WO2006050159A2

WO2006050159A2 - Methods of predicting responsiveness to treatment

Info

Publication number: WO2006050159A2
Application number: PCT/US2005/039070
Authority: WO
Inventors: Asifa S. Haider; James G. Krueger; Raj Bandaru; Humphrey Gardner
Original assignee: Astellas Us Llc; Rockefeller University, The
Priority date: 2004-10-28
Filing date: 2005-10-28
Publication date: 2006-05-11
Also published as: WO2006050159A3

Abstract

Methods and compositions are described for predicthing the responsiveness to an LFA-3-Ig fusion protein or other inhibitor of CD2/LFA-3 interaction.

Description

METHODS OF PREDICTING RESPONSIVENESS TO

TREATMENT

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/623,364 filed October 28, 2004, the entire contents of which are hereby incorporated by reference herein.

BACKGROUND

The invention relates to the prediction of responsiveness to drugs.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that the cellular, protein, and/or gene expression profile of a subject is indicative of the responsiveness (or lack of responsiveness) of the subject to the administration of an inhibitor of the CD2/LFA-3 interaction, and in particular, an LFA-3-Ig fusion protein, e.g., AMEVTVΕ® (alefacept). Accordingly, in one aspect, the invention features a method of evaluating a subject, e.g., a subject having a disorder described herein, e.g., a skin disorder described herein, e.g., psoriasis, for responsiveness, or lack thereof, to an inhibitor of the CD2/LFA-3 interaction. A preferred inhibitor is an LFA-3-Ig fusion protein, e.g., AMEVIVE. (Unless otherwise stated, an LFA-3-Ig fusion protein, preferably AMEVIVE, is the preferred inhibitor in all embodiments described herein. Similarly, unless stated otherwise, the preferred disorder is psoriasis.) The method includes evaluating the proportion of T cells able to cross into the epidermis. Li one embodiment, the method includes evaluating the level of cells, e.g., T cells, in the epidermis of the subject. The level of cells, e.g., T cells, can be evaluated by detecting the expression of one or more cellular markers, e.g., T cell markers, e.g., CD3, CD4, CD8 or CD 103. Alternatively, the level of cells in the epidermis can also be evaluated by detecting the expression of VLA-I in the epidermis. The method further includes comparing the level of cells, e.g., T cells, in the epidermis with a reference value, e.g., a first reference value, as defined herein. In some embodiments, a level of cells in the epidermis that is above the first reference value indicates nonresponsiveness. In some embodiments, a level of cells in the epidermis that is below the first reference value indicates responsiveness.

In another embodiment, the method includes evaluating the level of cells, e.g., T cells, in the dermis of the subject. The level of cells, e.g., T cells, can be evaluated by detecting the expression of one or more cellular markers, e.g., T cell markers, e.g., CD3, CD4, CD8 or CDl 03. The method further includes comparing the level of cells, e.g., T cells, in the dermis with a reference value, e.g., a second reference value, as defined herein. In some embodiments, a level of cells in the dermis that is above the second reference value indicates responsiveness. In some embodiments, a level of cells in the dermis that is below the second reference value indicates nonresponsiveness.

In one embodiment, the first reference value and the second reference value are the same. In another embodiment, the first reference value and the second reference value are different.

In another embodiment, the method includes evaluating both the level of cells, e.g., T cells, in the epidermis of the subject, and the level of cells, e.g., T cells, in the dermis of the subject. The level of cells, e.g., T cells, in the epidermis or dermis can be evaluated by detecting the expression of one or more cellular markers, e.g., T cell markers, e.g., CD3, CD4, CD8 or CD103. Alternatively, the level of cells in the epidermis can be evaluated by detecting the expression of VLA-I in the epidermis. The method further includes comparing the level of cells, e.g., T cells, in the epidermis with a first reference value and comparing the level of cells, e.g., T cells, in the dermis with a second reference value. In some embodiments, a level of cells in the epidermis that is above the first reference value and/or a level of cells in the dermis that is below the second reference value indicates nonresponsiveness. In some embodiments, a level of cells in the epidermis that is below the first reference value and/or a level of cells in the dermis that is above the second reference value indicates responsiveness.

In another embodiment, the level of cells, e.g., T cells, in the epidermis and the level of cells, e.g., T cells, in the dermis are evaluated and compared to one another. In one embodiment, a level of T cells in the epidermis that is equal to or less than 50%, e.g., equal to or less than 40, 30, 20, 10, 5, or 2.5%, of the level of T cells in the dermis indicates responsiveness to the LFA-3-Ig fusion protein. In another embodiment, a ratio of the level of cells, e.g., T cells, in the epidermis relative to the level of cells, e.g., T cells, in the dermis can be determined. In one embodiment, a ratio of the level of cells, e.g., T cells, in the epidermis relative to the level of cells, e.g., T cells, in the dermis, that is below a first selected value, indicates responsiveness. In one embodiment, a ratio of the level of cells, e.g., T cells, in the epidermis relative to the level of cells, e.g., T cells, in the dermis, that is above a second selected value, indicates nonresponsiveness. In some embodiments, the first selected value and the second selected value can independently be chosen from, e.g., values less than or equal to 1/2, e.g., less than or equal to 1/3, 1/4, 1/5, 1/10, or 1/20. In some embodiments, the first selected value and the second selected value are the same. In one embodiment, the first reference value is selected to be the level of cells, e.g., T cells, in the dermis.

In another embodiment, the method includes determining if an inhibitor of the CD2/LFA-3 interaction will be administered to the subject. The method can further include administering an inhibitor of the CD2/LFA-3 interaction to the subject.

Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably, between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. In one embodiment, the method includes evaluating the level of cells in the epidermis and/or dermis following the administration. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor.

In one embodiment, the method includes repeated administrations of an inhibitor of the CD2/LFA-3 interaction to the subject and repeated evaluations. For example, following an initial administration, the inhibitor can be administered at a regular interval, e.g., every 2, 4, 6, 12, 24 or more hours, and the level of cells, e.g., T cells, in the epidermis and/or dermis evaluated prior to or following each administration. The total number of cycles of evaluation and administration can be, e.g., 1, 2, 4, 6, 8, or more cycles. In another embodiment, the method includes determining that an inhibitor of the CD2/LFA-3 interaction will not be administered to the subject.

In one embodiment, the method also includes obtaining a sample, e.g., a biological sample, from the subject. A biological sample can be, e.g., blood, skin or urine. Preferably, the biological sample is skin, e.g., a skin biopsy. Preferably, the biological sample is a skin biopsy from uninvolved skin.

In one embodiment, the method includes evaluating the levels of cells, e.g., T cells, in the epidermis and/or dermis, comparing the levels of cells to one or more reference values, e.g., a first reference value and a second reference value, and repeating the method steps at least 1 time, e.g., at least 2, 3, 4, 5, 10, or more times. The method can further include determining whether an inhibitor of the CD2/LFA-3 interaction will be administered to the subject. For example, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will not be administered to the subject. Alternatively, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will be administered to the subject and, optionally, administering an inhibitor of the CD2/LFA-3 interaction to the subject.

In one embodiment, the method includes evaluating the levels of cells, e.g., T cells, in the epidermis and/or dermis by measuring RNA, e.g., mRNA, of VLA-I or of T cell markers, e.g., CD3, CD4, CD8 or CD 103. In another embodiment, the levels of cells, e.g., T cells, in the epidermis and/or dermis are evaluated by measuring protein levels of VLA-I, or of T cell markers, e.g., CD3, CD4, CD8 or CD 103.

In one embodiment, the method includes evaluating a subject that has been treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., AME VIVE. In another embodiment, the method includes evaluating a subject that has not been previously treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., AMEVIVE.

The subject can be between the ages of 6 and 18 years, 18 and 35 years, 35 and 65 years, or more than 65 years old. The subject can be male or female and can have various stages or levels of affliction of a condition described herein. Preferably, the subject has psoriasis. In some embodiments, the evaluation can be performed before or after, or before and after, administration of an inhibitor of the CD2/LFA-3 interaction.

In one embodiment, the method of determining responsiveness includes evaluating the likelihood of achieving a predetermined Psoriasis Area and Severity Index (PASI) response. For example, the PASI response can have a score of, e.g., at least 50, e.g., at least 60, 70, 75, 80, 90, or greater.

In some embodiments, the method further includes evaluating the weight of the subject and determining whether the subject has an increased or decreased propensity for responsiveness. For example, a subject's weight above a threshold value can indicate a propensity for nonresponsiveness, whereas a subject's weight below a threshold value can indicate a propensity for responsiveness to treatment. A threshold value can be the average weight of a group of subjects in which more than 40%, e.g., more than 50, 60, 70 or 80%, of the subjects are nonresponders.

In another aspect, the invention features a method of monitoring a subject being treated with an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVrVΕ. In one embodiment, the method includes evaluating the level of regulatory T cells in the epidermis and/or dermis of the subject. The level of regulatory T cells can be evaluated by detecting one or more regulatory T cell marker proteins, e.g., Foxp3 protein.

In one embodiment, the method can further include comparing the level of regulatory T cells, e.g., in the epidermis and/or dermis of the subject, with a regulatory T cell reference value to evaluate responsiveness to the inhibitor. For example, a higher level of regulatory T cells compared to the regulatory T cell reference value indicates responsiveness to the inhibitor, e.g., a level of regulatory T cells that is, e.g., 10%, 20%, 30%, 40%, 50%, or more, than the level of regulatory T cells before treatment with the inhibitor indicates responsiveness.

In one embodiment, the level of regulatory T cells is evaluated before treatment with the LFA-3-Ig fusion protein. In another embodiment, the level of regulatory T cells is evaluated after treatment with the LFA-3-Ig fusion protein. Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably, between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor.

In one embodiment, the method also includes obtaining a sample, e.g., a biological sample, from the subject. A biological sample can be, e.g., blood, skin or urine. Preferably, the biological sample is skin, e.g., a skin biopsy. Preferably, the biological sample is a skin biopsy from involved skin.

In one embodiment, the method includes evaluating a subject that has been treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., AMEVrVE. In another embodiment, the method includes evaluating a subject that has not been previously treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., AMEVIVE.

