956,281 to 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 intact LFA-3 molecules or intact CD2 molecules as starting materials. This can be accomplished by conventional chemical synthesis techniques or by well-known recombinant DNA techniques in which only those DNA sequences encoding the desired peptide are expressed in a transformed host. For example, a gene encoding a desired soluble LFA-3 polypeptide or soluble CD2 polypeptide may be chemically synthesized using an oligonucleotide synthesizer. The oligonucleotides are designed based on the desired amino acid sequence of a soluble LFA-3 polypeptide or soluble CD2 polypeptide. The specific DNA sequence encoding the desired peptide may also be derived from the full-length DNA sequence by isolating specific restriction endonuclease fragments or by PCR synthesis of specific regions.

Standard methods can be used to synthesize genes encoding soluble LFA-3 polypeptides or soluble CD2 polypeptides useful in the invention. For example, a back-translated gene can be constructed using the complete amino acid sequence. DNA oligomers comprising a nucleotide sequence encoding a soluble LFA-3 polypeptide or a soluble CD2 polypeptide useful in the present invention can be synthesized in a single step. Alternatively, some smaller oligonucleotides encoding portions of the desired polypeptide may be synthesized and then ligated. Preferably, a soluble LFA-3 polypeptide or soluble CD2 polypeptide useful in the present invention will be synthesized as separate oligonucleotides that are subsequently ligated together. The individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly.

Once assembled, preferred genes will be characterized by sequences recognized by a restriction endonuclease (including unique restriction sites for direct assembly into a cloning or expression vector), taking into account the preferred codons used by the host expression system, and sequences that, when transcribed, produce a stable, efficiently translated rnRNA. Proper assembly can be determined by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host.

It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, DNA molecules comprising many other nucleotide sequences are also capable of encoding a soluble LFA-3 polypeptide or a soluble CD2 polypeptide encoded by the specific DNA sequences described above. These degenerate sequences also encode polypeptides useful in the present invention.

The DNA sequence may be expressed in a unicellular host, or preferably in an isolated mammalian host cell. As is well known in the art, in order 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 expression host of choice. Preferably, the expression control sequences, as well as the gene of interest, will be contained in an expression vector that also includes a bacterial selection marker and an origin of replication. If the expression host is a eukaryotic cell, the expression vector may also include additional expression markers for the expression host.

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

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

A wide variety of expression host/vector combinations may be employed. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from e.coli (e.coli), including colE1, pCR1, pBR322, pMB9, and derivatives thereof, broader host range plasmids, such as RP4, phage DNAs, numerous derivatives of, for example, lambda phage, such as NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Vectors that may be used for yeast cells include 2 μ plasmid and derivatives thereof. Vectors useful for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences can be used in the plasmids. Such useful expression control sequences include those associated with the structural genes of the aforementioned expression vectors. Examples of useful expression control sequences include, for example, the early or late promoters of SV40 or adenovirus, the lactose system (lac system), the tryptophan system (trp system), the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases, such as Pho5, the yeast alpha-mating system, and promoters of other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of host cells are available. The host cell may be a unicellular organism, or may be obtained from a multicellular organism, e.g., a cell isolated from a multicellular host. These hosts may include widely known eukaryotic and prokaryotic hosts such as strains of Escherichia coli, Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), fungi, yeasts, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and murine cells, African green monkey cells such as COS 1, COS7, BSC 1, BSC 40 and BMT 10, and human cells, and plant cells in tissue culture. For animal cell expression, CHO cell COS7 cell is preferred.

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

Various factors should also be considered in selecting expression control sequences. These factors include, for example, the relative concentration of the sequences (relative strength), their ability to control, and their compatibility with the DNA sequences discussed herein, particularly with respect to potential secondary structures. The selection of unicellular hosts should take into account their compatibility with the chosen vector, the toxicity of the product encoded by the DNA sequence, their secretory characteristics, their ability to fold the soluble polypeptide correctly, their requirements of fermentation or culture, and the ease of purification of the product encoded by the DNA sequence.

Within these parameters, one skilled in the art can select various vector/expression control sequence/host combinations that will express the desired DNA sequence in fermentation or large-scale animal culture, e.g., using CHO cells or COS7 cells.

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

Although recombinant DNA technology is a preferred method for producing useful soluble CD2 polypeptides or soluble LFA-3 polypeptides having a sequence of more than 20 amino acids, shorter CD2 or LFA-3 polypeptides having less than about 20 amino acids are preferably produced by conventional chemical synthesis techniques. The synthetically produced polypeptides used in the present invention can advantageously be produced in very high yields and are easy to purify.

Preferably, such soluble CD2 polypeptide or soluble LFA-3 polypeptide is synthesized by liquid or solid phase polypeptide synthesis and, optionally, digested with carboxypeptidase (to remove the C-terminal amino acid) or degraded by artificial Edman degradation (to remove the N-terminal amino acid). The use of liquid phase synthesis advantageously allows the direct insertion of certain derivatized amino acids into growing polypeptide chains, such as the O-sulfate of tyrosine. This avoids the need for subsequent derivatization steps to modify any residues used in the polypeptides of the invention.

Correct folding of Polypeptides can be achieved under oxidative conditions that favor disulfide bond formation, as described by Kent, "Chemical Synthesis of Polypeptides and proteins", ann. rev. biochem., 57, pp.957-89 (1988). The polypeptide produced by this method can then be purified by separation techniques well known in the art.

anti-LFA-3 and anti-CD 2 antibody homologs

As used herein, an "antibody homolog" is a protein comprising one or more polypeptides selected from the group consisting of: immunoglobulin light chains, immunoglobulin heavy chains, and antigen-binding fragments thereof that are capable of binding to an antigen. The constituent polypeptides of an antibody homolog comprising more than 1 polypeptide may optionally be disulfide-bonded or covalently cross-linked. Thus, antibody homologues include intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM class (and subtypes thereof), wherein the light chains of the immunoglobulins may be of the kappa or lambda class. Antibody homologs also include portions of intact immunoglobulins which retain antigen binding specificity, e.g., Fab fragments, Fab 'fragments, F (ab')₂Fragments, F (v) fragments, heavy chain unimers or dimers, light chain unimers or dimers, from one heavy chainAnd a light chain, and the like. This term includes recombinant antibodies, chimeric, CDR-grafted and humanized antibodies, or other antibodies that have been modified to be less immunogenic in humans.

As used herein, a "humanized recombinant or humanized antibody homolog" is an antibody homolog produced by recombinant DNA techniques in which some or all of the amino acids of the light or heavy chain of a human immunoglobulin required for antigen binding have been substituted for the corresponding amino acids from the light or heavy chain of a non-human mammalian immunoglobulin.

As used herein, a "chimeric, recombinant antibody homolog" is an antibody homolog produced by recombinant DNA techniques in which all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both have been substituted for the corresponding regions of a light chain or heavy chain from another immunoglobulin.

