US00871.5677B2

(12) United States Patent (10) Patent No.: US 8,715,677 B2 Bartlett et al. (45) Date of Patent: May 6, 2014

(54) IMMUNOLOGICAL USES OF WO WOO145750 6, 2001 MMUNOMODULATORY COMPOUNDS FOR WO WOO2O59 106 38: VACCNE AND ANT-INFECTIOUS DISEASE W8 w885 358. THERAPY WO WO2004103274 12, 2004 WO WO2005OO7190 1, 2005 (75) Inventors: Justin B. Bartlett, Warren, NJ (US); George W. Muller, Bridgewater, NJ OTHER PUBLICATIONS (US); Peter H. Schafer, Randolph, NJ (US); Christine Galustian, Croydon Kaechet al., “Effector and Memory T-cell Differentiation: Implica Surrey (GB); Angus G. Dalgleish, tions for Vaccine Development.” Nature Reviews Immunology Cheam (GB); Brendan Meyer, London 2002:v2:251-262.* Bartlett et al., British Journal of Cancer, 90:955-961 (2004). (GB) Bielekova et al., The Journal of Immunology, 164: 1117-1124 (2000). (73) Assignee: Celgene Corporation, Summit, NJ (US) Cavanagh et al., Int. J. Cancer, 70: 98-105 (1997). Corral et al., The Journal of Immunology, 163:380-386 (1999). Davies et al., Blood, 98: 210-216 (2001). (*) Notice: Subject to any disclaimer, the term of this Dredge et al., Critical Reviews in Immunology, 22(5&6): 425-437 patent is extended or adjusted under 35 (2002). U.S.C. 154(b) by 714 days. Dredge et al., Cancer Immunol. Immunother. 51: 521-531 (2002). Dredge et al., The Journal of Immunology, 168: 4914-4919 (2002). (21) Appl. No.: 11/514,447 Gollob et al., J. Clin. Invest., 102(3): 561-575 (1998). Haslett et al. The Journal of Infectious Diseases, 187: 946-955 (22) Filed: Aug. 31, 2006 (2003). Marriott et al., Clin. Exp. Immunol. 130: 75-84 (2002). (65) Prior Publication Data Marriott et al., Expert Opin. Biol. Ther. 1(4): 1-8 (2001). US 2007/OO48327 A1 Mar. 1, 2007 Schafer et al., The Journal of Pharmacology and Experimental Therapeutics, 305: 1222-1232 (2003). Related U.S. Application Data Schey et al., Journal of Clinical Oncology, 22(16): 1-8 (2004). Galustian et al., “Lenalidomide (Revlimid(R), CC-5013) and (60) Provisional application No. 60/712,823, filed on Sep. Actimid TM (CC-4047) inhibit the function and expansion of Tregu 1, 2005. latory (Treg) cells in vitro: Implications for anti-tumor activity in vivo..” Proc. Am. Assoc. Cancer Res. Ann. Meeting, 47: 1147, Abstract (51) Int. Cl. No. 4882 (XP00 1248611), (2006). A6 IK39/00 (2006.01) Tsenova et al., Antimicrobial Agents and Chemotherapy, 46(6): (52) U.S. Cl. 1887-1895 (2002). USPC ...... 424/184.1; 424/277.1 (58) Field of Classification Search * cited by examiner USPC ...... 514/323, 417; 424/208.1; 435/4 See application file for complete search history. Primary Examiner – Walter Webb (74) Attorney, Agent, or Firm — Jones Day (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT Methods of enhancing immune response to an immunogen in 5,723,283 A * 3/1998 Classen ...... 435/4

6,281.230 B1* 8, 2001 Muller et al. .... 514/323 a Subject are disclosed. Also disclosed are methods of reduc 2003/0096841 A1* 5/2003 Robarge et al...... 514,323 ing the sensitivity to an allergen in a Subject. The methods 2004/002281.0 A1* 2, 2004 Rudenko et al...... 424,208.1 comprise the administration of an immunomodulatory com pound in specific dosing regimens that result in enhanced FOREIGN PATENT DOCUMENTS immune response or reduced sensitivity. WO WO901 1764 10, 1990 WO WO93 13772 7, 1993 15 Claims, 21 Drawing Sheets U.S. Patent May 6, 2014 Sheet 1 of 21 US 8,715,677 B2

List of Vaccines Product or trade Antigen(s) Manufacturer (country) name A D iphtheria, tetanus (adsorbed Commonwealth (Australia A iphtheria, tetanus, pertussis Fl eninqOCOccus (polysaccharide AEGEphtheria, tetanus, (acellular) pertussis WYE (US) A iphtheria, tetanus, pertussis, Hib A aemophilus influenzae type b (PRP-T Eter m H epatitis A (inactivated) Chemo-Sero-Therapeutic Reshinst (Japan) A iphtheria (absorbed S evac (Czechoslovakia iphtheria, tetanus (absorbed S evac (Czechoslovakia EGEE pertussis ellow fever AVP (France aemophilus influenzae, type b Anadifteral iphtheria (adsorbed etanus (adsorbed C ellow fever Attenuvax * easles (live, further attenuated nthrax epatitis A B-CAPSA* Haemophilus influenzae type b polysaccharide, 1987 to 1989 BayGam Human immunoglobulin BayHep B Hepatitis Bimmune globulin (human Bay Rab Rabies immune globulin aver Corporation (U.S. BayTet Tetanus immune globulin (human aver Corporation (U.S.

Countries

Botulism immune globulin (nota vaccine BIK (Japan Bimmugen epatitis B (recombinant, adsorbed, Chemo-Sero-Therapeutic Resh east derived inst (Japan FIG. 1 U.S. Patent May 6, 2014 Sheet 2 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) ae

Anthrax (adsorbed) BPT (U.S.) Biviraten Berna Measles, mumps (live) BER (Switzerland) Botulinum antitoxin (for U.S..mil military use ) C.D.T.C Diphtheria, tetanus (pediatric, adsorbed) Commonwealth (Australia Celluvax Pertussis (acellular CHR (tal ertiva Diphtheria, tetanus, (acellular) pertussis

S.D.P.T. ..T. Bis Rudivax Di Te Per Pol Impfstoff i-Te-Pol if-Tet-All Diphtheria, tetanus FTAVAX Diphtheria, tetanus, polio D Te Anatoxal itOxim ouble Aniaen B.l. Diphtheria, tetanus Bengal immunity Co. (India ?yVax

X Diphtheria, tetanus (for pediatric use) WYE (U.S. DT TAB Diphtheria, tetanus, Salmonella typhi, AVP (France) Paratyphi A & B TaP (generic Diphtheria, tetanus, (aCellular) pertussis AVP, WYE, GSK (U.S.

Measles, rubella (live) eIPV

Rubella (live Influenza WYE (U.S.) FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 3 of 21 US 8,715,677 B2

Product Or trade Antigen(s) Manufacturer (country) ae Fuad, Agrippal-S Influenza CHR (Ital FluMist influenza (live, attenuated, intranasal) MED (U.S. Fluoqen Influenza PD (U.S. Fluvirin influenza EVN (U.S.