The subject can be between the ages of 6 and 18 years, 18 and 35 years, 35 and 65 years, or more than 65 years old. The subject can be male or female and can have various stages or levels of affliction of a condition described herein. Preferably, the subject has psoriasis. In a preferred embodiment, the method further includes providing an additional administration of the LFA-3-Ig fusion protein, or withholding an additional administration of the LFA-3-Ig fusion protein. The decision to administer or withhold an additional administration can be based on the level of regulatory T cells.

In another aspect, the invention features a method of evaluating a subject, e.g., a subject having a disorder described herein, e.g., a skin disorder described herein, e.g., psoriasis, for responsiveness, or lack thereof, to an inhibitor of the CD2/LFA-3 interaction. The method includes evaluating the level of expression, e.g., mRNA or protein expression, of a gene that is differentially expressed in responders and nonresponders, e.g., a gene listed in Table 1, and comparing the level of expression with a reference value, e.g., a reference value described herein. A level of expression that is different, e.g., higher or lower, than the reference value can be indicative of responsiveness. In another embodiment, a level of expression that is different, e.g., higher or lower, than the reference value can be indicative of nonresponsiveness. The selection of particular genes for use in an embodiment will be evident from the Tables and other guidance provided herein.

In one embodiment, the level of expression of a gene, e.g., a gene listed in Table 1, is evaluated and if the level of expression is above the reference value, the level of expression indicates responsiveness, and the method evaluates the subject for responsiveness. The gene can be selected from a gene listed in Table 1 that has a fold difference of geometric means less than 1.

In one embodiment, the level of expression of a gene, e.g., a gene listed in Table 1, is evaluated and if the level of expression is below the reference value, the level of expression indicates responsiveness, and the method evaluates the subject for responsiveness. The gene can be selected from a gene listed in Table 1 that has a fold difference of geometric means less than 1.

In one embodiment, the level of expression of a gene, e.g., a gene listed in Table 1, is evaluated and if the level of expression is below the reference value, the level of expression indicates nonresponsiveness, and the method evaluates the subject for nonresponsiveness. The gene can be selected from a gene listed in Table 1 that has a fold difference of geometric means greater than 1.

In one embodiment, the level of expression of a gene, e.g., a gene listed in Table 1, is evaluated and if the level of expression is above the reference value, the level of expression indicates nonresponsiveness, and the method evaluates the subject for nonresponsiveness. The gene can be selected from a gene listed in Table 1 that has a fold difference of geometric means greater than 1.

In another embodiment, the method includes determining if an inhibitor of the CD2/LFA-3 interaction will be administered to the subject. The method can further include administering an inhibitor of the CD2/LFA-3 interaction to the subject. Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably, between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. The method can include evaluating the level of gene expression after administration. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor. In another embodiment, the method includes determining that an inhibitor of the

CD2/LFA-3 interaction will not be administered to the subject.

In one embodiment, the method includes obtaining a sample, e.g., a biological sample, from the subject, and evaluating the level of expression of a gene listed in. Table 1, e.g., the level of mRNA or protein, in the sample. A biological sample can be, e.g., blood, skin or urine. Preferably, the biological sample is blood.

In one embodiment, the method includes evaluating the level of expression, e.g., mRNA or protein expression, of a gene listed in Table 1 , comparing the level of expression with a reference value, e.g., a reference value described herein, and repeating the method steps at least 1 time, e.g., at least 2, 3, 4, 5, 10, or more times. The method can further include determining whether an inhibitor of the CD2/LF A-3 interaction will be administered to the subject. For example, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will not be administered to the subject. Alternatively, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will be administered to the subject and, optionally, administering an inhibitor of the CD2/LFA-3 interaction to the subject.

In one embodiment, the method includes evaluating the level of gene expression by measuring RNA₅ e.g., mRNA, levels. In another embodiment, the level of gene expression is evaluated by measuring protein levels.

In another embodiment, the method includes evaluating the levels of expression of a plurality of genes that have differential expression in responders and nonresponders, e.g., a plurality of genes listed in Table 1. For example, the plurality can include at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more, genes. The gene or genes can be evaluated using a probe or probes that specifically bind to the gene or genes being evaluated, or can bind to the product of the gene or genes being evaluated. The plurality of genes can be evaluated simultaneously or can be evaluated sequentially. The probe or probes can be nucleic acids and can be disposed on a substrate, e.g., glass, metal or nylon. Multiple probes can be disposed on the same substrate, e.g., disposed in a distinct address on the substrate. Alternatively, a substrate can contain only one probe.

In one embodiment, the method includes evaluating the levels of expression of a plurality of genes that have differential expression in responders and nonresponders, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, that have elevated levels of expression in responders. For example, the plurality of genes can be selected from the genes listed in Table 1 having a fold difference of geometric means less than one.

In another embodiment, the method includes evaluating a plurality of genes that have differential expression in responders and nonresponders, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, that have reduced levels of expression in responders. For example, the plurality of genes can be selected from the genes listed in Table 1 having a fold difference of geometric means greater than one.

In one embodiment, the method involves evaluating the levels of expression of at least two genes in which a first gene is selected from the genes listed in Table 1 with a fold difference of geometric means less than one, and a second gene is selected from tbxe genes listed in Table 1 with a fold difference of geometric means greater than one.

In yet another embodiment, the method includes evaluating a subject that has been treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVIVE. In another embodiment, the method includes evaluating a subject that has not been previously treated with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., an LF A3 -Ig fusion protein, e.g., AMEVIVE.

The subject can be between the ages of 6 and 18 years, 18 and 35 years, 35 and 65 years, or more than 65 years old. The subject can be male or female and can have various stages or levels of affliction of a condition described herein.

In preferred embodiments, the evaluation can be performed before or after, or before and after, administration of an inhibitor of the CD2/LFA-3 interaction.

In another aspect, the invention provides, a method of evaluating a subject, e.g., a subject having a disorder described herein, for responsiveness, or lack thereof, to an inhibitor of the CD2/LFA-3 interaction, following an initial administration of an inhibitor of the CD2/LFA-3 interaction. Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably, between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. The evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor. Preferably, the evaluation is performed 6 hours after administration of the inhibitor. The method includes evaluating the level of expression, e.g., mRNA or protein expression, of a gene listed in Table 2 or Table 3, and comparing the level of expression with a reference value. A level of expression that is different, e.g., higher or lower, than the reference value can be indicative of responsiveness. In another embodiment, a level of expression that is different, e.g., higher or lower, than the reference value can be indicative of nonresponsiveness.

In one embodiment, the level of expression of a gene listed in Table 2 is evaluated and if the level of expression is above the reference value, the level of expression indicates nonresponsiveness, and the method evaluates the subject for nonresponsiveness. In another embodiment, the level of expression of a gene listed in Table 2 is evaluated and if the level of expression is below the reference value, the level of expression indicates nonresponsiveness, and the method evaluates the subject for nonresponsiveness .

In one embodiment, the level of expression of a gene listed in Table 3 is evaluated and if the level of expression is below the reference value, the level of expression indicates responsiveness, and the method evaluates the subject for responsiveness. In another embodiment, the level of expression of a gene listed in Table 3 is evaluated and if the level of expression is above the reference value, the level of expression indicates responsiveness, and the method evaluates the subject for responsiveness. In a preferred embodiment, the subject was evaluated for expression of a gene listed in Table 1 prior to administration of an inhibitor of the CD2/LFA-3 interaction.

In another embodiment, the method includes determining that an inhibitor of the CD2/LFA-3 interaction will be subsequently administered to the subject. The method can further include subsequent administrations of an inhibitor of the CD2/LFA-3 interaction to the subject. Preferably, the inhibitor is subsequently administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. The method can include evaluating the level of gene expression after administration. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor.

In another embodiment, the method includes determining that an inhibitor of the CD2/LFA-3 interaction will not be subsequently administered to the subject. In one embodiment, the method includes obtaining a sample, e.g., a biological sample, from the subject, and evaluating the level of expression of a gene listed in Table 2 or Table 3, e.g., the level of mRNA or protein, in the sample. A biological sample can be, e.g., blood, skin or urine. Preferably, the biological sample is blood. In one embodiment, the method includes evaluating the level of expression, e.g., mRNA or protein expression, of a gene listed in Table 2 or Table 3, comparing the level of expression with a reference value, e.g., a reference value described herein, and repeating the method steps at least 1 time, e.g., at least 2, 3, 4, 5, 10, or more times. The method can be repeated at regular intervals, e.g., every 1, 2, 4, 8, 12, 24, 48 or more, hours. Alternatively, the method can be repeated at irregular intervals. The method can further include determining whether an inhibitor of the CD2/LFA-3 interaction will be subsequently administered to the subject. For example, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will not be subsequently administered to the subject. Alternatively, the method can further include determining that an inhibitor of the CD2/LFA-3 interaction will be subsequently administered to the subject and, optionally, subsequently administering an inhibitor of the CD2/LFA-3 interaction to the subject. The same genes or different genes can be evaluated in each repeated evaluation.

In one embodiment, the method includes repeated administrations of an inhibitor of the CD2/LFA-3 interaction to the subject and repeated evaluations. For example, following the initial administration, the inhibitor can be administered at a regular interval, e.g., every 2, 4, 6, 12, 24 or more hours, and gene expression evaluated prior to or following each administration. The total number of cycles of evaluation and administration can be, e.g., 1, 2, 4, 6, 8, or more cycles, e.g., at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight. The evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, before or after the administration of the inhibitor. Preferably, the evaluation is performed 6 hours after administration of the inhibitor. The method includes evaluating the level of expression, e.g., mRNA or protein expression, of a gene listed in Table 2 or Table 3, and comparing the level of expression with a reference value. The same genes or different genes can be evaluated in each cycle.

In one embodiment, the method includes evaluating the level of gene expression by measuring RNA, e.g., mRNA, levels. In another embodiment, the level of gene expression is evaluated by measuring protein levels. In another embodiment, the method includes evaluating the levels of expression of a plurality of genes listed in Table 2 and Table 3. For example, the plurality can include at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more, genes. The gene or genes can be evaluated using a probe or probes that specifically bind to the gene or genes being evaluated, or can bind to the product of the gene or genes being evaluated. The plurality of genes can be evaluated simultaneously or can be evaluated sequentially. The probe or probes can be nucleic acids and can be disposed on a substrate, e.g., glass, metal or nylon. Multiple probes can be disposed on the same substrate, e.g., disposed in a distinct address on the substrate. Alternatively, a substrate can contain only one probe.