Many types of anti-LFA-3 or anti-CD 2 antibody homologs may be used in the methods of the invention. These antibodies include monoclonal antibodies, recombinant antibodies, chimeric recombinant antibodies, humanized recombinant antibodies, and antigen-binding portions of the foregoing.

Among anti-LFA-3 antibody homologs, the use of monoclonal anti-LFA-3 antibodies is preferred. More preferably, a monoclonal anti-LFA-3 antibody produced by a hybridoma selected from the group of hybridomas having accession No. ATCC HB10693(1E6), ATCC HB 10694(HC-1B11), ATCC HB10695(7A6), and ATCC HB 10696(8B8), or a monoclonal antibody known as TS2/9 (Sanchez-Madrid et al, "Three diseases antibodies Associated with human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2 and LFA-3", Proc. Natl. Acad. Sci. USA, 79, p. 7489-93 (1982)) is used. Most preferably, the monoclonal anti-LFA-3 antibody is produced by a hybridoma selected from the group of hybridomas having accession numbers nos. ATCC HB10695(7a6) and ATCC HB10693(1E 6).

Among the anti-CD 2 antibody homologs, the use of monoclonal anti-CD 2 antibodies, such as the anti-CD 2 antibody known as the T111 epitope antibody, is preferredCD2 monoclonal antibodies, including TS2/18(Sanchez-Madrid et al, "Three diseases antibodies Associated with Human T-Lymphocyte-mediatedcells: LFA-1, LFA-2 and LFA-3',Proc.Natl.Acad.Sci.USA79, pages 7489-93 (1982)).

Techniques for producing monoclonal antibodies are well known. For general reference, 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 trading antibodies of Predefined Specificity", 256, pages 495-97 (1975). Immunogens that may be used for the purposes of the present invention include cells carrying CD2 or LFA-3(CD 2-or LFA-3-bearing cells), and cell free preparations (cell free preparations) containing LFA-3, CD2, or anti-receptor-binding fragments thereof (e.g., CD2 fragment bound to LFA-3 or LFA-3 fragment bound to CD 2).

Immunization can be accomplished using standard methods. The unit dose and immunization schedule (immunization regiment) depend on the species of mammal being immunized, its immune status, the weight of the mammal, and the like. Typically, blood is collected from the immunized mammal and serum from each blood sample is assayed for specific antibodies using an appropriate screening assay. For example, useful anti-LFA-3 antibodies or anti-CD 2 antibodies can be identified by testing immune sera for their ability to block rosetting of ovine red blood cells of Jurkat cells, which is caused by LFA-3 and CD2 being present on the surface of each of these cells. Lymphocytes used in the production of hybridomas are typically isolated from immunized mammals whose sera have been tested positive for the presence of the desired antibody using the screening assay.

The anti-CD 2 and anti-LFA-3 antibody homologs used 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 well-known genetic engineering techniques. See, for example, U.S. Pat. No.4,816,397, which is incorporated herein by reference. For example, recombinant antibodies can be produced by cloning cDNA and genomic DNA encoding the immunoglobulin light and heavy chains of a desired antibody from hybridoma cells that produce the antibody homologs for use in the invention. The cDNA and genomic DNA encoding the polypeptides are then inserted into an expression vector such that the 2 genes are operably linked to their respective transcriptional and translational expression control sequences. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. Typically, 2 genes are inserted into the same expression vector.

Prokaryotic or eukaryotic host cells may be used. Expression in eukaryotic host cells is preferred because such cells are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies. Possibly, the host cell will produce a portion of the complete antibody, such as a light chain dimer or a heavy chain dimer, which is also an antibody homolog according to the invention.

It should be understood that variations of the above-described methods may be used with the present invention. For example, it may be desirable to transform a host cell with DNA encoding the light or heavy chain (but not both) of an antibody homolog. Recombinant DNA techniques may also be used to remove some or all of the DNA encoding either or both the light and heavy chains, the removed portion not being necessary for CD2 or LFA-3 counter receptor binding. Molecules expressed from these truncated DNA molecules are used in the methods of the invention. Alternatively, the bifunctional antibody may be produced in a form in which 1 heavy and 1 light chain are anti-CD 2 or anti-LFA-3 antibody homologues, and the other heavy and light chain is specific for an antigen other than CD2 or LFA-3 or another epitope of CD2 or LFA-3.

Chimeric recombinant anti-LFA-3 or anti-CD 2 antibody homologs may be produced by transforming a host cell with a suitable expression vector comprising DNA encoding the desired immunoglobulin light and heavy chains, wherein all or some of the DNA encoding the hinge and constant regions of the heavy and/or light chains has been replaced with DNA from the corresponding region of an immunoglobulin light or heavy chain of a different species. When the original recombinant antibody is of non-human origin and the inhibitor is to be administered to a human, a substitution of the corresponding human sequence is preferred. An exemplary chimeric recombinant antibody has a mouse variable region and human hinge and constant regions. See, generally, U.S. Pat. nos. 4,816,397; morrison et al, "Chimerichman Antibody Molecules: mouse antibody-Binding Domains With human constant Domains ", Proc. Natl. Acad. Sci. USA, 81, pages 6851-55 (1984); robinson et al, International patent publication No. PCT/US 86/02269; akira, 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, 1987, 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-CD 2 antibodies can be generated by replacing the sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from the human Fv variable region. Conventional methods for generating humanized antibodies are described by Morrison, s.l., 1985, Science 229: 1202-1207, Oi et al, 1986, BioTechniques 4: 214, and Queen et al, US5,585,089, US5,693,761, and US5,693,762, all of which are incorporated herein by reference. The methods comprise isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable region from at least 1 heavy or light chain. The source of the nucleic acid is well known to those skilled in the art and may, for example, be obtained from hybridomas producing anti-LFA-3 or anti-CD 2 antibodies. The nucleic acid encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Humanized or CDR-grafted antibody molecules or immunoglobulins may be produced by CDR-grafting or CDR-substitution, in which 1, 2, or all of the CDR's of the immunoglobulin chain may be replaced. See, for example, U.S. Pat. nos. 5,225,539; jones et al, 1986 Nature 321: 552 to 525; verhoeyan et al, 1988 Science 239: 1534; beidler et al, 1988 J.Immunol.141: 4053-4060; winter US5,225,539, the contents of all of which are expressly incorporated herein by reference. Winter describes a CDR-grafting procedure that can be used to prepare the humanized antibodies of the present invention (UK patent application GB 2188638A, filed 3/26 of 1987; Winter US5,225,539), the contents of which are expressly incorporated by reference. All of the CDR's of a particular human antibody can be replaced with at least a portion of the non-human CDRs, or only some of the CDR's can be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding the humanized antibody to the predetermined antigen, e.g., LFA-3 or CD 2.