Genevac BPasteur HepatitisB

Hexavac Diphtheria, tetanus, pertussis, polio, AVP (Europe) hepatitis B, Hib HibTITER Haemophilus influenzae type b (HbOC) WYE (U.S. Hinkuys karokoe Pertussis (adsorbed) Natl. Public Health Institute Finland

Humotet-antiTetanus Hyper-Tet (now called Tetanus immune globulin Bayer Corporation (U.S.) "BayTet" Polio (inactivated Statens Seruminstitut (Denmark Immune Globulin Broad-spectrum immune globulins MA, BPT, New York Blood Ctr, Intramuscular (Human Bayer Corporation, CEN (U.S. Imogam Rabies - HT Rabies immune alobulin AVP (U.S. AVP (U.S. movax Parotiditis Mumps AVP (France) imovax Polio Polio AVP (France) imovax Sarampion Measles AVP (France) moVax D.T. Diphtheria, tetanus F.G. 1 (COnt.) U.S. Patent May 6, 2014 Sheet 4 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country)

name

movax R.O.R. Measles, rubella, mumps (live) VP (France) movax Rubeola Measles AVP (International) movax Mumps Mumps

( movax Rabies I.M Rabies vaccine (HDCV) AVP (U.S.) Infanrix Diphtheria, tetanus, (acellular) GSK (Belgium, U.S.) pertUSSis pad TP Tetanus, polio AVP (France) POL Polio (enhanced potency, AVP (U.S.)

inactivated

PV Polio (inactivated ) General term for inactivated Dolio Vaccine

stivaC Influenza - JE-VAX Japanese encephalitis AVP (U.S.)

Dubbelwaccin Finland

Norwa

Institute (Switzerland

Central Pub Health Lab (Finland CHIR (Italy) Measles, rubella ((Five) 4/74 to 6/78 DowlPitneyMoore (U.S.) Lirugen Measles AVPant) Measles (live) 2165 to 6/78 Dow (U.S.) LM - 3 RIT Measles, mumps, rubella (live) Dong Shin Pharm (Korea) LM-2 RIT Measles, mumps (live) Dong Shin Pharm (Korea) LTEANAS muna Tetanus (adsorbed lmuna S.p. (Slovakia LYMErix * GSK (U.S.) Measles (live, attenuated) IVIRK (U.S.) Rubella (live) MRK (U.S.)

Masern-mofstoff SSW Measles (live Measles Vaccine DK3* Measles (live) 1964 to 1972 FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 5 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) ae Measles Measles (inactivated) 1963 to 1966 Eli Lilly (U.S .) Measles (live) 12/64 to 1974 MenCevax A MenindOCOCCUS (polysaccharide) (Group A) SmithKline/RT (Belgium Meningitec Meningococcus (coniugate) (Group C WYE (U.K., Australia Menomune-A/C/YW-Meningococcus (polysaccharide) AVP (U.S.) 135 Groups A, C, Y, W435 Menpovax 4 Meningococcus (polysaccharide) (Groups A CHIR (Italy) & C Menpovax A+C ( Meruvax * .S.) Meruvax .S.) Mevilin-L* MMRX MMR (aeneric) * Measles, mumps, rubella (live) 4/74 to 61.78 (U.S.U.S.) Moniarix Mopavac Sevac Measles, mumps attenuated Institute of Sera and vaccines live, Czechoslovakia MOPVX Polio (live, Sabin, monovalent types , , Ill) WYE (U.S. Morbilwax Measles (live, attenuated) CHIR (Italy) Morubel ( Moruman Berne Measles immunglobulin ( MoruDar Movivac Measles Clive, attenuated) M-R VAX* Mumaten Berne BER (SWitzerland Mumps (aeneric) * Mumpsumps (live)474(live) 4,74 to 61.78 Mumps (generic) M (U.S. Mumps (generic) M E Mumpsvax * Mutagrip Influenza Nabi-HB Hepatitis B immune (lobulin

Nothaw CHI (Ital OmniHIB* Haemophilus influenzae type b (PRP-T GSK, AVP (U.S.

OPV General term for Oral polio vaccine FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 6 of 21 US 8,715,677 B2

Product or trade Manufacturer (country) name Orimune olio vaccine (oral, trivalent) U. S.) ariorix umps (live) SmithKline/RT (Belgium)( PavivaC-Sevac Mumps (live) Institute of Immunology Croatia PCV, PCV7 General term for pneumococcal Coniudate (7-Valent Pediarix Diphtheria, tetanus, (acellular) pertussis, GSK (U.S .) hepatitis B, PW edwaxHIB Haemophilus influenzae type b (PRP-OMP) MRK (U.S

Diphtheria, tetanus, (acellular) pertussis, AVP (Ca(Canada) entaCel Diphtheria,iphtheria, tetanus, pertussis, polio, Hib A VP (Canada entaCOC D iphtheria, tetanus, pertUSsis, polio, Hib ENTACt-HB iphtheria, tetanus, pertussis, polio, Hib entaVaC Einiphtheria, tetanus, Epertussis, polio, Hib e W al e nt e Diphtheria, tetanus, pertussis, hepatitis B, ttaa Hi b fizer Vax-Measles K* Measleseasles (inactivated)3763(inactivated) 3/63 to 1970 Pfizer (U.S fizerVax-Measles L* easles (live) 2.65 to 1970 Pfizer (U.S. uSerix easles, mumps, rubella neumoVax 23 neumococcal (polysaccharide) RK (U.S.) NU-IMUNE 23 * EEneumoCOCCal (polysaccharide EE (U.S. - OLIACe, D iphtheria, tetanus, pertussis, polio, HB AVP (Ardentina P P V, P P W2 3 eneral term for pneumococcal olysaccharide (23-valent Waf neumococcal (7-valent, Conjugate) E (U.S.)

riorix easles, mumps, rubella (live K (U.K. OHBTX aemophilus influenzae type b (PRP-D AVP (U.S. RP-OMP hemical abbreviation for PedwaxHIB Fl RP-T hemical abbreviation for ACtHB Fl urivax * olio (inactivated) 1956 to 1965

UADRACe D iphtheria, tetanus, pertussis, polio

UADRACe/Hibest iphtheria, tetanus, pertussis, polio, Hib uadriaen gigsTP + Dolio (1959-1968 pelo. He uatro-Virelon iphtheria, tetanus, polio uintuple Diphtheria,iphtheria, tetanus, pertussis, Hib, Polio SEE-HB Vaccine epatitis B (recombinant F.G. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 7 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) ae -VAC RA2713 RabAVert Rabies (PCEC ReCombivax HB epatitis B (recombinant) Respigam, RSV-IVIG Respiratory syncytial virus immune globulin IMEDI (U.S.) not a vaccine RIG (deneric abies immune qlobulin ayer Corporation, AVP (U.S.