In one embodiment, the method includes evaluating the levels of expression of a plurality of genes, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, listed in Table 3 that have elevated levels of expression in responders.

In another embodiment, the method includes evaluating a plurality of genes, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, listed in Table 3 that have reduced levels of expression in responders.

In one embodiment, the method includes evaluating the levels of expression of a plurality of genes, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, listed in Table 2 that have elevated levels of expression in nonresponders.

In another embodiment, the method includes evaluating a plurality of genes, e.g., at least 2, e.g., at least 3, 5, 10, 20, 50, 100 or more genes, listed in Table 2 that have reduced levels of expression in nonresponders. In one embodiment, the method involves evaluating the levels of expression of at least two genes in which a first gene is selected from the genes listed in Table 3 that has elevated levels of expression in responders, and a second gene is selected from the genes listed in Table 3 that has reduced levels of expression in responders.

In another embodiment, the method involves evaluating the levels of expression of at least two genes in which a first gene is selected from the genes listed in Table 2 that has elevated levels of expression in nonresponders, and a second gene is selected from the genes listed in Table 2 that has reduced levels of expression in nonresponders.

In another embodiment, the method involves evaluating the levels of expression of at least two genes in which a first gene is selected from the genes listed in Table 3 that has elevated levels of expression in responders, and a second gene is selected from the genes listed in Table 2 that has reduced levels of expression in nonresponders.

In another embodiment, the method involves evaluating the levels of expression of at least two genes in which a first gene is selected from the genes listed in Table 3 that has reduced levels of expression in responders, and a second gene is selected from the genes listed in Table 2 that has increased levels of expression in nonresponders. In yet another embodiment, the method includes evaluating a subject that, prior to the initial administration, has been treated previously with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVrVΕ. In another embodiment, the method includes evaluating a subject that, prior to the initial administration, has not been previously treated with a therapeutic agent, e.g., an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVIVE.

In another aspect, the invention features methods described herein in combination with methods of determining other measurements of responsiveness. For example, the methods described herein can be used in combination with measurements of epidermal thickness or clinical assessments, e.g., determinations of Psoriatic and Severity Index (PASI) scores.

In one embodiment, the method includes evaluating the level of cells, e.g., T cells, in the epidermis of the subject, and assessing the epidermal thickness and/or PASI score of the subject prior to treatment. The level of cells, e.g., T cells, can be evaluated by detecting the expression of one or more cellular markers, e.g., T cell markers, e.g., CD3, CD4, CD8 or CDl 03. Alternatively, the level of cells in the epidermis can also be evaluated by detecting the expression of VLA-I in the epidermis. The method can further include comparing the level of cells, e.g., T cells, in the epidermis with a reference value, e.g., a first reference value, as defined herein. In some embodiments, the method further includes assessing epidermal thickness and/or the PASI score of the subject. In another embodiment, the method includes evaluating the level of cells, e.g., T cells, in the dermis of the subject, and assessing the epidermal thickness and/or PASI score of the subject prior to treatment. The level of cells, e.g., T cells, can be evaluated by detecting the expression of one or more cellular markers, e.g., T cell markers, e.g., CD3 , CD4, CD8 or CD 103. The method further includes comparing the level of cells, e.g., T cells, in the dermis with a reference value, e.g., a second reference value, as defined herein. In some embodiments, the method further includes assessing epidermal thickness and/or the PASI score of the subject.

In another aspect, the invention features, a method of making a decision, e.g., a medical or financial decision. The method includes: generating or receiving data on the likelihood of a subject responding or not responding to an inhibitor of the CD2/LFA-3 interaction, e.g., AMEVIVE, for the treatment of psoriasis, e.g., receiving the protein, cell, or gene expression level data generated by a method described herein; and using the data to make the decision, e.g., selecting between a first course of action and a second course of action.

In a preferred embodiment, the decision includes comparing the data to a standard and making the decision based on the relationship of the data to the standard. For example, the data can be a value or other term for the likelihood of response and if the value or other term has a preselected relationship to the standard, e.g., if the value or term in the data is greater than a reference standard, selecting a first course of action, and if the data is less than a reference standard selecting a second course of action. A course of action can be, e.g., providing or not providing service or treatment, or paying for or not paying for all or part of a service or treatment.

In a preferred embodiment, the first course of action is suggesting or providing a first course of medical treatment, e.g., any treatment described herein, and the second course of action is suggesting or deciding that the treatment not be given or not providing the treatment. In a preferred embodiment the first course of action includes or results in the authorization or transfer of funds to pay for a service or treatment provided to a subject and the second course of action includes or results in the refusal to pay for a service or treatment provided to a subject. For example, an entity, e.g., a hospital, caregiver, government entity, or an insurance company or other entity, that pays for or reimburses medical expenses can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services or treatment provided to the patient. For example, a first entity, e.g., an insurance company, can use the outcome of a method described herein to determine whether to provide financial payment to, or on behalf of, a patient, e.g., whether to reimburse a third party, e.g., a vendor of goods or services, a hospital, physician, or other care-giver, for a service or treatment provided to a patient.

In another aspect, the invention features a method of providing a database, e.g., a database useful for establishing a reference value referred to herein or for otherwise evaluating responsiveness, or lack thereof, levels in one or more subjects. The method includes: generating or receiving data, e.g., the protein, cell, or gene expression level data generated by a method described herein, on the likelihood of responsiveness, or lack thereof, in a patient, which data is generated by a method described herein; and entering the data into the database.

In a preferred embodiment, one or more of an indicator (e.g., a value) for the disease state or responsiveness, or lack thereof, of a patient and a patient identifier are entered into the database.

In a preferred embodiment, the database includes a plurality of entries, each one of which includes one or more of: data (e.g., gene expression levels) on responsiveness, or lack thereof, in a patient; an indicator for the disease state or responsiveness, or lack thereof, of a patient; and a patient identifier.

In another aspect, the invention features a method of evaluating a patient that includes: comparing data from the patient on the likelihood of responsiveness, or lack thereof, in a patient, wherein the data was generated by a method described herein, with data from a database described herein. In another aspect, the invention features a method of constructing a reference standard that includes: including data from a database described herein in the standard. For example, one can take values from a database, perform a mathematical operation on them and use the result as a reference in arriving at a reference standard, e.g., take an average of a plurality of values selected from the database and use the average as the standard.

In another aspect, the invention features a method for the treatment of diabetes, e.g., insulin dependent (type-I) diabetes, in a subject. The subject can be an individual who has been determined to be at risk for diabetes, e.g., because of genetics or family history or other clinical evaluation. The subject can also be an individual who is prediabetic, who has suffered partial beta cell destruction, or who has diabetes, preferably a diabetic having some remaining beta cells. In particular, the invention provides a method for the prevention of insulin dependent diabetes. The method includes administering to a subject, an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVIVE. The method can delay the onset of diabetes, slow progression, or ameliorate one or more symptom of diabetes.

Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight.

In one embodiment, the subject is evaluated after treatment with the LFA-3-Ig fusion protein. In some embodiments, the evaluation is performed at least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after the administration of the inhibitor.

In one embodiment, the method includes treating a subject that has been treated previously with a therapeutic agent. In another embodiment, the method includes treating a subject that has not been treated previously with a therapeutic agent.

In one embodiment, the method includes administering an LFA-3-Ig fusion protein in combination with another therapeutic agent, e.g., a therapeutic agent for the treatment of diabetes, e.g., insulin. The subject can be between the ages of 6 and 18 years, 18 and 35 years, 35 and 65 years, or more than 65 years old. The subject can be male or female and can have various stages or levels of affliction.

Definitions

As used herein, "CD2" means a CD2 polypeptide that binds to a naturally occurring LFA-3 polypeptide and which is encoded by (a) a naturally occurring mammalian CD2 DNA sequence (e.g., SEQ ID NO:5) (this is the preferred embodiment); (b) a DNA sequence degenerate to a naturally occurring CD2 DNA sequence; or (c) a DNA sequence that hybridizes to one of the foregoing DNA sequences under conditions equivalent to about 20°C to 27°C below T_m and 1 M sodium chloride.

As used herein, "LFA-3" means an LFA-3 polypeptide that binds to a naturally occurring CD2 polypeptide and which is encoded by (a) a naturally occurring mammalian LFA-3 DNA sequence (e.g., SEQ ID NO: 1 or SEQ ID NO:3) (this is the preferred embodiment); (b) a DNA sequence degenerate to a naturally occurring LFA-3 DNA sequence; or (c) a DNA sequence that hybridizes to one of the foregoing DNA sequences under conditions to about 20°C to 27°C below T_m and 1 M sodium chloride.

As used herein, a "soluble LFA-3 polypeptide" or a "soluble CD2 polypeptide" is an LFA-3 or CD2 polypeptide incapable of anchoring itself in a membrane. Such soluble polypeptides include, for example, CD2 and LFA-3 polypeptides that lack a sufficient portion of their membrane-spanning domain to anchor the polypeptide or are modified such that the membrane-spanning domain is non-functional. As used herein soluble LFA-3 polypeptides include full-length or truncated (e.g., with internal deletions) PI- linked LFA-3. As used herein, an "antibody homolog" is a protein comprising one or more polypeptides selected from immunoglobulin light chains, immunoglobulin heavy chains and antigen binding fragments thereof which are capable of binding to one or more antigens. The component polypeptides of an antibody homolog composed of more than one polypeptide may optionally be disulfide-bound or otherwise covalently crosslinked. Accordingly, antibody homologs include intact immunoglobulins of types IgA, IgG, IgE,

IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. Antibody homologs also include portions of intact immunoglobulins that retain antigen-binding specificity, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like.

As used herein, a "humanized recombinant antibody homolog" is an antibody homolog, produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding have been substituted for the corresponding amino acids from a nonhuman mammalian immunoglobulin light or heavy chain.

As used herein, a "chimeric recombinant antibody homolog" is an antibody homolog, produced by recombinant DNA technology, 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 from another immunoglobulin light chain or heavy chain.

"Reference value", as used herein, refers to a value that, when applied to subjects, will partition subjects into responder and nonresponder classes. In a preferred embodiment, the reference value is selected such that at least 60, more preferably 70, 80, 90, or 95%, of the subjects identified as responders are responders. A reference value can be determined by evaluating a number of responders and nonresponders and selecting the value so as to provide the desired accuracy. A first reference value can thus be chosen such that a desired level of accuracy in terms of identifying nonresponders is achieved.