Humanized antibodies are also within the scope of the invention, and include immunoglobulins in which specific amino acids have been substituted, deleted or inserted. In particular, preferred humanized antibodies have amino acid substitutions within the framework regions, such as to improve binding to the antigen. For example, selected, small numbers of acceptor framework residues of the humanized immunoglobulin chain may be replaced with corresponding donor amino acids. Preferred positions for substitution include amino acid residues adjacent to or capable of interacting with the CDRs (see, e.g., US5,585,089). Criteria for selecting amino acids from donors are described in U.S. Pat. No.5,585,089, e.g., columns 12-16 of U.S. Pat. No.5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing immunoglobulin chains, including antibodies, are described in Padlan et al EP 519596A 1, published at 23.12.1992.

Transgenic mice carrying the intact human immune system, rather than the murine system, can be used to produce human monoclonal antibodies (mAbs) directed to human LFA-3 or CD 2. Splenocytes from transgenic mice immunized with an antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinity for an epitope of a protein from a human (see, e.g., 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, L.L. et al, 1994 Nature Genet.7: 13-21; Morrison, S.L. et al, 1994 Proc.Natl.Acad.Sci.USA 81: 6851-6855; Bruggeman et al, Yearmunol 7: 33-40; Imaillon et al, PNAS 90: 3720-3724; Brgeman et al, Eumul J1991: 1323-1993).

Monoclonal antibodies can also be produced by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, known as the "combinatorial antibody library display" method, has been developed to identify and isolate antibody fragments with specific antigen specificity and can be applied to the production of monoclonal antibodies (for a description of combinatorial antibody library display see, e.g., Sastry et al 1989 PNAS 86: 5728; Huse et al 1989 Science 246: 1275; and Orlandi et al 1989 PNAS 86: 3833). After immunization of the animals with the immunogen as described above, the antibody repertoire of the resulting B cell pool is cloned. Methods for obtaining variable region DNA sequences of immunoglobulin molecules from different populations by using oligomer primer mixtures and PCR are generally known (Larrick et al, 1991, Biotechnology 11: 152-156; Larrick et al, 1991, Methods: Companion to Methods in Enzymology 2: 106-110).

Examples of methods and reagents particularly suitable for generating a variegated antibody display library can be found, for example, in the following documents: ladner et al, U.S. Pat. Nos. 5,223,409; international publication No. WO 92/18619 to Kang et al; dower et al, International publication No. WO 91/17271; winter et al, International publication WO 92/20791; markland et al, International publication No. WO92/15679; breitling et al, International publication WO 93/01288; McCafferty et al, International publication No. WO 92/01047; garrrard 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 antibody hybrids 3: 81-85; huse et al (1989) Science 246: 1275-1281; griffhs et al, (1993) EMBO J12: 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 display libraries are commercially available (e.g., Pharmacia recombinant phage antibody System, catalog No. 27-9400-01; and Stratagene SurfZAPTM phage display kit, catalog No. 240612).

In certain embodiments, the V region domains of the heavy and light chains may be expressed on the same polypeptide, joined by a flexible linker to form a single chain Fv fragment, and the scFV genes subsequently cloned into a desired expression vector or phage genome. Generally, as in McCafferty et al, Nature (1990) 348: 552-554, the entire VH and VL domains of the antibody linked by a flexible (Gly4-Ser)3 linker can be used to produce single chain antibodies whose antigen-based affinity can confer a separable package to the display phage. The isolated scFV antibody that immunoreacts with the antigen can then be formulated into a pharmaceutical formulation for use in the subject methods.

Specific antibodies having high affinity for surface proteins can be prepared according to methods known to those skilled in the art, for example, methods involving library screening (Ladner, r.c., et al, U.S. patent 5,233,409; Ladner, r.c., et al, U.S. patent 5,403,484). Also, these library methods can be used in screening to obtain binding determinants, which are mimics of the structural determinants of the antibody. See, for example, baiorith, j, and s.sheiff, 1996, Proteins: struct, funct, and genet.24(2), 152-157; webster, D.M. and A.R.Rees, 1995, "Molecular modeling of binding-binding sites," S.Paul, Ed., Methods in Molecular biol.51, Antibody Engineering Protocols, Humana Press, Totowa, NJ, pages 17-49; and Johnson, G, Wu, t.t. and e.a.kabat, 1995, "Seqhunt: a program to sequenced nucleotides and amino acid sequences, "Methods in Molecular biol.51, op.cit., pages 1-15.

The invention may also be practiced with anti-CD 2 antibodies and anti-LFA-3 antibody homologs that are not intact antibodies. The homologue canDerived from any of the antibody homologs described above. For example, antigen-binding fragments derived from the antibodies described above, as well as full-length monomeric, dimeric or trimeric polypeptides, are useful per se. Useful antibody homologs of this type include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment comprising, at the hinge region, a bivalent fragment of 2 Fab fragments linked by a disulfide bond; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al, (1989) Nature341: 544-546) consisting of one VH domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that enables them to be made into a single protein chain using recombinant methods, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science)242: 423-426; and Huston et al (1988) Proc.Natl.Acad.Sci.USA85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for use in the same manner as intact antibodies. The anti-LFA-3 heavy chain is a preferred anti-LFA-3 antibody fragment.

Antibody fragments may also be produced chemically, for example, by cleaving intact antibodies with a protease such as pepsin or papain, and optionally treating the cleaved product with a reducing agent. Alternatively, useful fragments are produced by host cells transformed with truncated heavy and/or light chain genes. Production of heavy and light chain monomers can be achieved by treating the intact antibody with a reducing agent such as dithiothreitol, followed by purification to isolate the chains. Production of heavy and light chain monomers can also be achieved by host cells transformed with DNA encoding either the desired heavy or light chain, but not both. See, e.g., Ward et al, "Binding Activities of a Repertoireof Single Immunoglobulin Variable Domains Secreted from Escherichia coli″，Nature341 pages 544-46 (1989); sasty et al, "Cloning of a the immunogenic resin reagent inEscherichia colifor Generation of Monoclonal catalytic antibodies: the Construction of a Heavy Chain Variable Region-Specific cDNAlibrary ", Proc. Natl. Acad. Sci. USA, 86, pp 5728-32 (1989).

LFA-3 and CD-2 mimetic or small molecule agents

Also useful in the methods of the invention are LFA-3 and CD-2 mimetic agents. These agents are CD 2: an inhibitor of LFA-3 interaction, wherein the agent may be a peptide, a semi-peptide compound, or a non-peptide compound (e.g., a small organic molecule). Preferred CD2 and LFA-3 mimetic agents will inhibit CD 2: LFA-3 interaction, which inhibits at least as well as anti-LFA-3 monoclonal antibody 7A6 or anti-CD 2 monoclonal antibody TS2/18 (described above).

In a preferred embodiment, the test agent is a member of a combinatorial library, e.g., a peptide or organic combinatorial library, or a natural product library. In a preferred embodiment, the plurality of test compounds, e.g., library members, comprises at least 10, 10²，10³，10⁴，10⁵，10⁶，10⁷Or 10⁸The compound of (1). In a preferred embodiment, a plurality of test compounds, e.g., library members, share a structural or functional characteristic.