imevax easles (live SmithKline/RTmithKline/RT (Belgium)(Belgium imparix M S mithKline/RIT T - LM-2 T - LM-3 otaShield, RRV-TV* E (U.S.) OUV3X VP (FranceER ubeaten Berna R B

ubella (generic) R P ubellowaC R C HIR (Germany) ubeloqen P D (U.S. ubeoVax RK (U.S.) Udi-ROUVax easles, rubella (live) P (France) udivaX ubella (live, attenuated P (France VA (generic) abies Vaccine adsorbed (U.S.) S 3 bi n eneralGee term for oral (live) polio vaccine Sahia Polio (live, oral) Multiple manufacturers Salk General term for injectable (inactivated)

pO O Va CC e andovac erobacterin ertussis - 1945 to 1954 MRK (U.S.) iTriple Antigen iphtheria, tetanus, pertussis Serum Institute (India) tamaril EGellow fever (live, attenuated AVP (France) Synagis (palizivumab) R eS p r 3. tO ry S y Cyti al W S m U e g O b ul M EDI (U.S.S.) not a waCCine Polio Tet a n u S toxO id,d polio

T.A.B. ) - Institute Pasteur (Tunisia) - Pharmaceutical Industries Corp. (Burma FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 8 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) 3. T-Immun etanus (adsorbed T d (Generic etanus, diphtheria (adult formulation AVP,BPAVP, BP (U.S.(US) e/Vac/Ptap etanus e Anatoxal etanus BER (Europe elvacliptap etanuS P etagrip etanus, influenza AVP (France) e d Web Sachsisches Serumwerk fluid, German etamWn Bioclon, S.A. De C.V. (Mexico etanol etanus (adsorbed) CHR (Germany) Orb atS SSSW W 33. U SSS ( dO S2Od r bS eO Web Sachsisches Serumwerk d)rb German Tetavax etanus (adsorbed) A VP (France) etracoq 05 iphtheria, tetanus, pertussis, polio AVP (France) etract-HB iphtheria, tetanus, pertussis, Hib etramune * iphtheria, tetanus, pertussis, Hib WYE (U.S.) et ra W a X X Diphtheria, tetanus, pertussis, polio -1959 MRK (U.S.) to 1965 ice BCG acilius Calmette-Gudrin vaccine (for TB OTC (U.S.

etanus immune globulin (generic) Bayer Corporation (U.S.) TOPV Trivalent oral polio vaccine Multiple manufacturers and Countries itifica yphoid and paratyphoid H resivac LVOpholized easles, mumps, rubella S riace iphtheria, tetanus, (aCellular) pertussis F riaCelluVax iphtheria, tetanus, (aCellular) pertussis riHBit iphtheria, tetanus, (aCellular) pertussis, Hib ri-Immunol Diphtheria,iphtheria, tetanus, pertussis WY E (U. S rimOVax easles, mumps, rubella (live) AVP (France) rinivac * iphtheria, tetanus, pertussis - 1952 to 1964 MRK (U.S. ripace? iphtheria, tetanus, (acellular) pertussis F ripedia iphtheria, tetanus, (aCellular) pertussis AVP (U.S. Triple antigen iphtheria, tetanus, pertussis - Chowgule & Co. (India) - CSL Limited (Australia riple Sabin olio (live, oral) riple Ea.iphtheria, tetanus, pertussis FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 9 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) 28

riple Viral Measles, mumps, rubella rivacuna Leti Diphtheria, tetanus (adsorbed), pertussis Laboratory Leti (Spain rivaX Diphtheria, tetanus (plain), pertuSSis Wellcome (U.K. ivaX-ad D phtheria, tetanus (adsorbed), pertussis - EVN (UK) - Wellcome (UK rivax-Hib

rivb Diphtheria, tetanus, pertussis riviraten rivivac 8 easles, mumps, rubella (live, attenuated)institute of Sera &Vaccines Czechoslovakia Tetanus toxoid (oeneric AVP (U.S. WW CCC inS Tetanus toxoid (adsorbed F USSitrupin Forte Pertussis Staatliches Institut (German Twinrix Hepatitis A & B (adult formulation Hepatitis A & B (pediatric formulation 2la (Vivotif Berna Tuberculosis (BCG

pherix Ohim Vi (VCPS phoid Vaccine * phopara-typhoidique Typhoid and paratyphoid VA-Mengoc-BC Meningococcal (Groups B & C) Finlay Vacunas y Sueros Centro de Investidation (Cuba accin Difteric Adsorbit Diphtheria toxoid (adsorbed Cantacuzino institute (Romania Y at Diphtheria, tetanus (adsorbed) Cantacuzino Institute (Romania) Dift Cne cie O T et a C VaWa C Cl fXn U CJ mOU MbiO O bi o U r M e a Sl eS (live) MOSCOW Research Institute Vivum Russia acina Triplice Viral M acina Triplice D

acina DUpla D aksin CaCar S mallpox F aksin Serap aksin Campak Kerig Measles (live, attenuated aksin Kotipa holera, typhoid and paratyphoid A, B & CPerum Bio Farina (Indonesia) FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 10 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) ae

easles, mumps (live Institute of Immunology (Croatia epatitis A (inactivated) MRK (U.S. ) Varicellon aricella Zoster immunoglobulin B ehringwerke Aktienqesellischaft (German Varie S mallpox (lyophilized) Institute of Sera and Vaccine Czechoslovakia Varilrix V aricella (live, Oka strain) G SK (Australia, Belgium ) aricella (live) MRK U.S. V aemophilus influenzae type b CHRC (Ital V ertussis (adsorbed) AVP (France ) V nfluenza

V umps (live CHR (tal V iphtheria, tetanus CantaCuzino institute (Romania VDA Vaccin Difteric Diphtheria Cantacuzino Institute (Romaniaia) Adsorbit iCPs (Typhim Vi) Typhoid (inactivated, injectable AV P U S VG Variola (smallpox) immune Distributed by CDC alobulin (not a vaccine

Wielon T 20 Polio (live, oral, trivalent) Behringserke Aktiengesellischaft German

Virovac Massling, Measles, mumps, rubella Perotid, Rubella V ivotif Berna (TV21a phoid (Oral, live BER (Switzerland VT (Vacina Triplice iphtheria, tetanus, pertussis Instituto Butantan (Brazil VTV (Vacina Triplice Measles, mumps, rubella Viral

easles (live, attenuated Cantucuzino institute (Romania) VZIG aricella zoster immune globulin MA (U.S.) eneric Weltrivax trivalente Diphtheria, tetanus, pertussis Y Yellow fever A

Z Diphtheria anti-toxin Zaantite Tetanus anti-toxin Zaditeadvax Diphtheria, tetanus Zaditewax Diphtheria, tetanus

Zamevax A+C Mening0COCCuS (polysaccharide, Inst, of immunology (Croatia) roups A & C Z M easles (live FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 11 of 21 US 8,715,677 B2

Product or trade Antigen(s) Manufacturer (country) are

FIG. 1 (Cont.) U.S. Patent May 6, 2014 Sheet 12 of 21 US 8,715,677 B2

ACTIMID"