In another embodiment, the first reference value is less than or equal to 50%, e.g., less than or equal to 40, 20, 10, 5 or 1%, of the number of T cells in the dermis. The second reference value can be chosen such that a desired level of accuracy in terms of identifying responders is achieved.

In another embodiment, the second reference value is less than or equal to 50%, e.g., less than or equal to 40, 20, 10, 5 or 1%, of the number of T cells in the dermis.

In another embodiment, the first or second reference value can be equal to or less than 30%, e.g., equal to or less than 20, 16.6, 10, 5, or 2.5%, of the total number of T cells in the epidermis and dermis. When applied to methods of analyzing gene expression, a "gene reference value" can be the level of expression of the gene or genes in a group of subjects in which more than 40%, e.g., more than 50, 60, 70 or 80%, of the subjects are responders. "Fold difference of geometric means" can be determined by the ratio of the level of gene expression in a group of subjects in which more than 40%, e.g., more than 50, 60, 70 or 80%, of the subjects are nonresponders, relative to the level of gene expression in a group of subjects in which more than 40%, e.g., more than 50, 60, 70 or 80%, of the subjects are responders.

"Regulatory T cell reference value", as used herein, is a value selected such that, when applied to subjects, will partition subjects into responder and nonresponder classes. In a preferred embodiment, the reference value is selected such that at least 60, more preferably 70, 80, 90, or 95%, of the subjects identified as responders are responders. A. reference value can be determined by evaluating a number of responders and nonresponders and selecting the value so as to provide the desired accuracy. A first reference value can thus be chosen such that a desired level of accuracy in terms of identifying nonresponders is achieved. An exemplary regulatory T cell reference value can be the level of regulatory T cells prior to treatment or, e.g., less than 70%, e.g., less than 50, 30, 20, 10 or 5%, of the level of regulatory T cells prior to treatment.

"Responder", as used herein, is a subject that demonstrates a positive response following treatment, e.g., treatment with an inhibitor of the CD2/LFA-3 interaction, e.g., an LFA-3-Ig fusion protein, e.g., AMEVIVE. Response can be measured by, e.g., histological parameters, e.g., measurements of epidermal thickness, or by clinical parameters, e.g., PASI response. A positive response refers to an improvement as measured by a parameter described herein. "Nonresponder", as used herein, refers to a subject that demonstrates no response, or a negative response, following treatment.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a graph of the CD3 ratios in uninvolved skin from psoriatic patients prior to treatment. Fig. 2A is a graph of CD3 ratio versus PASI 75 responders. Fig. 2B is a graph of CD3 ratio versus PASI 50 responders. Fig. 2C is a graph of CD3 ratio versus histology responders. Nonresponders are shown on the left in each Figure.

Fig. 3 is a graph of the distribution of PASI 75, PASI 50, and histologically defined responders versus initial PASI. Nonresponders are shown on the left. Fig. 4 is a graph of CD3 ratio versus initial PASI. Fig. 5 is a chart of CD 103 and VLA-I expression in responders and nonresponders.

Fig. 6 is a graph of the classification of the PASI 50 based responders using uninvolved data and physical data, as determined by Matlab implementation of nonlinear tree based classification.

Fig. 7 is a graph of the classification of the PASI 50 based responders using involved pretreatment data and physical data, as determined by Matlab implementation of nonlinear tree based classification. Fig. 8 is a graph of the classification of the PASI 75 based responders using involved pretreatment data and uninvolved data, as determined by Matlab implementation of nonlinear tree based classification. Fig. 9 is a chart of PASI 50 response prediction.

Fig. 10 is a graph of uninvolved CD 103 ratio from different classes. Stars represent responders and circles represent nonresponders, as defined by the indicated parameters.

Fig. 11 is a graph of VLA-I cells in the epidermis of nonlesional skin from different classes. Stars indicate responders and circles represent nonresponders, as defined by the indicated parameters. Fig. 12 is a graph of CDl 03 ratio versus VLA-I cells in the epidermis of nonlesional skin.

Fig. 13 is a graph of the CD3 epidermis/dermis ratio from uninvolved skin. Stars indicate responders and circles indicate nonresponders, as defined by the indicated parameters. Fig. 14 is a graph of PASI 50 responders and nonresponders, and of PASI 75 responders and nonresponders. Fig. 15A is a graph of Foxp3 expression in nonresponders. Fig. 15B is a graph of Foxp3 expression in responders. Fig. 15C is a graph of Foxp3 expression following cyclosporin A treatment. Fig. 15D is a graph of Kl 6 expression following cyclosporin A treatment.

DETAILED DESCRIPTION

The invention is based, in part, on the discovery that the cellular, protein and/or gene expression profile of a subject can be used to predict the responsiveness, or lack of responsiveness, to the administration of an inhibitor of the CD2/LFA-3 interaction, and in particular, an LFA-3-Ig fusion protein, e.g., AMEVWE® (alefacept).

Protein and Gene Expression Profiling

The expression, level, or activity of a protein, e.g., VLA-I , or a T cell marker, e.g., CD3, CD4, CD8 or CD103, in a biological sample, e.g., a skin biopsy of the dermis or epidermis, can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes the protein, e.g., VLA-I, or a T cell marker, e.g., CD3, CD4, CD8 or CD103, such that the presence of the protein or nucleic acid is detected in the biological sample. The term biological sample includes tissues (e.g., blood and skin), cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject, e.g., serum and urine. The expression and level of a protein, e.g., VLA-I, or a T cell marker, e.g., CD3, CD4, CD8 or CD 103, can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by a gene, e.g., a CD3, CD4, CD8, CDl 03 or VLA-I gene; measuring the amount of protein encoded by the gene; or measuring the activity of the protein encoded by the gene.

The level of mRNA, e.g., CD3, CD4, CD8, CD103 or VLA-I mRNA, in a cell can be determined both by in situ and by in vitro formats.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA of, e.g., CD3, CD4, CD8, CD103 or VLA-I . The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the gene.

The level of mRNA in a sample that is encoded by a gene can be evaluated with nucleic acid amplification, e.g., by rtPCR (U.S. Patent No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), self sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)), transcriptional amplification system (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173- 1177 (1989)), Q-Beta Replicase (Lizardi et al.. Bio/Technology 6: 1197 (1988)), rolling circle replication (U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as a pair of nucleic acid molecules that can anneal to 5 ' or 3 ' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the gene being analyzed.

In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting mRNA, or genomic DNA, and comparing the presence of the mRNA or genomic DNA in the control sample with the presence of mRNA, e.g., CD3, CD4, CD8, CD103 or VLA-I mRNA, or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Patent No. 5,695,937, is used to detect transcript levels of, e.g., CD3, CD4, CD8, CDl 03 or VLA-I.

A variety of methods can be used to determine the level of a protein, e.g., VLA-I , or a T cell marker protein, e.g., CD3, CD4, CD8 or CD103. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled," with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

The detection methods can be used to detect a protein, e.g., VLA-I, or a T cell marker protein, e.g., CD3, CD4, CD8 or CD103, in a biological sample in vitro as well as in vivo. In vitro techniques for detection include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection include introducing into a subject a labeled antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an antibody positioned on an antibody array. The sample can be detected, e.g., with avidin coupled to a fluorescent label.

In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting a protein, e.g., CD3, CD4, CD8, CD103 or VLA-4, and comparing the presence of the protein, e.g., CD3, CD4, CD8, CD103 or VLA-4, in the control sample with the presence of the protein in the test sample.

The invention also includes kits for detecting the presence of a protein, e.g., CD3, CD4, CD8, CD 103 or VLA-4, in a biological sample. For example, the kit can include a compound or agent capable of detecting protein (e.g., an antibody) or mRNA (e.g., a nucleic acid probe) and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to evaluate a subject, e.g., for responsiveness to treatment with an inhibitor of the CD2/LFA-3 interaction, e.g., AMEVIVE.

Database Generation

A collection of biological samples from a subject or subjects can provide information that contributes to a database that correlates the gene expression of a subject to predictability of responsiveness, or lack thereof. Nucleic acid (DNA and/or RNA) can be harvested from a biological sample, e.g., skin, blood or urine, collected by a biopsy, such as a needle biopsy, by a tissue, scraping, such as from skin, by needle or other device for collecting blood, or by collecting a subject's urine. A biological sample is collected prior to initiation of therapy and can also be collected at a particular point following therapy. DNA and/or RNA can be isolated from the biological sample and labeled for analysis by array technology, such as array analysis or gene chip analysis. Optionally, the biological sample can be frozen and stored for a period of time (such as for a day, a week, a month, or a year, or any fraction thereof) before isolation of nucleic acid. DNA or RNA isolation from a biological sample can be accomplished by methods known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Micro array analysis can be performed by applying labeled nucleic acid to an array for comparative analysis (Schena et al., Science 270:467-70, 1995). The array can be any array described herein, or any other array that is functional in the described analysis. For example, an RNA sample isolated from a biological sample before the start of therapy can be labeled and mixed with RNA isolated from a biological sample after initiation of therapy (e.g., with an inhibitor of the CD2/LFA-3 interaction). The nucleic acid can be labeled with a fluorescent dye, such as Cy3 or Cy5. Preferably the nucleic acid samples from each biological sample are labeled with a different and distinguishable dye. For example, RNA isolated from a biological sample before administration of an inhibitor described herein can be labeled with Cy3, such as in the form of Cy3-dUTP (e.g., via a PCR reaction following reverse transcription), and RNA isolated from a biological sample following administration of an inhibitor described herein can be labeled with Cy5, such as in the form of Cy5-dUTP.

Labeled nucleic acids are hybridized to an array following labeling, and unbound nucleic acids are washed away. The bound, labeled nucleic acids are detected using an appropriate method. For example, to detect fluorescence intensity at each spot on an array, a laser confocal scanner or CCD-based scanner can be used. To detect spots hybridized with radioactively labeled nucleic acids, a phosphorimager can be used. Information from protein arrays (see below) can also be added to a database described herein as a supplement to the gene expression data. Protein expression and genomic information from protein arrays can be incorporated into algorithms that will predict whether a subject will respond to treatment with an inhibitor described herein.