In one embodiment, the invention provides libraries of LFA-3 and/or CD2 inhibitors. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e.g., e.m. gordon et al, j.med.chem. (1994)37：1385-1401；DeWitt，S.H.；Czarnik，A.W.Acc.Chem.Res.(1996) 29：114；Armstrong，R.W；Combs，A.P；Tempest，P.A.；Brown，S.D.；Keating，T.A.Acc.Chem.Res.(1996) 29：123；Ellman，J.A.Acc.Chem.Res.(1996) 29：132；Gordon，E.M.；Gallop，M.A.；Patel，D.V.Acc.Chem.Res.(1996) 29: 144, 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; U.S. patents 5,359,115, 5,362,899, and 5,288,514; PCT publication Nos. WO92/10092, WO93/09668, WO91/07087, WO93/20242, WO 94/08051).

Libraries of the compounds of the invention can be prepared according to various methods, some of which are known in the art. For example, the "split-pool" strategy can be implemented as follows: placing beads (beads) of the functionalized polymeric support into a plurality of reaction vessels; a variety of multimeric supports suitable for solid phase peptide Synthesis are known, and some are commercially available (see, e.g., M. Bodansky "Principles of Peptides Synthesis", second edition, Springer-Verlag, Berlin (1993)). A different solution of activated amino acid was added to each aliquot of beads and the reaction was allowed to proceed to produce a plurality of immobilized amino acids, one in each reaction vessel. Aliquots of the derived beads are then washed, "pooled" (i.e., recombined) and the bead pool is subdivided, placing each aliquot into a separate reaction vessel. Another activated amino acid was then added to each aliquot of beads. The cycle of synthesis is repeated until the desired peptide length is obtained. The amino acid residues added in each synthetic cycle can be randomly selected; alternatively, amino acids may be selected to provide a "biased" library, e.g., such a library: wherein certain portions of the inhibitor are non-randomly selected, e.g., to provide an inhibitor having known structural similarity or homology to a known peptide capable of interacting with the antibody, e.g., an anti-idiotypic antibody antigen binding site. It will be appreciated that a wide variety of peptidic, peptidomimetic, or non-peptidic compounds can be readily produced in this manner.

The "lysis pool" strategy results in the formation of peptide libraries, e.g., inhibitors, which can be used to prepare test compounds of the inventionThe library of (1). In another exemplary synthesis, a "diversity library" (proc.natl.acad.sci.us.a.) was made by the method of Hobbs DeWitt et al.90: 6909(1993)). Other synthetic methods, including Houghten's "tea-tag" technology (see, e.g., Houghten et al Nature354: 84-86(1991)), or can be used for the synthesis of libraries of compounds according to the objects of the invention.

Libraries of compounds can be screened to determine if any member of the library has the desired activity, and, if so, to identify active species. Methods of screening combinatorial libraries have been described (see, e.g., Gordon et al, J med. Libraries of soluble compounds can be screened by affinity chromatography using the appropriate receptor to isolate ligands for the receptor, followed by identification of the isolated ligands by conventional techniques (e.g., mass spectrometry, NMR, etc.). Immobilized compounds can be screened by contacting the compound with a soluble receptor; preferably, the soluble receptor is coupled to a detectable label (e.g., a fluorophore, colorimetric enzyme, radioisotope, luminescent compound, etc.) to indicate ligand binding. Alternatively, the immobilized compound may be selectively released and allowed to diffuse through the membrane to interact with the receptor. Exemplary assays for library screening of the invention are described below.

In one embodiment, compounds of the invention may be screened for the ability to interact with CD2 or LFA-3 polypeptides by determining whether each compound binds directly to the polypeptide or inhibits CD 2: LFA-3 interaction activity, for example, by incubating a test compound with CD2 or an LFA-3 polypeptide and a lysate, e.g., a T or APC cell lysate, e.g., in one well of a multi-well culture plate, such as a standard 96-well microplate. In this embodiment, the activity of each individual compound can be determined. One or more wells without test compound can be used as a control. After incubation, the activity of each test compound can be determined by assaying each well. Thus, the activity of a plurality of test compounds can be determined in parallel.

In another embodiment, the binding activity of a plurality of test compounds can be determined simultaneously. For example, in the "one bead-one compound" synthesis, the test compound can be synthesized on solid resin beads; the compound may be immobilized on a resin support via a photolabile linker. A plurality of beads (e.g., up to 100,000 beads or more) may then be combined with the yeast cells and sprayed to form a plurality of "nano-droplets" (each of which contains a single bead (and, therefore, a single test compound). The nanodrops were then exposed to UV light causing the compound to break apart from the beads. It will be appreciated that this assay format allows large libraries of test compounds to be screened in a rapid manner.

Combinatorial libraries of compounds can be synthesized using "tags" (tags) to encode the identity (identity) of each member of the library (see, e.g., w.c. still et al, U.S. patent No.5,565,324 and PCT publication nos. WO94/08051 and WO 95/28640). In general, this method focuses on the use of labels attached to solid supports or compounds that are inert but easy to detect. When an active compound is detected (e.g., by one of the techniques described above), the identity of the compound is determined by the identification of the unique companion tag. This tagging approach allows the synthesis of large libraries of compounds that can be identified at very low levels. This labeling scheme can be used, for example, to identify compounds released from the beads in the "millidroplet" screening assay described above.

In a preferred embodiment, the compound library of the invention contains at least 30 compounds, more preferably at least 100 compounds, and more preferably at least 500 compounds. In a preferred embodiment, the compound library of the invention contains less than 10⁹More preferably less than 10⁸Seed compound, and more preferably less than 10⁷A compound is provided.

Derivatized inhibitors

CD 2: inhibitors derived from LFA-3 interactions may also be used in the methods of the invention, where, for example, any of the antibody homologs described herein, soluble CD2 and LFA-3 polypeptides, or CD2 and LFA-3 mimetics, are functionally bound (by chemical coupling, genetic fusion, or other means) to one or more members independently selected from the group consisting of: anti-LFA-3 and anti-CD 2 antibody homologs, soluble LFA-3 and CD2 polypeptides, CD2 and LFA-3 mimetic agents, cytotoxic agents, and pharmaceutical agents.

One type of derivatized inhibitor is produced by crosslinking 2 or more inhibitors (of the same type or of different types). Suitable crosslinking agents include those heterobifunctional reagents (heterobifunctional) having 2 different reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or those homobifunctional reagents (e.g., disuccinimidyl suberate). The linker is available from Pierce Chemical Company, Rockford, Illinois.