EEE H H CD25-CELLS DMSO 0.01 uM 0.1 uM 1 uM 10 uM ONLY FG.2A

LENOLIDOMIDE

CD25- DMSO 0.001 uM 0.01 uM 0.1 uM 1 uM 10 uM ONLY FIG.2B

THALDOMIDE

CELLS CONTROL FG.2C U.S. Patent May 6, 2014 Sheet 13 of 21 US 8,715,677 B2

DMSO CONTROL TESTED CELLS

100 1 0 1 102 103 10 100 10 102 10 104 100 101 102 10 104 FoxP3 FITC FoxP3 FITC FoxP3 FITC FIG.3A FIG.3B FIG.3C

LENOLIDOMIDE 14M LENOLIDOMIDE 0.01.pl. M 10

10 O ". 2 f 3. 10 4 FoxP3 FITC FG3D FIG.3E

THALIDOMIDE 1.M

100 0 1 102 10 104 100 10 102 10 10 FoxP3 FITC FoxP3 FITC FIG.3F FIG.3G U.S. Patent May 6, 2014 Sheet 14 of 21 US 8,715,677 B2

TITT

O

mH

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rh no No on CN v- v UGOO GOOO 3)NISS3X3 STO 700 % U.S. Patent May 6, 2014 Sheet 15 of 21 US 8,715,677 B2

G/ 09

0 0 09 STO WIBO WWWW) 24 STO WIBO WNWW) 2. U.S. Patent May 6, 2014 Sheet 16 of 21 US 8,715,677 B2

U d - N

s s

s

O O O O O O TO O f no on es CA 32M) O- O 9Sas S2 st Luesc 2555e s N 8

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s S& s

O O O O Cd NO can U.S. Patent May 6, 2014 Sheet 17 of 21 US 8,715,677 B2

l 2 - N

e G ce S25 O N CO Deas CD 2 d 2 S2H

O O C C Co O Od o o f to ?o n) on CN v- v MD 32V - O Ol s D25 st Lu 9 DS555 a NO &

solo: 2a

Cde

Ol O co C CO > as CD U.S. Patent May 6, 2014 Sheet 18 of 21 US 8,715,677 B2

OCD3 ALONE aCD3 + ACTIMIDTM

m O 45.8

yo FITC FIG.7F

IPP + ACTIMIDTM s U.S. Patent May 6, 2014 Sheet 19 of 21 US 8,715,677 B2

RPM CELLS RPM CELLS WITHOUT PAMIDRONATE - WITHOUT PAMIDRONATE ACTIMIDTM

S S. 0.5:1 05:1 IFN-GAMMA FITC IFN-GAMMA EXPRESSION FIG.8A FIG.8B

RPM CELLS RPM CELLS WITHOUT PAMIDRONATE - WITHOUT PAMIDRONATE ACTIMDTM

3 a Cld O 1:1 a 1:1 ol 2 2 d IFN-GAMMA EXPRESSION IFN-GAMMA EXPRESSION FIG.8C FIG.8D

RPM CELLS RPM CELLS WITHOUT PAMIDRONATE + WITHOUT PAMIDRONATE ACTIMIDTM

Cda aO 2:1 all 2:1 a

C- Ol IFN-GAMMA EXPRESSION IFN-GAMMA EXPRESSION FIG.8E FIG.8F U.S. Patent May 6, 2014 Sheet 20 of 21 US 8,715,677 B2

RPM CELLS RPM CELLS WITH PAMIDRONATE -

WITH PAMIDRONATE ACTMDTM 3 0.5:1 O 2

IFN-GAMMA EXPRESSION IFN-GAMMA EXPRESSION FIG.8G FG.8H RPM CELLS RPM CELS WITH PAMIDRONATE + ACTIMDTM

WITH PAMIDRONATE

3 1:1 Ol 1:1 2 IFN-GAMMA EXPRESSION IFN-GAMMA EXPRESSION FIG.8 FIG.8J RPM CELS RPM CELLS WITH PAMIDRONATE - ACTIMIDTM WITH PAMIDRONATE

S 2:1 s 2:1

IFN-GAMMA EXPRESSION IFN-GAMMA EXPRESSION FG.8K FIG.8L U.S. Patent May 6, 2014 Sheet 21 of 21 US 8,715,677 B2

