US 2010O3O3835A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0303835 A1 Gocke et al. (43) Pub. Date: Dec. 2, 2010 (54) PEPTOID LIGANDS FOR ISOLATION AND Publication Classification TREATMENT OF AUTOMMUNE TCELLS (51) Int. Cl. (75) Inventors: Anne R. Gocke, Baltimore, MD A 6LX 39/395 (2006.01) (US); D. Gomika GOIN 33/53 (2006.01) Udugamasooriya, Flower Mound, CI2N 5/0783 (2010.01) TX (US); Thomas Kodadek, A6II 35/12 (2006.01) Jupiter, FL (US) A6II 35/14 (2006.01) C07K 2/00 (2006.01) Correspondence Address: A6IP37/00 (2006.01) FULBRIGHT & UAWORSK L.L.P. A6IP37/06 (2006.01) 600 CONGRESSAVE., SUITE 2400 AUSTIN, TX 78701 (US) (52) U.S. Cl. ..................... 424/178.1; 435/7.24:435/325; (73) Assignee: THE BOARD OF REGENTS OF 424/520; 424/529; 435/372.3; 435/352:435/375; THE UNIVERSITY OF TEXAS 530/300; 977/774 SYSTEM, Austin, TX (US) (21) Appl. No.: 12/789,711 (57) ABSTRACT (22) Filed: May 28, 2010 The present invention provides for the identification of autoreactive T cell populations from individuals having Related U.S. Application Data autoimmune diseases, such as multiple Sclerosis and EAE. (60) Provisional application No. 61/182,368, filed on May Peptoids recognized by autoreactive T cells can be used to 29, 2009, provisional application No. 61/260,608, identify various types of autoimmune disease, and can also be filed on Nov. 12, 2009. used to target therapies against Such populations. Screen Against 300,000 Peptoids Patent Application Publication Dec. 2, 2010 Sheet 1 of 14 US 2010/0303835 A1 Isolate CD4+ T Cells Screen Against 300,000 Peptoids FIG. 1A Patent Application Publication Dec. 2, 2010 Sheet 3 of 14 US 2010/0303835 A1 º, :::- 0 Patent Application Publication Dec. 2, 2010 Sheet 4 of 14 US 2010/0303835 A1 FIG. 1D Patent Application Publication Dec. 2, 2010 Sheet 5 of 14 US 2010/0303835 A1 Patent Application Publication Dec. 2, 2010 Sheet 6 of 14 US 2010/0303835 A1 s tex CR g cells + AG3A one W ais - ACS 2A 20 t 0.1 o 160 1000 Peptoid Concentration (M) FIG. 2B Patent Application Publication Dec. 2, 2010 Sheet 7 of 14 US 2010/0303835 A1 TG TG W T G TG 4. CD4+ CD4- CD4+ CD4 NA-R Arti-Wy2 TCR FIG. 2C Patent Application Publication Dec. 2, 2010 Sheet 8 of 14 US 2010/0303835 A1 A. MBP AC 1-1 1 CD4+ T Cels 30H AG12APeptoid Control peptoid C S. g 2 w C d U s C) 0.1 1 10 100 1000 B. B cells 60 ?a-74 .9 â40 w O CD S. 20 n 0 0.1 1 10 100 1000 C. MOG 35-55 CD4+ T cells 60 .9 D . s 40 S-N-2 w G9 9. 20 C A. 0.1 1 10 100 1000 Peptoid Concentration (uM) FIG 3A-C Patent Application Publication Dec. 2, 2010 Sheet 9 of 14 US 2010/0303835 A1 0 (!), W 0 WWW Patent Application Publication Dec. 2, 2010 Sheet 10 of 14 US 2010/0303835 A1 X light No Light as - 7 5 Patent Application Publication Dec. 2, 2010 Sheet 11 of 14 US 2010/0303835 A1 Light No Light © © FIG. 4C Patent Application Publication Dec. 2, 2010 Sheet 12 of 14 US 2010/0303835 A1 88 AG t2A-Ru2+'Ag. as a Control-Ru3. Ag. ^ No Peptoid + Ag. meter No Peptoid + No Ag. 25 Days Post-Transfer FIG. 4D Patent Application Publication Dec. 2, 2010 Sheet 13 of 14 US 2010/0303835 A1 A. 5 -- PBS/CFA -- MBPAC 1-111CFA 4 3- ---. O 5 O 15 2O 25 30 Days Post-immunization 262,144 (8') Compounds 1 O Y1 NH H2N-N-r Methoxyethylamine 1,4-Diarminobutane (Nmea) (Nys)Niys O NH 4 O rt. Cru,N Piperonylamine (R)-Methylbenzylamine (Npip) (Nmba) | \ N Fufurylamine Tryptamine (Nffa) (Ntrp) S1N NH r NH 2 Allyamine Isobutylamine (Nall) (Nieu) FIG 5A-B Patent Application Publication Dec. 2, 2010 Sheet 14 of 14 US 2010/0303835 A1 Control Peptoid / - O O O O O 2 O HN-N-N-N-N-N-N-N-N-N-) O ) O ) O O HNT2 4 HN2 '4 2 N 4 CaS FIG 6. US 2010/0303835 A1 Dec. 2, 2010 PEPTOID LIGANDS FOR ISOLATION AND able label; (b) providing a second T cell population from a TREATMENT OF AUTOMMUNE TCELLS Subject having an autoimmune disease, wherein said popula tion is labeled with a second detectable label; (c) contacting said first and second T cell populations with a plurality of said 0001. This application claims priority to U.S. Provisional candidate peptoid; and (d) assessing binding of said first and Patent Application Ser. No. 61/182,368 filed May 29, 2009 second T cell populations to said candidate peptoid, wherein and Ser. No. 61/260,608 filed Nov. 12, 2009, each of which is if said peptoid binds to said second T cell population but not incorporated herein by references in its entirety. to said first T cell population, the said peptoid is recognized 0002 This invention was made with