Another possibility for cross-linking is to use the PI bonding signal sequence in the PI-linked LFA-3 or a fragment thereof. Specifically, DNA encoding a PI bonding signal sequence (e.g., amino acids 162-212 of SEQ ID NO: 4) is ligated downstream of DNA encoding the LFA-3 polypeptide, which is preferably soluble, of the desired polypeptide. If this construct is expressed in an appropriate eukaryotic cell, the cell will recognize the PI bonding signal sequence and will covalently attach the PI to the polypeptide. The hydrophobicity of the PI can then be exploited to form micellar aggregates (micella aggregatates) of the polypeptide.

Inhibitors linked to 1 or more cytotoxic or pharmaceutical agents may also be used. Useful pharmaceutical agents include biologically active peptides, polypeptides and proteins, such as antibody homologs specific for human polypeptides other than CD2 or LFA-3, or fragments thereof. Useful pharmaceutical and cytotoxic agents also include UV radiation (e.g., UVB), cyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids, interferons, and nitrogen mustard.

Preferred inhibitors that are derivatized with a pharmaceutical agent include recombinantly produced polypeptides in which a soluble LFA-3 polypeptide, a soluble CD2 polypeptide, or a peptidyl CD2 or peptidyl LFA-3 mimetic agent is fused to all or part of an immunoglobulin heavy chain hinge region or all or part of a heavy chain constant region. Preferred polypeptides for preparing the fusion protein are soluble LFA-3 polypeptides. Most preferred are fusion proteins containing fusion to human IgG₁Amino acids 1-92 of mature LFA-3 (including the C-terminal 10 amino acids of the hinge region, which contains 2 cysteine residues believed to be involved in interchain disulfide bonds), and IgG, over a portion of the hinge region₁C of heavy chain constant domain_H2 and C_HAnd (3) zone. Such fusion proteins are expected to exhibit extended serum half-life and to be able to dimerize inhibitors.

The utility of specific soluble CD2 polypeptides, soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD 2 antibody homologs, or CD2 and LFA-3 mimetic agents in the methods of the invention can be readily determined by assaying their ability to inhibit LFA-3/CD2 interactions. This ability can be determined, for example, using a simple cell binding assay that allows a visual (under magnification) assessment of the ability of putative inhibitors to inhibit the interaction between LFA-3 and CD2 on cells carrying these molecules. Jurkat cells are preferred as CD2⁺Substrate, and sheep red blood cells or human JY cells are preferred as LFA-3⁺A substrate. The binding characteristics of soluble polypeptides, antibody homologs, and mimetic agents useful in the invention can be determined by a number of known methods, such as by radiolabelling (e.g., with) the antibody homolog, polypeptide, or agent³⁵S or¹²⁵I) The labelled polypeptide, mimetic or antibody homologue is then suitably conjugated to CD2⁺And LFA-3⁺And (4) contacting the cells. The binding characteristics can also be determined using a suitable enzymatically labelled secondary antibody. Rosette competition assays such as those described by Seed et al (Proc. Natl. Acad. Sci. USA, 84, pp. 3365-69 (1987)) may also be used.

Combination therapy

The agents, e.g., soluble CD2 binding LFA-3 polypeptides, may be used in combination with other therapies, e.g., other agents. The other agents are referred to herein as "second agents" or "additional agents" and include 1 or more of: immunosuppressants (e.g., methotrexate, cyclosporin, or chlorambucil), cyclophosphamide, prednisone, FK506, steroids, retinoids, interferons, nitrogen mustard, cytokine binding agents (e.g., type 2 cytokine binding agents, e.g., IL-2 or IL-8 binding agents, e.g., anti-IL-2 or IL-8 monoclonal antibodies (Abgenix)), inhibitors of ICAM/LFA-1 interactions, ICAM-binding agents (e.g., antibodies, e.g., monoclonal antibodies) against ICAM-1 (e.g., humanized, chimeric, or human anti-ICAM-1 antibodies); or an LFA-1 (also known as CDlla) binding agent (e.g., an antibody, e.g., a monoclonal antibody) against LFA-1 (e.g., a humanized, chimeric, or human anti-LFA-1 antibody, e.g., Raptiva (Genentech/Xoma)); costimulatory molecule binding agents, such as B7-1(CD80) binding agents (anti-B7-1 monoclonal antibody (IDEC)); vasodilators (e.g., ACE inhibitors or minoxidil); corticosteroid or penicillamine. In one embodiment, an agent, such as CD 2: an inhibitor of LFA-3 interaction, administered in combination with 1 or more interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-8, TGF- β, PDGF, granzyme A, or leukotriene B4 inhibitors. Such combination therapy may advantageously utilize lower doses of therapeutic or prophylactic agents.

"combined" administration, as used herein, means that two (or more) different treatments are administered to a subject during the course of the subject experiencing the disease, e.g., two or more treatments are administered after the subject has been diagnosed with the disease and before the disease has not yet cured or eliminated. In some embodiments, administration of one treatment also occurs at the beginning of administration of the second treatment, such that there is overlap. This is sometimes referred to herein as "simultaneous" or "concurrent administration". In other embodiments, administration of one treatment is discontinued before the other treatment is started. In some embodiments of another scenario, the treatment is more effective as a result of the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or the like seen with the first treatment. In some embodiments, the reduction in symptoms, or other parameters associated with the disease, resulting from such administration, e.g., a reduction in T cell levels or activity, is greater than would be observed if one treatment were administered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Administration can be such that the effect of the first treatment administered is also detectable when the second treatment is administered, e.g., when CD 2-or LFA-3 binding agent is first administered, a decrease in T cell levels or activity is also detectable when the second agent is administered. In preferred embodiments, the administration of the first treatment and the second treatment occurs within 1, 2,5, 10, 15, or 30 days of each other.

In preferred embodiments, the CD 2-binding agent (e.g., LFA-3/Ig fusion), the second agent (or both), or a pharmaceutical composition containing the same is administered systemically, e.g., intravenously, intramuscularly, subcutaneously, intraarticularly, transdermally, intrathecally, periosteally, intratumorally, intralesionally, perilesionally, by infusion (e.g., using an infusion device), orally, topically, or by inhalation. Preferably, the CD 2-binding agent is administered intramuscularly or intravenously. In other embodiments, the CD 2-binding agent is administered locally to the affected area, e.g., locally or by needle-free injection (needle injection).

Parenteral administration of the CD 2-binding agent (e.g., LFA-3/Ig fusion), the second agent (or both), or a pharmaceutical composition containing the same may be carried out by methods known in the art using a needle or needleless syringe. Examples of needleless injector systems and modes of administration are described in US6,132,395, US6,096,002, US5,993,412, US5,893,397, US5,520,639, US5,503,627, US5,399,163, US5,383,851, US5,312,577, US5,312,335, all of which are incorporated herein by reference.