-0- US -- ACTIMDTM -A - PAM -O-PAM/ACT

10:1 5:1 2.5:1 1.25:1 FIG.9A -0- US -- ACTIMIDTM -A PAM

-O-PAM/ACT US 8,715,677 B2 1. 2 IMMUNOLOGICALUSES OF Many experimental adjuvants have advanced to clinical trials IMMUNOMODULATORY COMPOUNDS FOR since the development of Alum, and some have demonstrated VACCNE AND ANT-INFECTIOUS DISEASE high potency but have proven too toxic for therapeutic use in THERAPY humans. Thus, an on-going need exists for safe and potent adjuvants. This application claims priority to U.S. provisional appli Cancer vaccines have been a subject of much attention. cation No. 60/712,823, filed Sep. 1, 2005, the entirety of Recently, there appears to be an emerging consensus that which is incorporated herein by reference. cancer vaccines are less likely to be successful in the context of high tumor buden/load (see, e.g., Nature Medicine Com 1. FIELD OF THE INVENTION 10 mentary, 10(12): 1278 (2004) and Cancer Immunol. Immu nother, 53(10): 844-54 (2004)). This is attributed to effective This invention relates to the use of certain non-peptide tumor-mediated immune Suppression due to the Secretion of Small molecules known as immunomodulatory compounds IL-10, TGF-b, and PGE-2, among others. or IMiDS(R) in various immunological applications, in particu On the other hand, recent evidence Suggests that immedi 15 ately after tumor resection or ablation, there is leakage of lar as vaccine adjuvants, particularly anticancer vaccine adju tumor cells in the peripheral blood. Therefore, the presence of vants. The invention also relates to the uses of IMiDs(R in tumor antigen in the context of low tumor burden, without combination with vaccines to treat or prevent cancer or infec associated immune Suppression, may enable re-priming of tious diseases. This invention also relates to other various uses the immune response. Thus, a need exists for an agent that of immunomodulatory compounds such as reduction or promotes the long-term anti-tumor immunity, possibly desensitization of allergic reactions. through Th1 type cellular immune responses. 2.2 Regulatory T Cells (T. Cells) 2. BACKGROUND T cells refer to a population of specialized T cells that express CD4 and CD25. T cells are exceptional in that their 2.1 Vaccines 25 main function appears to be suppression of function of other Vaccines have traditionally consisted of live attenuated cells. In this regard, T cells are also referred to as "suppres pathogens, whole inactivated organisms or inactivated toxins. sor cells.” It has been reported that a further defining charac In many cases, these approaches have been successful at teristic of T cells is their expression of the transcription inducing immune protection based on antibody mediated factor Foxp3. responses. However, certain pathogens, e.g., HIV. HCV. TB, 30 Due to the variety of their effect, T cells have been a and malaria, require the induction of cell-mediated immunity subject of a great deal of interest. It has been reported that T. (CMI). Non-live vaccines have generally proven ineffective cells may influence the outcome of infection, autoimmunity, in producing CMI. In addition, although live vaccines may transplantation, cancer and allergy. It has been suggested that induce CMI, Some live attenuated vaccines may cause disease the modes of suppression employed by T cells range from in immunosuppressed Subjects. As a result of these problems, 35 the cytokines IL-10 and TGF-B to cell-cell contact via the several new approaches to vaccine development have inhibitory molecule CTLA-4. Recently, it has been reported emerged, such as recombinant protein subunits, synthetic that dendritic cells (DC) may induce the activation and pro peptides, protein polysaccharide conjugates, and plasmid liferation of T cells, although DC are recognized as pow DNA. While these new approaches may offer important erful activators of immune response due, in part, to their safety advantages, a general problem is that vaccines alone 40 potency as antigen presentation cells (APC). See Yamazaki et are often poorly immunogenic. Therefore, there is a continu al., J. Exp. Med., 198: 235 (2003). ing need for the development of potent and safe adjuvants that Generally, it is believed that T cells suppress the immu can be used in vaccine formulations to enhance their immu nity of the host, and thus preventing an immunogen (e.g., a nogenicity. See, e.g., Edelman, Molecular Biotech. 21: 129 vaccine) from invoking effective immune response in the 148 (2002); O'Hagan et al., Biomolecular Engineering, 18: 45 host. On the other hand, the absence of T cells can lead to 69-85 (2001); Singh et al., Pharm. Res. 19(6): 715-28 (2000) an outburst of immune response, often resulting in inflamma for detailed review of the state of the art in vaccine develop tion or autoimmunity. Therefore, to maximize the immunity ment. acquired from an immunogen, a balance needs to be achieved Traditionally, the immunogenicity of a vaccine formula with regard to the level or functionality of T cells. tion has been improved by injecting it in a formulation that 50 2.3 Gamma Delta (yo) T Cells includes an adjuvant. Immunological adjuvants were initially Human T cells bearing the Yö T cell receptor represent a described by Ramon (1924, Ann. Inst. Pasteur, 38: 1) “as unique lymphocyte population with characteristic tissue dis Substances used in combination with a specific antigen that tribution, being present in organized lymphoid tissue as well produced a more robust immune response than the antigen as skin- and gut-associated lymphoid tissue. Yö T cells are alone.” A wide variety of substances, both biological and 55 activated in a non-MHC restricted manner by small phospho synthetic, have been used as adjuvants. However, despite rylated non-peptidic metabolites, including the prototypic extensive evaluation of a large number of candidates over ligand isopentenyl pyrophosphate (IPP). Some Yo T cell many years, the only adjuvants currently approved by the ligands are microbial intermediates from the farnseylpyro U.S. Food and Drug administration are aluminum-based min phosphate synthesis pathway, which is ubiquitous and essen erals (generically called Alum). Alum has a debatable safety 60 tial for cell Survival. This unique antigen specificity has been record (see, e.g., Malakoff, Science, 2000, 288: 1323), and Suggested to be best Suited for activation of sentinel cells comparative studies show that it is a weak adjuvant for anti independently of antigens derived from individual microbes body induction to protein subunits and a poor adjuvant for (De Libero, Immunology Today, 18: 22-26 (1997)). Recent CMI. Moreover, Alum adjuvants can induce IgE antibody data Suggest that yo T cells play a role in tumor Surveillance, response and have been associated with allergic reactions in 65 for example, of spontaneous B cell lymphomas (Street etal, J some subjects (see, e.g., Gupta et al., 1998, Drug Deliv Rev. Exp Med, 199: 879-884(2004)), since these cells have been 32: 155-72; Relyveld et al., 1998, Vaccine 16: 1016-23). shown to recognize intermediates of the melavonate pathway, US 8,715,677 B2 3 4 an essential pathway leading to cholesterol biosynthesis Pharmaceutical compositions, dosing regimen, and com (Gober et al. J Exp Med, 197: 163-168 (2003)). These yö T bination therapies using an immunomodulatory compound cell tumorligands can be enhanced by treatment with amino are also encompassed by the invention. bisphosphonates (nitrogen containing bisphosphoante drugs include pamidronate and Zolodronate and are used in 4. BRIEF DESCRIPTION OF FIGURES myeloma treatment), Suggesting that pretreatment with these drugs could sensitize tumor cells to Yö T cell-mediated kill FIG. 1 is a non-limiting list of vaccines that may be used in ing. Yö T cells may also be able to augment anti-tumor immu connection with methods of this invention. nity by enhancing dendritic cell maturation (Ismailietal, Clin FIG. 2A illustrates the effects of 4-(amino)-2-(2,6-dioxo Immunol, 103:296-302 (2002)). 