The data generated by the methods featured in the invention can be stored in a database, such as a computer-accessible medium. The database can be a storehouse for the information pertaining to each subject and gene expression. The database can store personal information, including demographic data (e.g., weight, gender, or age). The database can generate information regarding the responsiveness to treatment with an inhibitor described herein. Nucleic Acid Arrays

A nucleic acid array is a substrate, such as a glass, wafer (e.g., a silica wafer) or membrane, to which is tethered a designated set of nucleic acid molecules, called capture probes, each representing a specified gene or nucleic acid sequence. Placement of the nucleic acid probes onto the substrate can be accomplished by methods known in the art. For example, a drop (e.g., spray) method, or other mechanical method, such as the directed-flow method described in U.S. Patent No. 5,384,261, or the pin-based method described in U.S. Patent No. 5,288,514.

A nucleic acid array can contain a set of probes that represents the entire genome of an organism, such as a mouse or human, or an array can contain a subset of gene- specific probes. For example, the subset can include a group of genes whose expression has been determined to be differentially expressed in responders or nonresponders, such as in pilot experiments, or as reported in the literature. The subset of probes can also represent genes determined to be amplified or deleted. An array can include probes that will serve as controls, including positive control probes and negative control probes. A positive control probe can include a housekeeping gene, such as an RNA polymerase gene, the beta actin gene, the glyceraldehyde-3- phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein Ll 3a, the TATA binding protein gene, and/or the ubiquitin C gene. The nucleic acid sequences of these genes are known in the art. A synthetic positive control will hybridize to a control nucleic acid that is added to the test sample from the tumor before hybridization to the array. The synthetic positive control probe should have a sequence that is not substantially identical to any of the genes of the biological sample being assayed, such that the labeled nucleic acid from the test sample will not hybridize to the control probe. A negative control probe should have a sequence that is not substantially identical to any of the genes of the biological sample being assayed or to the positive control sequence. Other optional control probes include a polyA, polyT, polyG, and polyC probe, useful for measuring the effects of non-specific hybridization. A gene array can contain tens, hundreds, or thousands of individual probes immobilized at discrete, predetermined locations (addresses or "spots") on a solid, planar support, such as a glass, metal, or nylon support. An array can be a macroarray or microarray, the difference being in the size of the spots. Macroarrays contain spots of about 300 microns in diameter or larger and can be imaged using gel or blot scanners. Microarrays contain spots less than about 300 microns, typically less than about 200 microns, in diameter. The array can have a density of at least about 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more probes/cm², and ranges between. The capture probes can be single stranded, or the probes can have a structure comprising a double stranded portion and a single stranded portion.

To generate data from an array, a population of labeled cDNA representing total mRNA from a biological sample is contacted with the DNA array under suitable hybridization conditions. Hybridization of cDNAs with sequences in the array is detected, such as by fluorescence at particular addresses on the solid support. Thus, a pattern of fluorescence representing a gene expression pattern in the biological sample of a particular subject or group of subjects is obtained, for example, before administration of an inhibitor described herein, and/or after administration of inhibitor. These patterns of gene expression can be digitized and stored electronically, such as in a digital database, for computerized analysis and comparison.

By some methods, cDNAs can be used as capture probes to form the array. Suitable cDNAs can be obtained by conventional polymerase chain reaction (PCR) techniques, such as reverse transcription coupled to PCR (RT-PCR). The length of the cDNAs can be from about 20 to 2,000 nucleotides, e.g., from about 100 to 1,000 nucleotides. Other methods known in the art for producing cDNAs can be used. The cDNA probes can be attached to a suitable solid substrate, such as a coated glass microscope slide, at specific, predetermined locations in a two-dimensional grid. For example, the substrate can be coated with polylysine, which will facilitate attachment of the cDNA. A small volume (e.g., about 5 nanoliters) of a concentrated DNA solution can be placed in each spot. Spotting can be carried out using a commercial microspotting device (sometimes called an arraying machine or gridding robot) according to the vendor's instructions. Commercial vendors of solid supports and equipment for producing DNA arrays include BioRobotics Ltd., Cambridge, UK; Corning Science Products Division, Acton, MA; GENPAK Inc., Stony Brook, NY; SciMatrix, Inc., Durham, NC; and TeleChem International, Sunnyvale, CA. The cDNAs can be attached to the solid support by any suitable method. In general, the linkage is covalent. Suitable methods of covalently linking DNA molecules to the solid support include amino cross-linking and UV crosslinking. For guidance concerning construction of cDNA arrays according to the invention, see, e.g., DeRisi et al. (Nature Genetics 14:457-460, 1996), Khan et al. (Electrophoresis 20:223-229, 1999), Lockhart et al. (Nature Biotechnol. 14:1675-1680, 1996).

In an alternative method, immobilized DNA probes of an array are synthetic oligonucleotides. Preformed oligonucleotides can be spotted to form a DNA array, using techniques described herein with regard to cDNAs. In yet another alternative, the oligonucleotides are synthesized directly on the solid support. Methods for synthesizing oligonucleotide arrays are known in the art. See, for example, Fodor et al., U.S. Patent No. 5,744,305. The sequences of the oligonucleotides represent portions of the sequences of a particular gene to be detected above. Generally, the lengths of oligonucleotides are about 10 to 50 nucleotides (e.g., about 15, 20, 25, 30, 35, 40, or 45 nucleotides).

Protein Arrays

Protein arrays can be used to assay protein expression levels or genomic integrity to verify gene expression generated from nucleic acid arrays. Information from protein arrays can be added to a database described herein, and the information can be incorporated into algorithms that will predict a subject's responsiveness to treatment with an inhibitor described herein.

To generate a protein array, biological samples can be obtained and fixed, such as formalin-fixed, and paraffin-embedded. More than one biological sample per subject can be arrayed. For example, 2, 3, 4, 5, or more biological samples can be arrayed to account for heterogeneity in the samples.

Immunohistochemical analysis (IHC) of a protein array can be customized and optimized for each antibody. Standard indirect immunoperoxidase procedures can be used for immunohistochemistry. A target specific primary antibody and a secondary antibody visualized by, for example, diaminobenzidine as a chromogen. The primary antibodies can be omitted for negative staining controls. The intensity of the cytoplasmic staining can be classified into groups, such as negative, weak, intermediate, and strong staining groups. Alternatively, or in addition, FISH analysis can be performed to validate gene copy number change. A bacterial artificial chromosome (BAC) clone or another large insert clone can be used in addition to or instead of IHC. IHC and FISH data can be analyzed by statistical methods. For example, contingency table analyses and chi-square tests can be performed to assess changes in gene expression. The information gained from a protein array can be stored in a database, such as a database dedicated to the storage of protein array data, or any database described herein.

Proteomics

In addition to, or in an alternative to, the approaches discussed above, changes in gene expression can be identified through proteomic methods. Proteomic methods are useful for the identification of proteins in cells and/or tissues. A-ccordingly, a protein profile of a subject can be determined before the administration of an inhibitor described herein and, optionally, again after administration of the inhibitor. Protein microarrays (or protein microchips) are useful for this purpose. As described above for nucleic acid arrays, a protein microarray can include a subset or collection of^" proteins previously found to be differentially expressed in a responder or nonresponder. A protein microarray suitable for use in the methods described herein can be prepared by a number of methods known in the art. See, for example, methods disclosed in MacBeath and Schreiber (Science 289: 1760-1763, 2000), PCT Publication Nos. WO 00/4389A2, WO 00/04382, WO 99/60156, WO 99/39210, WO 00/54046, and WO 99/36576, and U.S. Pat. Nos. 6,087,102, 6,139,831, and 6,087,103. Detection of the proteins can be by the use of peptidic probes, such as antibodies

(e.g. polyclonal, monoclonal, and binding fragments thereof); peptides with high affinity to a target protein, as well as analogues and mimetics thereof; ligands, receptors, and the like. Peptidic probes may be obtained from naturally occurring sources or synthesized using available technologies. Probes can be directly detectable labels including isotopic and fluorescent moieties incorporated into (e.g., covalently bonded to) a moiety of the probe. Isotopic moieties or labels of interest include ³²P, ³³P, ³⁵S, ¹²⁵I, and the like. Fluorescent moieties or labels of interest include coumarin and its derivatives, e.g., 7-amino-4- methylcoumarin, aminocoumarin, bodipy dyes, such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g., fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g., Texas red, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g., Cy3 and Cy5, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, TOTAB, etc. Labels may also be members of a signal producing system that act in concert with one or more additional members of the same system to provide a detectable signal. Illustrative of such labels are members of a specific binding.pair, such as ligands, e.g., biotin, fluorescein, digoxigenin, antigen, polyvalent cations, chelator groups and the like, where the members specifically bind to additional members of the signal producing system, where the additional members provide a detectable signal either directly or indirectly, e.g., antibody conjugated to a fluorescent moiety or an enzymatic moiety capable of converting a. substrate to a chromogenic product, such as alkaline phosphatase conjugate antibody and the like. Additional labels of interest include those that provide for signal only when the probe with which they are associated is specifically bound to a target molecule, such as "molecular beacons" (see Tyagi & Kramer, Nature Biotechnology 14:303, 1996; and EP 0 070 685 Bl). Other useful labels are known in the art.

Skin Conditions

The methods of this invention are useful to predict the effectiveness of inhibitors of the CD2/LFA-3 interaction to prevent or treat mammalian, including human, slcin conditions characterized by increased T cell activation and abnormal antigen presentation in the dermis and epidermis. Such conditions include psoriasis, UV damage, atopic dermatitis, cutaneous T cell lymphoma such as mycosis fungoides, allergic and irritant contact dermatitis, lichen planus, alopecia areata, pyoderma gangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria. It is to be understood that methods of predicting treatment skin conditions such as pyoderma gangrenosum and urticaria are included within the scope of the present invention. These latter skin conditions are also cyclosporin A sensitive dermatoses and therefore involve T cell activation. Preferably, the methods of the invention are used in predicting treatment of psoriasis. The methods of the invention may be practiced on any mammal, preferably on humans.