Pharmaceutical composition

Preferably, an effective amount of CD 2: LFA-3 inhibitors (e.g., soluble, CD 2-binding LFA-3 polypeptides described herein). By "effective amount" is meant an amount capable of reducing the spread or severity of the disease described herein. In therapeutic embodiments, an effective amount of the agent refers to an amount of the agent that is effective to inhibit, ameliorate, or ameliorate the disease (e.g., increase the PASI score or PGA score in a patient with psoriasis) or to prolong the life of a patient with the disease beyond that expected in the absence of such treatment, when administered in a single or multiple doses to the subject. For example, improvement in psoriasis is expected to result in an improvement in quality of life, as assessed, for example, by the SF-36 Health questionnaire conducted by RAND Health of one department of RAND corporation (Santa monaca, ca). An effective amount of a dose does not necessarily indicate complete elimination of the disease. In a prophylactic embodiment, an effective amount of a CD 2-or LFA-3 binding agent described herein refers to an amount of the agent that is effective to prevent or delay the onset of an episode or the recurrence of a disease when administered to a subject in a single or multiple doses.

It will be understood by those skilled in the art that an effective amount of an agent will depend upon, among other things, the disease being treated (e.g., T cell mediated skin disease versus T cell mediated organ disease other than skin disease), the dosing schedule, the unit dose administered, whether the agent is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic or prophylactic activity of the particular agent administered, and the half-life of the serum. The medicament may be packaged differently depending on the disease to be treated.

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

Unit doses are typically administered until an effect is observed. The effect can be measured by various methods, including in vitro T cell activity assays and clearance or improvement of affected skin areas, or improvement in other affected body areas that may be associated with a particular disease. Preferably, the unit dose is administered periodically, such as once per week, during the treatment cycle. More preferably, it is administered periodically, e.g. at weekly intervals for an administration period of several weeks, e.g. 12 weeks. More frequent administration, e.g., 2 or 3 times per week, is also contemplated and may be appropriate if the subject's disease is severe or if urgent intervention is indicated. Less frequent administration, e.g., 1 or 2 times per month, is also contemplated and may be employed if the subject responds well to treatment so as to be suitable for maintaining the dose. However, it should be recognized that lower or higher doses and other dosing schedules may be employed during any one particular cycle of administration.

The agent, e.g., CD 2-binding LFA-3 polypeptide (e.g., amevie), is also preferably administered in the form of a composition comprising a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that does not elicit an allergic reaction or other untoward effect in the patient to whom it is administered.

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

Formulations of CD 2-binding agents, e.g., pharmaceutical formulations, may be prepared in aqueous or non-aqueous phase, e.g., lyophilized forms. Preferred pharmaceutical formulations are suitable for injection. Examples of aqueous phase formulations encompassed by the present invention include Phosphate Buffered Saline (PBS) frozen liquid formulations. Examples of lyophilized formulations include one or more of: citric acid, glycine and sucrose. For example, one preferred lyophilized formulation includes 1-5% sucrose, preferably 2.5% sucrose, and 0.5% -2% glycine, preferably 1% glycine, buffered to a pH of at least about 4, preferably 5, more preferably about 6 (or even more preferably 6.8) in a sodium citrate-citric acid buffer (at least 10mM, preferably 25 mM).

The second agent may be administered in a single dose form with the CD 2-binding agent (i.e., as part of the same pharmaceutical composition), in multiple dose forms administered separately from but simultaneously with the CD 2-binding agent, or in multiple dose forms wherein the two components are administered separately and sequentially. Alternatively, the CD 2-binding agent and the other active agent may be in the form of a single conjugated molecule. Coupling of the two components can be achieved by standard crosslinking techniques well known in the art. The single molecule may also take the form of a recombinant fusion protein. In addition, the pharmaceutical compositions used in the present invention may be used in combination with other therapies, such as PUVA, chemotherapy and UV light. Such combination therapy may advantageously utilize lower doses of therapeutic or prophylactic agents.

The CD 2-binding agent, or pharmaceutical composition, may be in various forms. These forms include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable solutions, infusions (infusible), and topical formulations. The preferred form depends on the mode of administration and the therapeutic application needs. The preferred form is an injection or infusion solution.

The present invention includes formulations suitable for use as sunscreens or UV protectants for topical application. Preferred embodiments include AMEVIVE formulations. The active ingredient may be formulated as liposomes. The product may be applied before, during, or after UV exposure, or before, during, or after redness (redness) develops.

Sequence of

The following is a summary of the sequences described in US6,162,432 and referred to throughout this application:

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

SEQ ID NO: amino acid sequence of LFA-3 with 2 membrane-spanning

SEQ ID NO: DNA sequence of 3 PI-linked LFA-3

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

SEQ ID NO: DNA sequence of 5 CD2

SEQ ID NO: amino acid sequence of 6 CD2

SEQ ID NO: 7 AMEVIVE DNA sequence

SEQ ID NO: 8 AMEVIVE amino acid sequence

Examples

Example 1: multiple course treatment of psoriasis using AMEVIVE (alefacept)

This example examines efficacy and safety in patients receiving multiple courses of treatment with amevie _ for up to 9 treatment cycles of up to 4.5 years.

Treatment of

The initial treatment cycle in the open-label (open-label) study is referred to as cycle a. Subsequent cycles are labeled cycles B, C, D, etc. During each cycle, patients receive weekly treatments for 12 weeks (dosing period), followed by 12 weeks of observation (rest period).

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

Prior to initial treatment for the extension study, based on the physician's assessment of disease severity, and CD4+ T cell count at or above the lower limit of normal (LLN; 404 cells/mm)³) Establishing the need for systemic treatment.

Eligibility for subsequent cycles is based on the aforementioned criteria, and, in addition: during the 12-week treatment period of the previous cycle, the patient must have received an AMEVIVE dose of ≧ 8, and for cycle C and subsequent cycles, the lymphocyte count requires ≧ 75% of the counts recorded at the screening visit (screening visit) of the study.

If the patient has a CD4+ T cell count < 300 but > 200 cells/mm during any given cycle³The dose of AMEVIVE was reduced by 50% (3.75 mg). If CD4+ T cell count < 200 cells/mm³The planned dose is maintained. If CD4+ T cell count < 200 cells/mm³AMEVIVE was maintained persistently for 4 consecutive follow-ups. The dose of AMEVIVE was maintained for 2 weeks when signs of clinically significant infection were seen.

Evaluation of

Efficacy was assessed by the Psoriasis Area and Severity Index (PASI) and by physician comprehensive assessment (PGA). For cycle a, evaluations were performed at 1, 3, 5, 7, 9,11, and 12 weeks during treatment and at 2,4, 6, 8, and 12 weeks after treatment. For subsequent cycles, evaluations were performed at 1 and 7 weeks during treatment and at 2 and 12 weeks after treatment. The proportion of patients who obtained > 50% and > 75% improvement in PASI relative to baseline (PASI 50 and PASI 75, respectively) and patients who obtained "clear" or "almost clear" PGA was reported.

At each study follow-up, analysis of all lymphocytes and lymphocyte subtypes was performed except 4 weeks after cycle a treatment and 4 and 8 weeks after all subsequent cycles of treatment. Patients with new or ongoing viral, bacterial, or fungal infections were monitored throughout the follow-up. Adverse events were monitored throughout the study.

