Paul I Freimuth, 71106 Old Post Rd, East Setauket, NY 11733

6737234, May 18, 2004

Sep 3, 1999

09/389603

Paul I. Freimuth - East Setauket NY

Brookhaven Science Associates, LCC - Upton NY

C12Q 168

435 5, 4352351, 4353201, 435 6, 435 71, 435 914, 435 9141, 435 9142, 536 231, 536 2372, 530350

Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

7157266, Jan 2, 2007

Aug 14, 2002

10/218419

Paul I. Freimuth - East Setauket NY, US

Brookhaven Science Associates LLC - Upton NY

C12N 7/01
C12N 7/04
C12N 15/861
A61K 48/00
C12Q 1/70

4352351, 4353201, 435236

Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

7691627, Apr 6, 2010

Oct 17, 2006

11/581689

Paul I. Freimuth - East Setauket NY, US

Brookhaven Science Associates, LLC - Upton NY

C12N 15/63
C07H 21/04
C12N 1/21
C07K 14/705
A61K 39/125
A61K 39/235

4353201, 536 235, 4352523, 530350, 4242161, 4242331

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

8535908, Sep 17, 2013

Jan 8, 2010

12/684195

Paul I. Freimuth - East Setauket NY, US
Jason Howitt - London, GB

Brookhaven Science Associates, LLC - Upton NY

C12P 21/06
C07H 21/04
C12N 5/02

435 691, 536 231

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

2002015, Oct 17, 2002

May 24, 2002

10/155282

Paul Freimuth - East Setauket NY, US

Brookhaven Science Associates LLC

C07K014/705
C12Q001/70
C07H021/04
C12P021/02
C12N005/06

530/350000, 536/023500, 435/005000, 435/320100, 435/325000, 435/069100

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

2003002, Feb 6, 2003

Jul 22, 2002

10/199290

Paul Freimuth - East Setauket NY, US

Brookhaven Science Associates

C12N015/861
C12N007/00

435/456000, 435/235100

The present invention is based on the solving of the crystal structure of adenovirus fiber protein knob domain bound to domain 1 of the coxsackie-adenovirus receptor. One aspect of the present invention relates to a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain, the viral particle encoded by the genome being characterized by a significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Another aspect of the present invention is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type., from an adenovirus which binds CARD1. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus, and whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is determined. Mutants produced by this method include those which under physiological conditions, have significantly weakened binding affinity for CARD1 relative to wild type adenovirus and those which bind a receptor other than CARD1, including an engineered receptor. Introduced mutations may produce an amino acid insertion, deletion or substitution in the encoded viral particle, and may serve to alter the conformation of one or more residues of knob which participate directly in D1 binding. Such residues include residues of the AB loop, the CD loop, the DE loop, the FG loop, the E strand and the F strand. Alternatively, the mutation may be directly introduced in a codon encoding the residue of knob which participates directly in D1 binding. Specific residues in the AB loop, the CD loop, the FG loop, the E strand, the F strand, and the DE loop which participate directly in binding are identified. Another aspect of the present invention is a method for identifying an inhibitor of adenovirus binding to CAR. In the method, a three-dimensional structure derived by X-ray diffraction from a crystal of adenovirus knob trimer bound to CARD1 is provided and then employed to design or select a potential inhibitor. The potential inhibitor is synthesized and then whether or not the potential inhibitor inhibits adenovirus binding to CAR is determined. Preferred crystal structures and space group symmetry is listed. A set of atomic coordinates which define the three dimensional structure is provided. The potential inhibitor may be designed to interact non-covalently with one or more residues of the adenovirus fiber knob protein domain. Alternatively, the potential inhibitor is designed to interact non-covalently with one or more residues of CARD1. Specific residues for covalent and non-covalent interaction are listed. The potential inhibitor may also be designed to interact non-covalently with residues which line a cavity formed during adenovirus knob trimer/CARD1 binding. The potential inhibitor can be designed by identifying chemical entities or fragments capable of associating with the adenovirus knob trimer, and assembling the identified chemical entities or fragments into a single molecule to provide the structure of said potential inhibitor. Such an inhibitor may be designed de novo or from a known inhibitor. Methods of inhibition include competitive inhibition, non-competitive inhibition and uncompetitive inhibition.

2003013, Jul 17, 2003

Jan 4, 2002

10/037243

Paul Freimuth - East Setauket NY, US
Jason Howitt - Wading River NY, US

C12P021/02
C12N005/06
C07H021/04
C12N015/85

435/069100, 435/455000, 435/320100, 435/325000, 536/023200

Disclosed herein are novel compositions and methods for enhancing the solubility and promoting the adoption of native folding conformation of a protein or polypeptide expressed by recombinant DNA techniques. One embodiment of the present invention relates to a protein or polypeptide of interest is modified through either carboxyl- or amino-terminal peptide extension, so as to promote folding within host cells. Another embodiment relates to a method for enhancing the in vitro renaturation of a protein or polypeptide of interest expressed by recombinant DNA techniques, in circumstances where, following expression, a substantial percentage of the expressed protein or polypeptide of interest is localized within inclusion bodies. Yet another embodiment of the present invention relates to an expression vector comprising a nucleic acid sequence encoding a peptide extension and a multiple cloning site for inserting, in-frame with the peptide extension, a nucleic acid sequence encoding a protein or polypeptide of interest. The peptide extensions of the present invention comprise different amino acid sequences and intrinsic net charges, depending upon the specific species. The total length of the peptide extensions comprise 61 amino acid residues or less, whereas the net intrinsic charges of the peptide extensions range from about -20 to about -2 and from about -20 to about +2, for peptide extensions fused to carboxyl- and amino-termini, respectively. Primary objectives of the present invention include: (i) enhancing the solubility, while concomitantly optimizing the folding, of proteins of interest into their biologically-active conformations in host cells ; (ii) characterizing the features of the carboxyl- and amino-terminal peptide extension that are necessary for their protein folding activity within host cells; (iii) determining whether these carboxyl- and amino-terminal peptide extensions can promote renaturation of mis-folded proteins in vitro; and (iv) identifying protein characteristics which determine behavior of the protein as a substrate for the peptide extension-mediated folding described herein.

6395875, May 28, 2002

Jan 25, 1999

09/236423

Paul I. Freimuth - East Setauket NY

Brookhaven Science Associates LLC - Upton NY

C07K 14705

530324, 530326, 530350

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.