While not wishing to be bound by theory, applicants believe that inhibitors of the CD2/LFA-3 interaction used in accordance with the methods of this invention are prophylactic and therapeutic for the treatment of the aforementioned skin conditions because they inhibit the interaction between T cells and antigen presenting cells, resulting in, among other things, an inhibition of T cell proliferation and activation. Applicants believe that adverse effects of skin conditions of the type discussed herein are due to such T cell proliferation and activation.

Inhibitors of the CD2/LFA-3 Interaction

The methods described herein are useful in predicting the effectiveness of treatment with any inhibitor of the CD2/LFA-3 interaction. Such inhibitors include anti- LFA-3 antibody homologs, anti-CD2 antibody homologs, soluble LFA-3 polypeptides, soluble CD2 polypeptides, small molecules, e.g., carbohydrates, LFA-3 and CD2 mimetic agents and derivatives thereof. Preferred inhibitors are soluble LFA-3 polypeptides and anti-LFA-3 antibody homologs. Predicting the effectiveness of treatment with LFA-3 -Ig fusion proteins, e.g., LFA-3-IgG fusion proteins, e.g., AMEVTVΕ® (alefacept), is the most preferred method.

Anti-LFA-3 and Anti-CD2 Antibody Homologs

The methods described herein are useful in predicting the effectiveness of treatment with many types of anti-LFA-3 or anti-CD2 antibody homologs. These include monoclonal antibodies, recombinant antibodies, chimeric recombinant antibodies, humanized recombinant antibodies, as well as antigen-binding portions of the foregoing. Anti-LFA-3 antibody homologs include, e.g., monoclonal anti-LFA-3 antibodies produced by hybridomas having Accession N_.os. ATCC HB 10693 (1E6), ATCC HB 10694 (HC-IBl 1), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8), or the monoclonal antibody known as TS2/9 (Sanchez-Madrid et al., Proc. Natl. Acad. Sci. USA, 79, pp. 7489-93 (1982)). Anti-CD2 antibody homologs include, e.g., anti-CD2 monoclonal antibodies known as the Tl l ₁ epitope antibodies, including TS 2/18 (Sanchez-Madrid et al., Proc.

Natl. Acad. Sci. USA, 79, pp. 7489-93 (1982)).

The methods described herein can be used to predict the effectiveness of treatment with anti-CD2 and anti-LFA-3 antibody homologs such as recombinant antibodies produced by host cells transformed with DNA encoding immunoglobulin light and heavy chains of a desired antibody. Recombinant antibodies may be produced by well-known genetic engineering techniques. See, e.g., U.S. Patent No. 4,816,397, which is incorporated herein by reference. It will be understood that variations on the above procedure are useful in the present invention. For example, it may be desired to transform a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody homolog. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for CD2 or LFA-3 counter receptor binding. The molecules expressed from such truncated DNA molecules are useful in the methods of this invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are anti-CD2 or anti-LFA-3 antibody homologs and the other heavy and light chain are 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-CD2 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 in which all or some of the DNA encoding the hinge and constant regions of the heavy and/or the light chain have been substituted with DNA from the corresponding region of an immunoglobulin light or heavy chain of a different species. An exemplary chimeric recombinant antibody has mouse variable regions and human hinge and constant regions. See generally, U.S. Patent No. 4,816,397 and Morrison et al., "Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains With Human Constant Region Domains", Proc. Natl. Acad. Sci. USA, 81, pp. 6851-55 (1984). Humanized recombinant anti-LFA-3 or anti-CD2 antibodies may be produced by transforming a host cell with a suitable expression vector comprising DNA encoding the desired nonhuman immunoglobulin light and heavy chains in which all or some of the DNA encoding amino acids not involved in antigen binding have been substituted with DNA from the corresponding region of a desired human immunoglobulin light or heavy chain. See generally, Jones et al., "Replacing the Complementarity-Determining Regions in a Human Antibody with Those from a Mouse", Nature, 321 , pp. 522-25 (1986). Other useful antibody homologs include Fab fragments, Fab' fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. Antibody fragments may be produced, e.g., by chemical methods or by using host cells transformed with truncated heavy and/or light chain genes. See, e.g., Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli", Nature, 341, pp. 544-46 (1989); Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library", Proc. Natl. Acad. Sci. USA, 86, pp. 5728-32 (1989).

Soluble CD2 and LFA-3 Polypeptides

The methods described herein can also be used to predict the effectiveness of treatment with soluble LFA-3 polypeptides or soluble CD2 polypeptides that inhibit the interaction of LFA-3 and CD2, e.g., soluble LFA-3 polypeptides.

Soluble LFA-3 polypeptides may be derived from the transmembrane form of LFA-3, particularly the extracellular domain (e.g., AA₁-AA₁₈₇ of SEQ ID NO:2). Such polypeptides are described in U.S. Patent No. 4,956,281. Preferred soluble LFA-3 polypeptides include polypeptides consisting of AA₁-AA₉₂ of SEQ ED NO:2, AA₁- AA₈₀ of SEQ ID NO:2. AA₅₀-AA₆₅ of SEQ ID NO:2 and AA₂₀-AA₈₀ of SEQ ID NO:2. A vector comprising a DNA sequence encoding SEQ ID NO:2 (i.e., SEQ ID NO:1) is deposited with the American Type Culture Collection under Accession No. 75107.

The most preferred fusion proteins of this type contain the amino terminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acids of a human IgGl hinge region containing the two cysteine residues thought to participate in interchain disulfide bonding, and the C_H2 and C_H3 regions of a human IgGj heavy chain constant domain

(e.g., SEQ ID NO:8). This fusion protein is referred to herein as "LFA3TIP." A plasmid, pSAB152, encoding an exemplary LFA3TIP, is deposited with American Type Culture Collection under the accession number ATCC 68720. The DNA sequence of the pSAB152 insert is SEQ ID NO:7.

Another preferred fusion protein consists of the first and second LFA-3 domain fused to the hinge C_H2 and C_H3 regions of human IgGl, herein referred to as LLFA3-Ig.

Soluble LFA-3 polypeptides may also be derived from the Pi-linked form of LFA-3 , such as those described in PCT Patent Application Serial No. WO 90/02181. A vector comprising a DNA sequence encoding Pi-linked LFA-3 (i.e., SEQ ID NO:3) is deposited with the American Type Culture Collection under Accession No. 68788. It is to be understood that the Pi-linked form of LFA-3 and the transmembrane form of LFA-3 have identical amino acid sequences through the entire extracellular domain. Accordingly, the preferred Pi-linked LFA-3 polypeptides are the same as for the transmembrane form of LFA-3.

Soluble CD2 polypeptides may be derived from full length CD2, particularly the extracellular domain (e.g., AA₁-AA₁₈₅ of SEQ ID NO:6). Such polypeptides may comprise all or part of the extracellular domain of CD2. Exemplary soluble CD2 polypeptides are described in PCT WO 90/08187, which is herein incorporated by reference.

The production of the soluble polypeptides described herein may be achieved by a variety of methods known in the art, e.g., by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation, or both; by purification from its natural source using conventional methods; or by known recombinant DNA techniques using cDNAs (see, e.g., U.S. Patent 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)).

While recombinant DNA techniques are the preferred method of 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. Synthetically produced polypeptides useful in this invention can advantageously be produced in extremely high yields and can be easily purified.

LFA-3 and CD2 Mimetic Agents

The methods described herein can also be used to predict the effectiveness of treatment with LFA-3 and CD2 mimetic agents. These agents that may be peptides, semi-peptidic compounds or non-peptidic compounds, are inhibitors of the CD2/LFA-3 interaction. The most preferred CD2 and LFA-3 mimetic agents will inhibit the CD2/LFA-3 interaction at least as well as anti-LFA-3 monoclonal antibody 7A6 or anti-CD2 monoclonal antibody TS2/ 18 (described supra).

Such mimetic agents may be produced by synthesizing a plurality of peptides (e.g., 5-20 amino acids in length), semi-peptidic compounds or non-peptidic, organic compounds, and then screening those compounds for their ability to inhibit the CD2/LFA-3 interaction. See generally U.S. Patent No. 4,833,092, Scott and Smith, "Searching for Peptide Ligands with an Epitope Library", Science, 249, pp. 386-90 (1990), and Devlin et al., "Random Peptide Libraries: A Source of Specific Protein Binding Molecules", Science, 249, pp. 404-07 (1990), which are herein incorporated by reference.

Derivatized Inhibitors

The methods described herein can also be used to predict the effectiveness of treatment with derivatized inhibitors of the CD2/LFA-3 interaction in which, for example, any of the antibody homologs, soluble CD2 and LFA-3 polypeptides, or CD2 and LFA-3 mimetic agents described herein are functionally linked (by chemical coupling, genetic fusion or otherwise) to one or more members independently selected from the group consisting of anti-LFA-3 and anti-CD2 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 two or more inhibitors (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Illinois. Another possibility for cross-linking takes advantage of the PI linkage signal sequence in Pi-linked LFA-3, or fragments thereof. Specifically, DNA encoding the PI- linkage signal sequence (e.g., AA₁₆₂-AA₂₁₂ of SEQ ID NO:4) is ligated downstream of DNA encoding a desired polypeptide, preferably a soluble LFA-3 polypeptide. If this construct is expressed in an appropriate eukaryotic cell, the cell will recognize the PI linkage signal sequence and will covalently link PI to the polypeptide. The hydrophobic property of the PI may then be exploited to form micellar aggregates of the polypeptides.

Also useful are inhibitors linked to one or more cytotoxic or pharmaceutical agents. Useful pharmaceutical agents include biologically active peptides, polypeptides and proteins, such as antibody homologs specific for a human polypeptide other than CD2 or LFA-3, or portions thereof. Useful pharmaceutical agents and cytotoxic agents also include cyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids, interferon, and nitrogen mustard.

Preferred inhibitors derivatized with a pharmaceutical agent include recombinantly-produced polypeptides in which a soluble LFA-3 polypeptide, 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 and all or part of a heavy chain constant region. Preferred polypeptides for preparing such fusion proteins are soluble LFA-3 polypeptides. Most preferred are fusion proteins containing AA₁-AA₉₂ of LFA-3

(e.g., SEQ ID NO:2) fused to a portion of a human IgG₁ hinge region (including the C-terminal ten amino acids of the hinge region containing two cysteine residues thought to participate in interchain disulfide bonding) and the C_H2 and C_H3 regions of an IgG₁ heavy chain constant domain. Such fusion proteins are expected to exhibit prolonged serum half-lives and enable inhibitor dimerization. Pharmaceutical Compositions

The above-mentioned skin conditions in a mammal can be treated by administering to the mammal one or more inhibitors of the CD2/LFA-3 interaction, or derivatized form(s) thereof. Preferably, an effective amount of the inhibitor or derivatized form thereof is administered. By "effective amount" is meant an amount capable of lessening the spread or severity of the skin conditions described herein.