Results

At the time of this analysis, patients had received repeated cycles of treatment with amevie for up to 4.5 years. In this study, 175 patients had received ≧ 1 cycle of AMEVIVE; 126 accept more than or equal to 2 cycles; 96 accepts more than or equal to 3 cycles; 71 accepts ≧ 4 cycles. Some patients have received up to 9 cycles of AMEVIVE as a result of exposure to AMEVIVE in the previous phase 2 study.

Efficacy of

The proportion of patients who obtained PASI50 at 2 or 12 weeks after cycle a-D treatment is shown in figure 2. The response rates for cycles C and D were significantly increased compared to cycles a and B. In cycles A, B, C, and D, the proportion of patients who achieved PASI 75 was 29%, 33%, 34%, and 52%, respectively. The response rates for the "clear" or "almost clear" PGA were 24%, 29%, 33%, and 34%, respectively.

Incremental benefit (incremental benefit) and repeated response of additional treatment cycles using AMEVIVE are shown in fig. 3 for cycles a-D. For cycle a responders who received additional cycles of AMEVIVE (i.e., patients who acquired PASI50 in cycle a), the proportion of patients who acquired PASI50 increased with each subsequent cycle. Generally, patients continue to respond to repeated treatments with amevie without signs of tachyphylaxis. Of those patients who acquire PASI50 in a given cycle, 75% -90% acquire PASI50 in subsequent cycles (i.e., repeated responses).

Adverse events

Generally, the incidence of adverse events does not change much in the cycle. The overall safety profile of AMEVIVE following multi-cycle (multi-course treatment) administration was similar to the results reported in the phase 3 study. The incidence of severe adverse events in any cycle was 7% or less, and the profile of severe adverse events was similar to the previous phase 2 and 3 studies.

Due to adverse events, 2 patients in each of cycles a and B and 1 patient in cycle E stopped treatment. The incidence of toxicity is low: 3% or less in any cycle; most are skin cancers.

Duration of Treatment-Free response

The slowing effect of AMEVIVE was demonstrated in a phase 3 study with patients maintaining a median PASI50 response duration of 7 months. In this example, some patients have been tracked for an extended period following a successful treatment cycle. Figure 4 shows the maximum length of response time in 4 of these patients. In some patients, response to treatment with amevie remained 18-24 months.

Lymphocyte count

The decrease in lymphocyte counts was consistent throughout the multi-cycle treatment with AMEVIVE. The decrease in lymphocyte counts observed with each cycle is not cumulative.

The mean count of CD4+ T cells remained above LLN for all cycles and was not reduced by multiple course exposure to AMEVIVE (fig. 5).

In summary, the present study shows that multiple course therapy (3 treatment cycles or more) provides more significant results than single course therapy without the additional risk of significant side effects. Patients tolerate multiple cycles of amevie well and the incidence of adverse events varies little throughout the cycle.

Example 2: multiple course treatment of psoriasis using AMEVIVE

This example examines the clinical response to treatment with AMEVIVE second cycle and the efficacy of multi-cycle treatment in psoriatic patients who failed to achieve > 50% reduction in psoriasis area and severity index (PASI 50) or > 25% reduction (PASI 25) during treatment with AMEVIVE first cycle.

Patient's health

Patients are ≥ 16 years old and have chronic plaque psoriasis for a period of ≥ 12 months, involving ≥ 10% of body surface area. The CD4+ T cell count requirement is above the lower normal limit (LLN). Phototherapy, systemic retinoids, systemic corticosteroids, systemic fumarate, immunosuppression (methotrexate, cyclosporin, azathioprine, and thioguanine), and high potency topical corticosteroids were prohibited within 4 weeks prior to treatment with AMEVIVE and throughout the study. Within 2 weeks prior to treatment with AMEVIVE and throughout the course of the study, moderate-acting topical corticosteroids, topical retinoids, coal tar, keratolytic agents, and vitamin D analogs were contraindicated except on the scalp, palm, groin, and sole. To be eligible for participation in this extended study, patients were asked to have received AMEVIVE at doses ≧ 8 and to complete the final follow-up visit (follow-up visit) of the previous treatment cycle. These patients were excluded from this extension study if they were involved in any other investigational study with drug or non-drug treatment, or initiated an alternative systemic psoriasis treatment, phototherapy, or other prohibited treatment 8 weeks prior to the previous cycle.

Treatment of

Phase 3 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, patients received 2 cycles of treatment, where each cycle consisted of the following phases: (i) a 12-week dosing period, AMEVIVE (7.5mg) or placebo once a week, and (ii) a 12-week follow-up (follow-up) (rest period). In a phase 3 study of Intramuscular (IM) treatment with AMEVIVE, patients received a single cycle of treatment consisting of a 12-week dosing period followed by a 12-week observation period (rest period), with AMEVIVE (10mg or 15mg) or placebo once a week during the dosing period.

In a multi-session treatment study, patients receiving additional cycles of treatment with AMEVIVE (same dose regimen) were those in whom the investigator determined that their disease had progressed to a point where systemic treatment or phototherapy was required and had a circulating CD4+ T cell count at or above LLN (Gordon et al, j. drugs dermatol.2: 624-628, 2003).

Evaluation of

PASI and physician combined assessment (PGA) were assessed at baseline during the phase 3 study, 1 every 2 weeks during treatment, and 1 every 2-4 weeks during follow-up. PASI and PGA were also evaluated at some time points during the IV multi-session study, but only PGA was evaluated during the IM multi-session study.

Analysis of

Data from a 2-cycle, 3-phase study of IV treatment with AMEVIVE was applied to determine the efficacy of the second cycle of treatment with AMEVIVE in 2 groups of patients: (a) those patients who failed to obtain PASI50 in the first cycle and (b) those patients who failed to obtain PASI 25 in the first cycle. Patients who failed to achieve PASI50 and PASI 25 during cycle 1 of treatment with AMEVIVE were determined to achieve a proportion of patients who achieved PASI50 or a PASI reduction of > 75(PASI 75) at any time during the second cycle of treatment. The likelihood ratio and corresponding 95% Confidence Intervals (CIs) were calculated to compare the response rates between patients receiving the second cycle of treatment with amevie and patients receiving placebo. Patient proportions to obtain PASI50, PASI 75 and a "clear" or "almost clear" PGA at any time during each cycle of IV therapy, and a "clear" or "almost clear" PGA at any time during each cycle of IM therapy, are also determined.

Results

The average age of the patients was-45 years, and-70% were males. The duration of psoriasis was-19 years and the average body surface area involved was-22%. The number of patients receiving each cycle of IV and IM treatment with amevie is summarized in table 1.

Treatment cycle
							1	2	3	4	5
IV AMEVIVE	521	327	217	158	39
						IM AMEVIVE	457	320	156	100	50

Table 1: the number of patients receiving multiple cycles of AMEVIVE.