10 (3-piperidyl))-isoindoline-1,3-dione on the function of regu In non-cancer settings, Yö T cells play a role in protection latory T cells. from viral infection, e.g., West Nile virus (Wang et al., J FIG. 2B illustrates the effects of 3-(4-amino-1-oxo-1,3- Immunol, 171: 2524-2531(2003)). Also, intraepithelial y&T dihydro-isoindol-2-yl)-piperidine-2,6-dione on the function cells play a protective role in intestinal inflammation (Chenet 15 of regulatory T cells. al, Proc. Natl. Acad. Sci. U.S.A., 99: 14338-14343 (2002); FIG. 2C illustrates the effects of thalidomide on the func and Inagaki-Ohara etal, J Immunol, 173: 1390-1398 (2004)). tion of regulatory T cells. Furthermore, Yö TCR-bearing dendritic epidermal cells play FIG. 3 illustrates the effects of immunomodulatory com a role in wound repair (Jameson et al. Science, 296: 747-749 pounds of the invention and thalidomide on the expression of (2002)). T. marker Foxp3 (FIG. 3A-DMSO control; FIG. 3B-1 uM 2.4 Immunomodulatory Compounds 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; A number of studies have been conducted with the aim of FIG. 3C-0.01 uM 4-(amino)-2-(2,6-dioxo(3-piperidyl))- providing compounds that can safely and effectively be used isoindoline-1,3-dione: FIG. 3D-1 uM3-(4-amino-1-oxo-1,3- to treat diseases associated with abnormal production of dihydro-isoindol-2-yl)-piperidine-2,6-dione; FIG. 3E-0.01 TNF-C. See, e.g., Marriott, J. B., eral. Expert Opin. Biol. 25 uM 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperi Ther: 1(4): 1-8 (2001); G. W. Muller, et al., Journal of Medici dine-2,6-dione: FIG.3F-1 uMthalidomide; and FIG.3G-0.01 nal Chemistry, 39(17): 3238-3240 (1996); and G. W. Muller, uM thalidomide). et al., Bioorganic & Medicinal Chemistry Letters, 8: 2669 FIG. 4 illustrates the effects of 4-(amino)-2-(2,6-dioxo(3- 2674 (1998). Some studies have focused on a group of com piperidyl))-isoindoline-1,3-dione and 3-(4-amino-1-oxo-1, pounds selected for their capacity to potently inhibit TNF-C. 30 3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on the num production by LPS stimulated PBMC. L. G. Corral, et al., ber of regulatory T cells. Ann. Rheum. Dis., 58 (suppl I): 1107-1113 (1999). These FIG. 5A illustrates the effects of 3-(4-amino-1-oxo-1,3- compounds, which are referred to as IMiDs(R (Celgene Cor dihydro-isoindol-2-yl)-piperidine-2,6-dione on the expres poration) or Immunomodulatory Drugs, show not only potent 35 sion of Yö T cells in PMBC activated with IL-2 and IPP inhibition of TNF-C. but also marked inhibition of LPS FIG. 5B illustrates the effects of 4-(amino)-2-(2,6-dioxo induced monocyte IL1B and IL12 production. LPS induced (3-piperidyl))-isoindoline-1,3-dione on the expression of Yö IL6 is also inhibited by immunomodulatory compounds, T cells in PMBC activated with IL-2 and IPP. albeit partially. These compounds are potent stimulators of FIG. 5C illustrates the effects of 3-(4-amino-1-oxo-1,3- LPS induced IL10. Id. Particular examples of IMiDs(R) 40 dihydro-isoindol-2-yl)-piperidine-2,6-dione on the expres include, but are not limited to, the substituted 2-(2,6-dioxopi Sion of NKG2D in PMBC activated with IL-2 and IPP. peridin-3-yl)phthalimides and substituted 2-(2,6-dioxopip FIG.5D illustrates the effects of 4-(amino)-2-(2,6-dioxo eridin-3-yl)-1-oxoisoindoles described and claimed in U.S. (3-piperidyl))-isoindoline-1,3-dione on the expression of Pat. Nos. 6,281.230 and 6,316,471, both to G. W. Muller, etal. NKG2D in PMBC activated with IL-2 and IPP. 45 FIG. 6 illustrates the effects of 4-(amino)-2-(2,6-dioxo(3- 3. SUMMARY OF THE INVENTION piperidyl))-isoindoline-1,3-dione on the apoptosis in Yö T cells on day 4 (FIG. 6A), day 5 (FIG. 6B), day 6 (FIG. 6C), This invention relates to immunological and other uses of and day 7 (FIG. 6D) after the treatment. IMiDs(R). In particular, this invention encompasses the use of FIGS. 7A and 7B illustrate the comparison of IFN-Y pro IMiDS(R) in combination of an immunogen (e.g., a vaccine) in 50 duction in cells treated with CCD3 alone (FIG. 7A) and those specific dosing regimen, providing an enhanced immune treated with C.CD3 and 4-(amino)-2-(2,6-dioxo(3-pip responses from the immunogen as compared to the responses eridyl))-isoindoline-1,3-dione (FIG. 7B) in freshly prepared obtained when IMiDs(R) are not used. yö T cells. This invention also encompasses methods of reducing or FIGS. 7C and 7D illustrate the comparison of TNF-C. pro 55 duction in cells treated with CCD3 alone (FIG.7C) and those inhibiting proliferation or immuno-Suppressive activity of treated with C.CD3 and 4-(amino)-2-(2,6-dioxo(3-pip T cells comprising contacting the T cell with an immu eridyl))-isoindoline-1,3-dione (FIG. 7D) in freshly prepared nomodulatory compound of the invention. yö T cells. This invention also encompasses methods of eliciting an FIGS. 7E and 7F illustrate the comparison of IFN-Y pro enhanced immune response from an immunogen. This inven 60 duction in cells treated with IPP alone (FIG. 7E) and those tion also encompasses methods of eliciting a reduced allergic treated with IPP and 4-(amino)-2-(2,6-dioxo(3-piperidyl))- response from an allergen. The methods comprise adminis isoindoline-1,3-dione (FIG.7F) in freshly prepared YöT cells. tering an immunomodulatory compound of the invention to a FIGS. 7G and 7H illustrate the comparison of TNF-C. pro Subject prior to the exposure of the Subject to an immunogen duction in cells treated with IPP alone (FIG. 7G) and those or an allergen. It should be noted that IMiDs(R) can be addi 65 treated with IPP and 4-(amino)-2-(2,6-dioxo(3-piperidyl))- tionally administered during and/or after the Subject’s expo isoindoline-1,3-dione (FIG. 7H) in freshly prepared Yö T Sure to the immunogen or allergen. cells. US 8,715,677 B2 5 6 FIG. 8A illustrates the IFN-Y production in response to host. In addition, without being limited by a particular theory, co-culture with RPMI cells with (tumor:yö T=0.5:1 ratio) immunomodulatory compounds of the invention augment the without preincubation with pamidronate. innate anti-tumor activity of Yö T cells. Furthermore, without FIG. 8B illustrates the IFN-Y production in response to being limited by a particular theory, it is also believed that the co-culture with RPMI cells with (tumor:yö T=0.5:1 ratio) immunomodulatory compounds of the invention promote without preincubation with pamidronate, but with treatment Successful Th1 type cellular immune responses necessary for with 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3- efficient long-term anti-tumor activity, thereby delaying or dione. prevent tumor recurrence. FIG. 8C illustrates the IFN-Y production in response to Accordingly, this invention encompasses methods of co-culture with RPMI cells with (tumor:yö T=1:1 ratio) with 10 reducing or inhibiting proliferation and/or immuno-Suppres out preincubation with pamidronate. sive activity of regulatory T cells comprising contacting the FIG. 8D illustrates the IFN-Y production in response to regulatory T cells with an immunomodulatory compound of co-culture with RPMI cells with (tumor:yö T=1:1 ratio) with the invention for a time sufficient for the reduction or the out preincubation with pamidronate, but with treatment with inhibition of proliferation and/or immuno-Suppressive activ 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. 15 ity. FIG. 8E illustrates the IFN-Y production in response to As used herein, and unless otherwise specified, the term co-culture with RPMI cells with (tumor:yö T-2:1 ratio) with “reducing or inhibiting the proliferation, when used in con out preincubation with pamidronate. nection with regulatory T cells, means that the number of FIG. 8F illustrates the IFN-Y production in response to regulatory T cells in a cell culture or a host treated with an co-culture with RPMI cells with (tumor:yö T-2:1 ratio) with immunomodulatory compound of the invention is less than out preincubation with pamidronate, but with treatment with the number of regulatory T cells in a cell culture or a host 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. without the treatment with an immunomodulatory compound FIG. 8G illustrates the IFN-Y production in response to of the invention, as determined by methods known in the art, co-culture with RPMI cells with (tumor:yö T=0.5:1 ratio) some of which are described herein. A typical method with preincubation with pamidronate. 