It will be apparent to those of skill in the art that the effective amount of inhibitor will depend, inter alia, upon the administration schedule, the unit dose administered, whether the inhibitor is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic or prophylactic activity of the particular inhibitor administered and the serum half-life.

Preferably, the inhibitor is administered at a dose between about 0.001 and about 50 mg inhibitor per kg body weight, more preferably, between about 0.01 and about 10 mg inhibitor per kg body weight, most preferably between about 0.1 and about 4 mg inhibitor per kg body weight.

Unit doses should be administered until an effect is observed. The effect may be measured by a variety of methods, including in vitro T cell activity assays and clearing of affected skin areas. Preferably, the unit dose is administered about one to three times per week or one to three times per day.. More preferably, it is administered about one to three times per day for between about 3 and 7 days, or about one to three times per day for between about 3 and 7 days on a monthly basis. It will be recognized, however, that lower or higher dosages and other administrations schedules may be employed. The methods described herein can be used to predict the effectiveness of treatment with inhibitor(s) described herein, or derivatized form(s) thereof, in a composition including a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered.

Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the inhibitor. The methods described herein can also be used to predict the effectiveness of a pharmaceutical composition or inhibitor when administered in conjunction with other therapeutic or prophylactic agents. These include, for example, cyclosporin A, steroids, retinoids, nitrogen mustard, interferon, methotrexate, antibiotics and antihistamines.

These agents may be administered in single dosage form with the inhibitor (i.e., as part of the same pharmaceutical composition), a multiple dosage form separately from the inhibitor, but concurrently, or a multiple dosage form wherein the two components are administered separately but sequentially. Alternatively, the inhibitor and the other active agent may be in the form of a single conjugated molecule. Conjugation of the two components may be achieved by standard cross-linking techniques well known in the art. A single molecule may also take the form of a recombinant fusion protein. In addition, the inhibitors, or pharmaceutical compositions, described herein may be used in combination with other therapies such as PUVA, chemotherapy and UV light. Such combination therapies may advantageously utilize lower dosages of the therapeutic or prophylactic agents.

The inhibitor, or pharmaceutical composition, may be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable infusible, and topical preparations. The preferred form depends on the intended mode of administration and therapeutic application. The preferred forms are injectable or infusible solutions.

The inhibitor or pharmaceutical composition may be administered intravenously, intramuscularly, subcutaneously, intra-articularly, intrathecally, periostally, intratumorally, intralesionally, perilesionally by infusion, orally, topically or by inhalation. Preferably it is administered subcutaneously, intramuscularly or intravenously. Most preferably, it is administered subcutaneously.

The inhibitors described herein can also be formulated for use as topically applied sunscreens or UV-protectants. Preferred embodiments include LF A3 TIP preparations. The active ingredient can be formulated in a liposome. The following invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application, are hereby expressly incorporated by reference.

EXAMPLES

Example 1 - Ratio of CD3 cells in the epidermis relative to the dermis as a pretreatment predictor of response to treatment with AMEVIVE® (alefacept)

Subjects

AMEVrVΕ was administered to 20 psoriatic subjects for 13 weeks. At the end of the 13 -week treatment period, subjects were identified as responders or nonresponders by the epidermal thickness of biopsies taken at the end of 13 weeks. Twelve of the subjects were identified as responders and eight of the subjects were identified as nonresponders.

Biopsies

Skin biopsies (5mm) from the 20 subjects were taken of uninvolved and involved skin prior to treatment with AMEVIVE. Uninvolved skin was taken approximately

10 cm from a plaque on either abdomen, back or thigh. Skin biopsies were also taken of involved skin 2, 6 and 13 weeks after the start of treatment. The skin biopsies were analyzed for epidermal thickness and counts of a variety of lymphocyte markers (CD4, CD8, CD3, CD103 and VLA-4), as well as Taqman assay of a variety of cytokines (IL-4, IFN-gamma, IL-2, CD69 and ILlO), as discussed below.

Immunohistochemistry

Tissue sections were stained with hematoxylin (Fisher, Fair Lawn, New Jersey) and eosin (Shandon, Pittsburgh, Pennsylvania) (H&E) and with purified mouse anti- human monoclonal antibodies to K16 (Sigma Aldrich), CD3 , CD4 (Becton Dickinson, San Jose, California), CD8 (BD Pharmingen, San Diego, California), CD 103 (Biodesign

International, Kennebunk, Maine) and VLA-I (provided by Biogen Inc., Cambridge, MA). Biotin-labeled horse anti-mouse antibody (Vector Laboratories, Burlingame, California) was amplified with avidin-biotin complex (Vector Laboratories) and developed with chromogen 3-amino-9-ethylcarbazole (Sigma Aldrich). Epidermal thickness measures were computed using National Institute of Health software (NIH Image 6.1), and positive cells were counted manually using computer-assisted image analysis.

Results

The levels of CD3 cells in the epidermis and dermis were determined from both uninvolved and involved skin. The ratios of CD3 cells in the epidermis relative to the dermis from both uninvolved and involved skin were then determined. As shown in Fig. 1, the best biopsy discriminator between responders and nonresponders in pretreatment samples was the ratio of CD3⁺ cells in the epidermis relative to the dermis of involved skin. At a cutoff ratio of approximately 0.2, 10 of the 12 responders were identified and all but 1 nonresponders were rejected (see Fig. 1 ; light gray bars indicate the one nonresponder and the two responders who would be predicted to fall into the opposite category). The only parameter that competed with this was the change in epidermal thickness relative to baseline, in lesional skin, after two weeks of treatment (which is a sensitive visualization of clinical response, data not shown). The CD3 ratio from lesional (involved) skin at any time point was not useful in discriminating between responders and nonresponders (data not shown). This finding suggests that response to treatment with AMEVIVE has a genetic predisposition.

Example 2 - Ratio of CD3 cells in the epidermis relative to the dermis and PASI analysis

The ratio of CD3 cells in the epidermis relative to the dermis was determined as described in Example 1, and the PASI responses of subjects before and after treatment were determined. As shown in Fig. 2, the CD3 ratio is predictive of response, as determined both by histological and PASI response parameters. Initial PASIs were distributed fairly evenly between responders and nonresponders (see Fig. 3). Further,

CD3 ratio and initial PASI did not correlate with one another (see Fig. 4). This indicates that the correlation between CD3 ratio and responsiveness (seen in Fig. 2) was not an artifact of responders having a less severe condition.

Example 3 - PASI score and histological predictors of response to treatment with AMEVIVE

As with histologic response as an endpoint (see Example 1), variables in uninvolved skin were evaluated for predicting PASI response to treatment with AMEVrVE. The expression of several proteins in the epidermis and dermis of twenty patients was analyzed (see Fig. 5). Other parameters were analyzed by FACS, and involved skin samples were also analyzed (see Fig. 7).

As shown in Fig. 6 and summarized in Fig. 9, tree analysis of PASI 50 predictors revealed that a combination of CD 103 and VLA-I stains could predict PASI 50 attainment from nonattainment in 100% of cases. The performance of CD3 ratio was similar to its performance in predicting histologic score.

While not wishing to be bound by theory, the CD 103/VLA-l finding can be summarized in biological terms such that nonresponders tend to have higher proportions of cells able to traverse into the epidermis using these receptors. The fact that the CD3 ratio is also a predictor (see Fig. 13) suggests that the cells in question are T cells.

Example 4 - Effect of weight on PASI response

Analysis of the weights of PASI 50 nonresponders and responders who also attained PASI 75 was performed. As shown in Fig. 14, PASI 50 nonresponders and responders had the same weight distribution. However, the PASI 50 responders who also attained PASI 75 were lighter in weight than the PASI 50 responders who did not attain PASI 75. This suggests that weight may be a factor in predicting whether a subject will respond to treatment with AMEVIVE. Example 5 - Treatment with AMEVIVE increases Foxp3 expression in responders

In these experiments, biopsies from nonlesional and lesional skin before and after treatment (as described in Example 1) were analyzed for Foxp3 expression. Figs. 15A and 15B depict Foxp3 expression in nonlesional skin (NL) and lesional skin (LS) before treatment, and in LS after treatment for the indicated times. Foxp3 mRNA was measured by real-time RT-PCR, and expression was normalized to total mRNA with hARP mRNA (also by real-time RT-PCR). As shown in Fig. 15B, responding patients developed a relative increase in Foxp3 mRNA expression in lesional skin, especially 13 weeks after treatment.

To look for relative expression in T cells, Foxp3 expression was normalized to the number of T cells counted in the same biopsies. The increase in Foxp3 was not just a function of improving psoriasis/reduced numbers of T cells, as cyclosporin (CSA) treatment produced little, if any, increase in Foxp3 mRNA (see Fig. 15C). For the sake of comparison with alef acept data, reduced expression of Kl 6 in CSA treated patients established the response (see Fig. 15D). While not wishing to be bound by theory, as Foxp3 expression is a marker for regulatory T cells, the induction of regulatory T cells could explain the relatively long remissions seen with alefacept (but not cyclosporin) and provides a potential immunoregulatory mechanism that is less immunosuppressive than deletion of memory T cells.

Example 6 - Gene expression analysis of the pretreatment blood of responders and nonresponders Blood was drawn (pretreatment) from 20 psoriatic subjects and AMEVIVE was . then administered to the subjects for 13 weeks. Blood was again drawn at 6 hours and at 13 weeks after the start of treatment. At the end of the 13 -week treatment period, subjects were identified as responders or nonresponders by the epidermal thickness of biopsies taken at the end of 13 weeks. Twelve of the subjects were identified as responders and eight of the subjects were identified as nonresponders. The blood of 12 subjects who responded to treatment with AMEVIVE and 8 subjects who did not respond to treatment with AMEVIVE was analyzed. FACS analysis was performed for a variety of parameters (including CD8, CD4, CCR7, CXCR3, CD45RO and CD45RA). White blood cells were collected from all the blood samples of all the subjects and gene profiling was performed on the Affymetrix platform, using probe sets available from Affymetrix as listed in Tables 1-3. RNA amplifications and hybridizations were performed on a subset of four responders and four nonresponders, at pretreatment, six hour, and 13 week time points.