Efficacy of the second treatment cycle in patients previously not achieving PASI50 or PASI 75

During the second period of IV treatment with AMEVIVE, an improvement in PASI was observed in 93% of patients who failed to achieve PASI50 during the first period and 89% of patients who failed to achieve PASI 25 during the first period. Treatment with amevie during the second period resulted in a significantly higher proportion of patients achieving PASI50 and PASI 75 than placebo treatment (table 2). Of the patients who failed to obtain PASI50 during the initial period of treatment with AMEVIVE, 19% obtained PASI 75 during the second period, and 53% obtained PASI 50. 14% of patients who failed to obtain PASI 25 during the initial period of treatment with amevie obtained PASI 75 during the second period, and 47% obtained PASI50 (table 2). The likelihood ratios indicate that patients who failed to achieve PASI50 or PASI 25 in cycle 1 are 2-3 times more likely to achieve PASI50 or PASI 75 in cycle 2 than patients who did not receive another cycle (table 2).

Treatment of
					AMEVIVE-AMEVIVE	AMEVIVE-placebo	Ratio of likeability (95% CI)
Patients who did not acquire PASI50 during 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 (%)	90(92.8)	74(86.0)	2.08(0.78-5.56)
				Patients who did not acquire 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 (%)	59(89.4)	38(82.6)	1.77(0.59-5.29)

Table 2: PASI improvement at any time during the second period of AMEVIVE in patients who failed to obtain PASI50 or PASI 25 during the first period.

The efficacy of multiple treatment courses

During each subsequent treatment cycle, patients receiving multi-cycle IV treatment with amevie showed an incremental improvement in PASI. The proportion of patients who achieved PASI 75 increased from 29% during cycle 1 to a maximum of 54% during cycle 5 (fig. 6A). Likewise, the proportion of patients who acquired PASI50 increased from 56% during cycle 1 to a maximum of 74% during cycle 5 (fig. 6B).

PGA results further supported the incremental clinical improvement observed in multi-cycle treatment of psoriasis with amevie. During cycle 1 of IV treatment with AMEVIVE, 23% of patients had a "clear" or "nearly clear" PGA. During cycle 5, 44% of patients achieved this level of response (fig. 7A). For IM treatment with AMEVIVE, the PGA "clear"/"almost clear" response rate increased from 21% during cycle 1 to a maximum of 41% during cycle 4 (fig. 7B).

In summary, an increasing clinical improvement was seen with successive cycles of treatment with AMEVIVE, indicating its efficacy for long-term treatment of patients with chronic plaque psoriasis. Data from multiple courses of treatment indicate that patients respond with increasing benefit to additional treatment with AMEVIVE regardless of response to initial treatment.

Claims (30)

1. A method of treating a subject having psoriasis, comprising administering to said subject a multi-course treatment of a soluble CD 2-binding LFA-3 polypeptide, wherein said multi-course treatment comprises a plurality of treatment cycles, and wherein each cycle comprises 1 dosing period and 1 rest period.

2. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide is an LFA-3 fusion protein.

3. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide is an LFA-3/immunoglobulin (Ig) fusion protein.

4. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide comprises a soluble LFA-3 polypeptide fused to all or part of an Ig heavy chain hinge region and all or part of a heavy chain constant region.

5. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide comprises a fusion protein consisting of the N-terminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acids of the human IgG1 hinge region, the CH2 region of the human IgG1 heavy chain, and at least a portion of the CH3 region of the human IgG1 heavy chain.

6. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide is AMEVIVE (fig. 1).

7. The method of claim 1, wherein the soluble CD 2-binding LFA-3 polypeptide is encoded by an insert contained in plasmid pSAB152, plasmid pSAB152 being deposited with the american type culture collection with accession number ATCC 68720.

8. The method of any one of claims 1-7, wherein the multiple treatment course comprises at least 4 treatment cycles.

9. The method of any one of claims 1-7, wherein the multiple treatment sessions comprise at least 5 treatment cycles.

10. The method of any one of claims 1-7, wherein the multiple treatment course comprises at least 6 treatment cycles.

11. The method of any one of claims 1-7, wherein the multiple treatment course comprises at least 7 treatment cycles.

12. The method of any one of claims 1-7, wherein the multiple treatment sessions comprise at least 8 treatment cycles.

13. The method of any one of claims 1-7, wherein the rest period of each successive cycle of the multiple course is longer than the rest period of the cycle preceding in the multiple course.

14. The method of any one of claims 1-7, wherein the rest period of the last cycle of the multiple course is at least 2 years.

15. The method of any one of claims 1-7, wherein the rest period of the last cycle of the multiple course is at least 3 years.

16. The method of any one of claims 1-7, wherein the administration period for each cycle of the multiple course is at least 8 weeks.

17. The method of any one of claims 1-7, wherein the administration period for each cycle of the multiple course is at least 10 weeks.

18. The method of any one of claims 1-7, wherein the administration period for each cycle of the multiple course is at least 12 weeks.

19. The method of any one of claims 1-7, wherein the polypeptide is administered intramuscularly.

20. The method of any one of claims 1-7, wherein the polypeptide is administered intravenously.

21. The method of any one of claims 1-7, wherein the polypeptide is administered in a unit dose in the range of 2-30 mg.

22. The method of any one of claims 1-7, wherein the method further comprises administering to the subject an additional therapeutic or prophylactic agent during the multiple course of treatment.

23. A method of treating a subject in need of treatment for psoriasis, comprising administering to the subject a multiple course of treatment of AMEVIVE (fig. 1), wherein the multiple course of treatment comprises at least 3 treatment cycles, each treatment cycle comprising 1 administration per week of AMEVIVE (fig. 1) for a dosing period of 12 weeks, followed by a rest period of at least 12 weeks.

24. The method of claim 23, wherein said multiple course of treatment comprises at least 4 treatment cycles.

25. The method of claim 23, wherein said multiple course of treatment comprises at least 5 treatment cycles.

26. The method of claim 23, wherein the method comprises evaluating the subject for the effect of AMEVIVE (figure 1) during one or both of the dosing period and the rest period of each cycle in the multiple course of therapy.

27. The method of claim 23, wherein the method further comprises administering to the subject an additional therapeutic or prophylactic agent during the multiple course of treatment.

28. A method of treating a patient having psoriasis, the method comprising (a) selecting a subject based on having had at least 2 treatment cycles with a soluble CD 2-binding LFA-3 polypeptide and (b) administering to the subject a3 rd treatment cycle of a soluble CD 2-binding LFA-3 polypeptide.

29. The method of claim 28, wherein the soluble CD 2-binding LFA-3 polypeptide is AMEVIVE (fig. 1).

30. A kit comprising a pharmaceutical composition comprising AMEVIVE and instructions for administering the pharmaceutical composition to a patient who has previously had 2 cycles of treatment with AMEVIVE (fig. 1).