25 involves the staining of a marker and analysis of the stain FIG. 8H illustrates the IFN-Y production in response to using, for example, FACS analysis. Preferably, reduced pro co-culture with RPMI cells with (tumor:yö T=0.5:1 ratio) liferation means the number of T cells in immunomodulatory with preincubation with pamidronate and treatment with compound treated culture or host is about 10%, 20%, 30%, 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. 50%, 70%, or 90% or less than those in the culture or host FIG. 8I illustrates the IFN-Y production in response to 30 without such treatment. co-culture with RPMI cells with (tumor:yo T=1:1 ratio) with As used herein, and unless otherwise specified, the term preincubation with pamidronate. “reducing or inhibiting the immuno-suppressive activity.” FIG. 8J illustrates the IFN-Y production in response to when used in connection with regulatory T cells, means that co-culture with RPMI cells with (tumor:yo T=1:1 ratio) with the immuno-Suppressive activity of regulatory T cells, when preincubation with pamidronate and treatment with 35 treated or contacted with an immunomodulatory compound 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. of the invention, is lower than those without such treatment or FIG. 8K illustrates the IFN-Y production in response to contact. The immuno-Suppressive activity can be determined co-culture with RPMI cells with (tumor:yo T=2:1 ratio) with using methods known in the art including those described preincubation with pamidronate. herein. Typically, the immuno-Suppressive activity of regula FIG. 8L illustrates the IFN-Y production in response to 40 tory T cells can be assessed by monitoring the proliferation of co-culture with RPMI cells with (tumor:yo T=2:1 ratio) with for example, anti-CD3 stimulated CD25-cells in response to preincubation with pamidronate and treatment with TCR signal. Preferably, reduced immuno-suppressive activ 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. ity means the activity of regulatory T cells treated with an FIG.9A illustrates the effects of immunomodulatory com immunomodulatory compound of the invention is about 10%, pounds of the invention on the cytotoxicity of Yö T cells on 45 20%, 30%, 50%, 70%, or 90% or less than the activity of those MM cell lines where the compounds are preincubated with without such treatment. tumor cells. This invention also encompasses methods of eliciting an FIG.9B illustrates the effects of immunomodulatory com enhanced immune response from an immunogen in a subject pounds of the invention on the cytotoxicity of Yö T cells on (e.g., human) comprising administering to the Subject an MM cell lines where the compounds are not preincubated 50 immunomodulatory compound of the invention prior to the with tumor cells, but are added during the chromium release administration of the immunogen to the Subject. assay only. As used herein, and unless otherwise specified, the term “immunogen' means any Substance or organism that pro 5. DETAILED DESCRIPTION OF THE Vokes an immune response (produces immunity) when intro INVENTION 55 duced to the body. In some embodiments, an immunogen can be used in therapeutic settings in a form of a vaccine. This invention is based, in part, on the inventors discovery As used herein, and unless otherwise specified, the term that pre-treatment with immunomodulatory compounds of "enhanced immune response' means that, when an immuno the invention, before the introduction of an immunogen (e.g., gen is administered in combination with an immunomodula a vaccine) results in an enhanced immune response in a host, 60 tory compound according to methods of this invention, there as determined by the experiments described herein. Without is an increased antibody formation, measured using any stan being limited by a particular theory, the invention encom dard methods known in the art or described herein, in a passes administration of an immunomodulatory compound to Subject that receives such an administration as compared to a a host, preferably prior to the introduction of an immunogen, Subject to which same amount of the immunogen alone is to enhance the function of dendritic cells as antigen presen 65 administered. As used herein, the term "administration in tation cells and/or suppress the proliferation and/or function combination with used in connection with two or more of T cells, resulting in an enhanced immune response in the therapeutic agents, means that such agents are administered US 8,715,677 B2 7 8 simultaneously, concurrently, or sequentially using the same from about 10 days to about 2 hours, from about 7 days to or different routes. Preferably, an enhanced immune response about 2 hours, from about 5 days to about 2 hours, from about means about 10%, 20%, 30%, 50%, 70%, or 100% or greater 5 days to about 1 hour, from about 1 day to about 30 minutes, increase in antibody formation. or from about 1 day to about 2 hours prior to the subjects In specific embodiments, an immunomodulatory com exposure to an allergen. pound is administered to a subject about 30 days, 20 days, 15 In other embodiments, methods of the invention further days, 12 days, 10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, comprises a second administration of an immunomodulatory or 5 hours prior to the administration of the immunogen. In compound of the invention after the Subject’s exposure to an other embodiments, an immunomodulatory compound is allergen. Without being limited by a particular theory, it is administered from about 30 days to about 5 hours, from about 10 believed that administering an immunomodulatory com 20 days to about 5 hours, from about 15 days to about 12 pound after the Subject’s exposure to an allergen can generate hours, from about 12 days to about 5 hours, from about 10 long term memory (e.g., Th1 type) immune response. In these days to about 12 hours, from about 7 days to about 12 hours, embodiments, there are at least two administrations of an from about 5 days to about 12 hours, from about 5 days to immunomodulatory compound of the invention—one pre about 1 day, from about 3 days to about 12 hours, or from 15 allergen and one post-allergen. about 3 days to about 1 day prior to the administration of an In specific embodiments, an immunomodulatory com immunogen. pound of the invention is administered to a subject about 30 In other embodiments, methods of the invention further days, 20 days, 15 days, 12 days, 10 days, 7 days, 5 days, 3 comprises a second administration of an immunomodulatory days, 1 day, 12 hours, or 5 hours after the subject’s exposure compound of the invention after the administration of an to an allergen. In other embodiments, an immunomodulatory immunogen. Without being limited by aparticular theory, it is compound of the invention is administered from about 5 believed that administering an immunomodulatory com hours to about 30 days, from about 5 hours to about 20 days, pound after the administration of an immunogen can enhance from about 12 hours to about 15 days, from about 5 hours to the immune response obtained from the immunogen by about 12 days, from about 12 hours to about 10 days, from improving antigen presentation of host cells, enhancing the 25 about 12 hours to about 7 days, from about 12 hours to about activity of T cells (e.g., C. Band Yö TCR positive), and gener 5 days, from about 1 day to about 5 days, from about 12 hours ating cytotoxic effector response and long term memory (e.g., to about 3 days, or from about 1 day to about 3 days after the Th1 type) immune response. In these embodiments, there are Subject’s exposure to an allergen. at least two administrations of an immunomodulatory com 5.1 Immunogens and Vaccines pound of the invention—one pre-immunogen and one post 30 Various immunogens may be used in connection with immunogen. methods of this invention. The immunogens are usually In specific embodiments, an immunomodulatory com administered to a subject in a form of an immunogenic com pound of the invention is administered to a subject about 30 position (e.g., a vaccine), but may be administered in any days, 20 days, 15 days, 12 days, 10 days, 7 days, 5 days, 3 form that is acceptable for use in animals, in particular, days, 1 day, 12 hours, or 5 hours after the administration of the 35 humans. immunogen. In other embodiments, an