Co-crystal structure of monoclonal antibody 5C8 and CD154, and use thereof in drug design

A computer for producing a three-dimensional representation of:

• a) a molecular complex comprising a first binding site defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50A;
• wherein said computer comprises:
• (i) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4;
• and
• (ii) instructions for processing said machine-readable data into said three-dimensional representation.

A computer for producing a three-dimensional representation of:

• a) a molecular complex comprising a first binding site, defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4, that associates with one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said CD154 amino acids and said one or more anti-CD154 amino acids between 0.00 Å and 1.50 Å;
• wherein said computer comprises:
• (i) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4 and the structure coordinates of one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• and
• (ii) instructions for processing said machine-readable data into said three-dimensional representation.

A computer for producing a three-dimensional representation of:

• a) a molecular complex defined by structure coordinates of one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue has a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å;
• wherein said computer comprises:
• (i) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• and
• (ii) instructions for processing said machine-readable data into said three-dimensional representation.

A computer for producing a three-dimensional representation of:

• a) a molecular complex defined by at least a portion of the structure coordinates of all the CD154 and anti-CD154 antibody amino acids set forth in FIG. 4, or
• b) a homologue of said molecular complex, wherein said homologue has a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å than 1.50 Å;
• and
• wherein said computer comprises:
• (i) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates of all of the CD154 and anti-CD154 antibody amino acids set forth in FIG. 4;
• and
• (ii) instructions for processing said machine-readable data into said three-dimensional representation.

A computer for determining at least a portion of the structure coordinates corresponding to X-ray diffraction data obtained from a molecular complex, wherein said computer comprises:

• a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates of CD154 or anti-CD154 antibody according to FIG. 4;
• b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises X-ray diffraction data obtained from said molecular complex;
• and
• c) instructions for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates.

A method for evaluating the potential of a chemical entity to associate with:

• a) a molecular complex comprising a first binding site defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the chemical entity and said first binding site of the molecular complex or said second binding site of said homologue of said molecular complex;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the chemical entity and said first binding site or said second binding site.

A method for evaluating the potential of a chemical entity to associate with:

• a) a molecular complex comprising a first binding site, defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4, that associates with one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31;
• Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said CD154 amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the chemical entity and said first binding site or said second binding site;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the chemical entity and said first binding site or said second binding site.

A method for evaluating the potential of a chemical entity to associate with:

• a) a molecular complex defined by at least a portion of the structure coordinates of all the CD154 and anti-CD154 antibody amino acids, as set forth in FIG. 4;
• or
• b) a homologue of said molecular complex having a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the chemical entity and a first binding site of said molecular complex or a second binding site of said homologue of said molecular complex;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the chemical entity and said first binding site or said second binding site.

A method for identifying a potential agonist or antagonist of CD154 comprising the steps of:

• a) using the structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4±a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å, to generate a three-dimensional structure of a molecular complex comprising a binding site;
• b) employing said three-dimensional structure to design or select said potential agonist or antagonist;
• c) synthesizing said potential agonist or antagonist;
• and
• d) contacting said potential agonist or antagonist with CD154 to determine the ability of said potential agonist or antagonist to bind to CD154.

A method for identifying a potential agonist or antagonist of CD154 comprising the steps of:

• a) using the structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4, wherein said CD154 amino acids associate with one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4±a root mean square deviation from the backbone atoms of said CD154 amino acids between 0.00 Å and 1.50 Å, to generate a three-dimensional structure of a molecular complex comprising a binding site;
• b) employing said three-dimensional structure to design or select said potential agonist or antagonist;
• c) synthesizing said potential agonist or antagonist;
• and
• d) contacting said potential agonist or antagonist with CD154 to determine the ability of said potential agonist or antagonist to bind to CD154.

A method for identifying a potential agonist or antagonist of CD154 comprising the steps of:

• a) using at least a portion of the structure coordinates of all the amino acids of CD154 and anti-CD154 antibody according to FIG. 4±a root mean square deviation from the backbone atoms of said CD154 amino acids between 0.00 Å and 1.50 Å, to generate a three-dimensional structure of a molecular complex comprising a binding site;
• b) employing said three-dimensional structure to design or select said potential agonist or antagonist;
• c) synthesizing said potential agonist or antagonist;
• and
• d) contacting said potential agonist or antagonist with CD154 to determine the ability of said potential agonist or antagonist to bind to CD154.

A method for evaluating the potential of a variant of monoclonal antibody 5c8, or a variant of an antigen binding fragment thereof, or a variant of humanized 5c8 mAb, or a variant of an antigen binding fragment thereof, to associate with:

• a) a molecular complex comprising a first binding site defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the variant and said first binding site or said second binding site;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the variant and said first binding site or said second binding site.

A method for evaluating the potential of a variant of monoclonal antibody 5c8, or a variant of an antigen binding fragment thereof, or a variant of humanized 5c8 mAb, or a variant of an antigen binding fragment thereof, to associate with:

• a) a molecular complex comprising a first binding site, defined by structure coordinates of CD154 amino acids Glu129, Ala130, Ser132, Glu142, Lys143, Gly144, Tyr146, Cys178, Cys218, Ser245, Gln246, Ser248, His249 and Gly250 according to FIG. 4, that associates with one or more anti-CD154 antibody amino acids Ser31, Tyr32, Tyr33, Asn52, Ser54, Asp57, Asn59, Arg102, Asn103 of the heavy chain and amino acids Ser31, Ser32, Tyr36, Ser95 and Trp96 of the light chain according to FIG. 4;
• or
• b) a homologue of said molecular complex, wherein said homologue comprises a second binding site that has a root mean square deviation from the backbone atoms of said CD154 amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the variant and said first binding site or said second binding site;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the variant and said first binding site or said second binding site.

A method for evaluating the potential of a variant of monoclonal antibody 5c8, or a variant of an antigen binding fragment thereof, or a variant of humanized 5c8 mAb, or a variant of an antigen binding fragment thereof, to associate with:

• a) a molecular complex defined by at least a portion of the structure coordinates of all the CD154 and anti-CD154 antibody amino acids, as set forth in FIG. 4;
• or
• b) a homologue of said molecular complex having a root mean square deviation from the backbone atoms of said amino acids between 0.00 Å and 1.50 Å;
• comprising the steps of:
• (i) employing computational means to perform a fitting operation between the variant and a first binding site of the molecular complex or a second binding site of the homologue of the molecular complex;
• and
• (ii) analyzing the results of said fitting operation to quantify the association between the variant and said first binding site or said second binding site.

A computer for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecular complex, wherein said computer comprises:

• a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates according to FIG. 4;
• b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises an X-ray diffraction pattern of said molecular complex;
• c) a working memory for storing instructions for processing said machine-readable data of a) and b);
• d) a central processing unit coupled to said working memory and to said machine-readable data of a) and b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates;
• and
• e) a display coupled to said central processing unit for displaying said structure coordinates of said molecular complex.