government support by autoimmune but not healthy T cells. under grant no. NO1-HV28.185 from the National Heart, Lung and Blood Institute, and grant no. DP10D00066301 0010. The autoimmune disease may be multiple sclerosis from the National Institutes of Health. The government has or rheumatoid arthritis. The ligand or peptoid may be a 3-mer, certain rights in the invention. a 4-mer, a 5-mer, a 6-mer, a 7-mer, an 8-mer, a 9-mer or a 10-mer. The first and second labels may be fluorescent or BACKGROUND OF THE INVENTION chemiluminescent, or quantum dots. The peptoid may be bound to a Support, Such as a bead, a chip, a filter, a dipstick, 0003 1. Field of the Invention a membrane, a polymer matrix or a well. The contacting step 0004. The present invention relates generally to the fields may comprise bringing said Support into contact with said of molecular biology, immunology and medicine. More par first and second T cell populations at the same time. The T cell ticularly, it concerns the identification of peptoids that are population may comprise CD4 T cells. The subjects may be recognized by autoimmune T-cells. These peptoids can be human or murine. used to identify subjects suffering from or at risk of autoim 0011. In another embodiment, there is provided a method mune disease, as well as to target these cells for removal, of removing an autoimmune T cell from a subject Suffering inhibition or destruction. from an autoimmune disease comprising (a) providing a 0005 2. Description of Related Art ligand or peptoid that binds specifically to autoimmune T 0006. The molecular basis of many autoimmune diseases cells, wherein said ligand or peptoid is bound to a Support; (b) remains unknown. Due in part to this lack of a molecular-level contacting a T cell-containing sample from said Subject with understanding, the state of the art in the development of said Support-bound peptoid for a sufficient time to permit diagnostic agents and effective therapies for autoimmune binding of autoimmune T cells to said Support-bound ligand diseases is far from optimal. For example, there is no highly or peptoid; and (c) separating said Support from said sample. reliable serum protein marker for diagnosis of most autoim The method may further comprise returning the sample of mune diseases. Almost without exception, drugs employed to step (c) to said subject. The autoimmune disease may be treat these conditions either inhibit an event downstream of multiple sclerosis or rheumatoid arthritis. the autoimmune response itself. Such as inflammation, or 0012. The ligand or peptoid may be a 3-mer, a 4-mer, a attempt to modulate or Suppress the entire immune system 5-mer, a0-mer, a 7-mer, an 8-mer, a 9-mer or a 10-mer. The non-selectively (Hemmer & Hartung, 2007), with significant Support may be a bead, a chip, a filter, a dipstick, a membrane, undesirable side effects. For both diagnostic and therapeutic a polymer matrix or a well. The sample may be blood, cere applications, one would ideally like to have molecules that brospinal fluidorsemen. Where the sample is blood, it may be target autoreactive B cells (and the antibodies they produce) obtained from said subject, treated ex vivo, and returned to and T cells directly, but ignore B and T cells that recognize said subject, and further, the blood may be perfused across foreign antigens. Such molecules could be employed as diag said Support-bound ligand or peptoid and returned to said nostic agents and research tools for the detection and enrich subject in a closed circuit. The method may further comprise ment of autoimmune antibodies, B cells and T cells. In addi obtaining said sample from said subject. The Subject may be tion, these molecules could serve as the foundation for a novel drug development program aimed at eradicating these autore human or murine. active cells without affecting the proper function of the 0013. In still another embodiment, there is provided a method of killing an autoimmune T cell obtained from a immune system. Subject Suffering from an autoimmune disease comprising (a) 0007 Thus, there remains a need for diagnostic proce providing a ligand or peptoid that binds specifically to dures for both of these diseases that are (i) accurate and autoimmune T cells, wherein said ligand or peptoid is conju objective, (ii) simple and reproducible, and (iii) useful in both gated to a toxin; and (b) contacting a T cell-containing sample early and late stage case.