As demonstrated in Table 1, there is a set of genes in pretreatment blood that distinguish the four responders from the four nonresponders. These profile differences cannot be easily fit with the FACS marker data, which do not provide an easy split between responders and nonresponders. This finding is not incompatible with there being a subpopulation of cells present in responders and not nonresponders or vice versa, which was not specifically detected by the FACS markers. Possibly, the subpopulation could be a circulating population of (potentially target) dendritic cells.

Although the responder and nonresponder WBC RNAs were prepared and hybridized at different times, and the raw data showed that the overall hybridization intensity was lower in the responder population, the distribution of hybridization intensities was the same for all samples, and the data was subject to extremely stringent (RMA) normalization prior to analysis.

Example 7 - Gene expression analysis of the blood of responders and nonresponders after 6 hours of treatment with AMEVTVE

The blood from responders and nonresponders was collected as described in Example 6. RNA amplifications and hybridizations were performed on a subset of four responders and four nonresponders, at pretreatment, six hour, and 13 week time points. The changes in gene profiles after 6 hours of initial therapy are dramatic and are strikingly different between responders and nonresponders.

In brief, nonresponders show only a small subset of the gene expression changes in responders. In both, upregulation of Stat 1, and downregulations of granzyme B, and perforin are seen; in the responders, many other changes are seen including upregulation of Fc gamma receptor Ia, and downregulations of CD8 and IL2 receptor (see Table 2 and Table 3). These observations demonstrate that pretreatment gene profiles are significantly different in responders and nonresponders.

Table 1

Number of classes: 2

Number of genes: 12625

Number of genes that passed filtering criteria: 12625

Type of univariate test used: Two-sample T-test (with random variance model)

Column of the Experiment Descriptors sheet that defines class variable : class

Exact Multivariate Permutations test was computed based on 35 available permutations

Nominal significance level of each univariate test: 0.001 Confidence level of false discovery rate assessment: 90 % Maximum allowed number of false-positive genes: 10 Maximum allowed proportion of false-positive genes: 0.1

Summary of Results:

Number of genes significant at 0.001 level of the univariate test: 931

Probability of getting at least 931 genes significant by chance (at the 0.001 level) if there are no real differences between the classes: 0.02857

Genes which discriminate among classes:

Table 1 - Sorted by p-vaJue of the univariate test.

The first 931 genes are significant at the nominal 0.001 level of the univariate test

With probability of 90 % the first 473 genes contain no more than 10 false discoveries.

With probability of 90% the first 1 100 genes contain no more than 10% of false discoveries. Further extension of the list was halted because the list would contain more than 100 false discoveries

Filtering parameters:

D Spot Filtering: OFF D Gene-Screening: OFF Normalization: OFF

'Observed v. Expected' table of GO classes and parent classes, in list of 1100 genes shown above;

Only GO classes and parent classes with at least 5 observations in the selected subset and with an Observed vs. Expected' ratio of at least 2 are shown.

Table 2

Description of the problem:

Number of classes: 2

Number of genes: 12625

Number of genes that passed filtering criteria: 12625

Type of univariate test used: Paired T-test (with random variance model)

Column of the Experiment Descriptors sheet that defines class variable : class

Exact Multivariate Permutations test was computed based on 8 available permutations

Nominal significance level of each univariate test: 0.001 Confidence level of false discovery rate assessment: 85 % Maximum allowed number of false-positive genes: 10 Maximum allowed proportion of false-positive genes: 0.1 Summary of Results:

Number of genes significant at 0.001 level of the univariate test: 9

Probability of getting at least 9 genes significant by chance (at the 0.001 level) if there are no real differences between the classes: 0.125

Genes which discriminate among classes:

Table 2 - Sorted by p-value of the univariate test.

The first 9 genes are significant at the nominal 0.001 level of the univariate test With probability of 85 % the first 11 genes contain no more than 10 false discoveries. With probability of 85% the first 1 genes contain no more than 10% of false discoveries.

'Observed v. Expected' table of GO classes and parent classes, in list of 11 genes shown above:

Only GO classes and parent classes with at least 5 observations in the selected subset and with an 'Observed vs. Expected' ratio of at least 2 are shown.

Filtering parameters:

O Spot Filtering: OFF D Normalization: OFF

D Gene-Screening: OFF Table 3

Description of the problem:

Number of classes: 2

Number of genes: 12625

Number of genes that passed filtering criteria: 12625

Type of univariate test used: Paired T-test (with random variance model)

Column of the Experiment Descriptors sheet that defines class variable : class

Nominal significance level of each univariate test: 0.001 Confidence level of false discovery rate assessment: 85 % Maximum allowed number of false-positive genes: 10 Maximum allowed proportion of false-positive genes: 0.1

Summary of Results:

Number of genes significant at 0.001 level of the univariate test: 196

Probability of getting at least 196 genes significant by chance (at the 0.001 level) if there are no real differences between the classes: 0.125

Genes which discriminate among classes:

Table 3 - Sorted by p-value of the univariate test.

The first 196 genes are significant at the nominal 0.001 level of the univariate test With probability of 85 % the first 11 genes contain no more than 10 false discoveries. With probability of 85% the first 1 genes contain no more than 10% of false discoveries. 'Observed v. Expected' table of GO classes and parent classes, in list of 196 genes shown above:

Biological Process

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of evaluating a subject for responsiveness, or lack thereof, to an LFA-3-Ig fusion protein comprising evaluating the level of T cells in the epidermis of the subject and comparing the level of T cells in the epidermis with a first reference value, thereby evaluating responsiveness to the LFA-3-Ig fusion protein.

2. The method of claim 1 , wherein the method further comprises evaluating the level of T cells in the dermis of the subject and comparing the level of T cells in the dermis with a second reference value, thereby evaluating responsiveness to the LFA-3-Ig fusion protein.

3. The method of claim 1, wherein if the level of T cells in the epidermis of the subject is above the first reference value, the level of T cells indicates nonresponsiveness to the LFA-3-Ig fusion protein.

4. The method of claim 1, wherein if the level of T cells in the epidermis of the subject is below the first reference value, the level of T cells indicates responsiveness to the LFA-3-Ig fusion protein.

5. The method of claim 1, wherein if the level of T cells in the dermis of the subject is above the second reference value, the level of T cells indicates responsiveness to the LFA-3-Ig fusion protein.

6. The method of claim 1 , wherein if the level of T cells in the dermis of the subject is below the second reference value, the level of T cells indicates nonresponsiveness to the LFA-3-Ig fusion protein.

7. The method of claim 1 , further comprising comparing the level of T cells in the epidermis to the level of T cells in the dermis.

8. The method of claim 7, wherein a level of T cells in the epidermis that is equal to or less than 50% of the level of T cells in the dermis indicates responsiveness to the LFA-3-Ig fusion protein.

9. The method of claim 1 , wherein the level of T cells in the epidermis is evaluated, the first reference value is the level of T cells in the dermis and a ratio of the level of T cells in the epidermis relative to the level of T cells in the dermis below a first selected value indicates responsiveness and a ratio a ratio of the level of T cells in the epidermis relative to the level of T cells in the dermis above a second selected value indicates nonresponsiveness.

10. The method of claim 9, wherein said first selected value and said second selected value are the same.

11. The method of claim 9, further comprising administering the LF A-3 fusion protein to the subject.

12. The method of claim 11 , further comprising determining that the LF A-3 - Ig fusion protein is not to be administered to the subject.

13. The method of claim 1 , wherein the subject has psoriasis.

14. The method of claim 1 , further comprising obtaining a skin biopsy from the subject.

15. The method of claim 14, wherein the skin biopsy is a nonlesional skin biopsy.

16. The method of claim 1, wherein the subject has not previously been treated with an LF A-3 -Ig fusion protein.

17. The method of claim 1, wherein the level of T cells is evaluated by detecting expression of a T cell marker.

18. The method of claim 1 , wherein the level of T cells is evaluated by detecting CD3 expression.

19. The method of claim 1 , wherein the level of T cells is evaluated by detecting CD4 expression.

20. The method of claim 1 , wherein the level of T cells is evaluated by detecting CD8 expression.

21. The method of claim 1 , wherein the level of T cells is evaluated by detecting CD 103 expression.

22. The method of claim 1, wherein the level of T cells in the epidermis is evaluated by detecting VLA-I expression.

23. The method of claim 1 , wherein the level of T cells is evaluated by detecting the expression of two T cell markers.

24. The method of claim 23, wherein the level of T cells is evaluated by detecting the expression of CD4 and CD8.

25. The method of claim 21 , wherein the level of T cells in the epidermis and/or dermis is evaluated by detecting the expression of CD 103 and the level of T cells in the epidermis is evaluated by detecting the expression of VLA-I.

26. The method of claim 1 , further comprising evaluating the weight of said subject to provide a further indication of responsiveness, or lack thereof, wherein weight above a threshold value indicates a propensity for nonresponsiveness and weight below a threshold value indicates a propensity for responsiveness.

27. The method of claim 1 , wherein evaluating responsiveness comprises evaluating the likelihood of achieving a predetermined Psoriasis Area and Severity Index (PASI) response.

28. The method of claim 27, wherein the predetermined PASI response is greater than 50.

29. A method of evaluating a subject for responsiveness, or lack thereof, to an LFA-3-Ig fusion protein comprising: evaluating the level of expression of a gene listed in Table 1 ; and comparing the level of expression with a reference value, wherein a level of expression that is different than the reference value is indicative of responsiveness.

30. A method of evaluating a subject for responsiveness, or lack thereof, to an inhibitor of the CD2/LFA-3 interaction comprising: evaluating the level of expression of a gene listed in Table 1 ; and comparing the level of expression with a reference value, wherein a level of expression that is higher or lower than the reference value is indicative of responsiveness.

31. A method of making a decision between a first course of action and a second course of action, the method comprising: receiving data on the likelihood of a subject to respond to an LFA-3-Ig fusion protein generated by the method of claim 1, 29 or 30; and using the data to make the decision between a first course of action and a second course of action.