immunomodulatory 5.1.1 Immunogens compound of the invention is administered from about 5 Immunogens that may be used in the immunogenic com hours to about 30 days, from about 5 hours to about 20 days, positions include antigens from an animal, a plant, a bacteria, from about 12 hours to about 15 days, from about 5 hours to a protozoan, a parasite, a virus or a combination thereof. about 12 days, from about 12 hours to about 10 days, from 40 Immunogens may be any substance that under appropriate about 12 hours to about 7 days, from about 12 hours to about conditions results in an immune response in a subject, includ 5 days, from about 1 day to about 5 days, from about 12 hours ing, but not limited to, polypeptides, peptides, proteins, gly to about 3 days, or from about 1 day to about 3 days after the coproteins, lipids, nucleic acids (e.g., RNAS and DNAS) and administration of an immunogen. polysaccharides. In another aspect, this invention encompasses methods of 45 An immunogenic composition may comprise one or more eliciting a reduced allergic response in a Subject comprising immunogens. The amount of the immunogen used in the administering to the Subject an immunomodulatory com compositions may vary depending on the chemical nature and pound of the invention prior to the Subject’s exposure to an the potency of the immunogen. allergen. As used herein, the term "subject's exposure to Immunogens may be any viral peptide, protein, polypep allergen’ encompasses a Subject’s exposure to an allergen 50 tide, or a fragment thereof, derived from a virus. which is foreseeable (e.g., intake of food or exposure to the Immunogens used in methods of the invention may be an naturally occurring allergens), as well as allergy vaccination antigen of a pathogenic virus such as, but are not limited to: where an allergen is administered to a Subject according to a adenovirdiae (e.g., mastadenovirus and aviadenovirus), her dosing scheme over a period of time. Without being limited pesviridae (e.g., herpes simplex virus 1, herpes simplex virus by a particular theory, it is believed that immunomodulatory 55 2, herpes simplex virus 5, and herpes simplex virus 6), compounds not only preferentially induce Th1 immune leviviridae (e.g., levivirus, enterobacteria phase MS2, response, but also inhibit and/or reverse Th2 differentiation, allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvi resulting in milder, non-acute immune response to an allergen rus, avipoxvirus, capripoxvirus, leporipoxvirus, Suipoxvirus, mediated by Th1 cells. molluscipoxvirus, and entomopoxyirinae), papovaviridae In specific embodiments, an immunomodulatory com 60 (e.g., polyomavirus and papillomavirus), paramyxoviridae pound is administered to a subject about 30 days, 20 days, 15 (e.g., paramyxovirus, parainfluenza virus 1, mobillivirus days, 12 days, 10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, (e.g., measles virus), rubulavirus (e.g., mumps virus), pneu 5 hours, 2 hours, or 30 minutes prior to the subject’s exposure monovirinae (e.g., pneumovirus, human respiratory syncytial to an allergen. In other embodiments, an immunomodulatory virus), and metapneumovirus (e.g., avian pneumovirus and compound is administered from about 30 days to about 30 65 human metapneumovirus), picornaviridae (e.g., enterovirus, minutes, from about 20 days to about 1 hour, from about 15 rhinovirus, hepatovirus (e.g., human hepatitis. A virus), car days to about 1 hour, from about 12 days to about 30 minutes, diovirus, and apthovirus, reoviridae (e.g., orthoreovirus, US 8,715,677 B2 10 orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and herpesvirus type 1 glycoprotein B, and equine herpesvirus oryzavirus), retroviridae (e.g., mammalian type B retrovi type 1 glycoprotein D, antigen of bovine respiratory syncytial ruses, mammalian type C retroviruses, avian type C retrovi virus or bovine parainfluenza virus (e.g., bovine respiratory ruses, type D retrovirus group, BLV-HTLV retroviruses, len syncytial virus attachment protein (BRSVG), bovine respi tivirus (e.g. human immunodeficiency virus 1 and human ratory syncytial virus fusion protein (BRSV F), bovine res immunodeficiency virus 2), spumavirus), flaviviridae (e.g., piratory syncytial virus nucleocapsid protein (BRSV N), hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), bovine parainfluenza virus type 3 fusion protein, and the togaviridae (e.g., alphavirus, e.g., Sindbis virus) and rubivirus bovine parainfluenza virus type 3 hemagglutinin neuramini (e.g., rubella virus), rhabdoviridae (e.g., vesiculovirus, lys dase), bovine viral diarrhea virus glycoprotein 48 or glyco savirus, ephemerovirus, cytorhabdovirus, and necleorhab 10 protein 53. dovirus), arenaviridae (e.g., arenavirus, lymphocytic chori Immunogens used in methods of this invention may also be omeningitis virus, Ippy virus, and lassa virus), and a cancer antigen or a tumor antigen. Any cancer or tumor coronaviridae (e.g., coronavirus and torovirus). antigen known to one skilled in the art may be used in accor Immunogens used in methods of this invention may be an dance with the immunogenic compositions of the invention infectious disease agent including, but not limited to, influ 15 including, but not limited to, KS /4 pan-carcinoma antigen enza virus hemagglutinin (Genbank Accession No. J02132: (Perez and Walker, 1990, J. Immunol. 142: 3662-3667; Air, 1981, Proc. Natl. Acad. Sci. USA 78: 7639-7643; Newton Bumal, 1988, Hybridoma 7(4): 407-415), ovarian carcinoma et al., 1983, Virology 128: 495-501), human respiratory syn antigen (CA125) (Yu et al., 1991, Cancer Res. 51(2): 468 cytial virus Giglycoprotein (Genbank Accession No. Z33429; 475), prostatic acid phosphate (Tailoret al., 1990, Nucl. Acids Garcia et al., 1994, J. Virol.: Collins et al., 1984, Proc. Natl. Res. 18(16): 4928), prostate specific antigen (Henttu and Acad. Sci. USA 81: 7683), core protein, matrix protein or any Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2): 903 other protein of Dengue virus (Genbank Accession No. 910; Israeli et al., 1993, Cancer Res. 53: 227-230), mela M19197; Hahn et al., 1988, Virology 162: 167-180), measles noma-associated antigen p97 (Estin et al., 1989, J. Natl. Can virus hemagglutinin (Genbank Accession No. M81899; Rota cer Instit. 81 (6): 445-446), melanoma antigen gp75 et al., 1992, Virology 188: 135-142), herpes simplex virus 25 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4): 1375-1380), type 2 glycoprotein gB (Genbank Accession No. M14923; high molecular weight melanoma antigen (HMW-MAA) Bzik et al., 1986, Virology 155:322–333), poliovirus I VP1 (Natali et al., 1987, Cancer 59:55-63; Mittelman et al., 1990, (Emini et al., 1983, Nature 304:699), envelope glycoproteins J. Clin. Invest. 86: 2136-2144), prostate specific membrane of HIV I (Putney et al., 1986, Science 234: 1392-1395), antigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, hepatitis B surface antigen (Itoh et al., 1986, Nature 308: 19: 30 Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic epithelial Neurathet al., 1986, Vaccine 4:34), diptheria toxin (Audibert mucin antigen, human milk fat globule antigen, colorectal et al., 1981, Nature 289: 543), streptococcus 24M epitope tumor-associated antigens such as: CEA, TAG-72 (Yokata et (Beachey, 1985, Adv. Exp. Med. Biol. 185:193), gonococcal al., 1992, Cancer Res. 52: 3402-3408), CO17-1A (Ragnham pilin (Rothbard and Schoolnik, 1985, Adv. Exp. Med. Biol. mar et al., 1993, Int. J. Cancer 53: 751-758); GICA 19-9 185:247), pseudorabies virus g50 (gpD), pseudorabies virus 35 (Herlyn et al., 1982, J. Clin. Immunol. 2: 135), CTA-1 and II (gpB), pseudorabies virus g|II (gpC), pseudorabies virus LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et glycoprotein H. pseudorabies virus glycoprotein E, transmis al., 1994, Blood 83: 1329-1336), human B-lymphoma anti sible gastroenteritis glycoprotein 195, transmissible gastro gen-CD20 (Ref et al., 1994, Blood 83: 435-445), CD33 enteritis matrix protein, Swine rotavirus glycoprotein 38, (Sgouros et al., 1993, J Nucl. Med. 34: 422-430), melanoma Swine parvovirus capsid protein, Serpulina hydodysenteriae 40 specific antigens such as ganglioside GD2 (Saleh et al., 1993, protective antigen, bovine viral diarrhea glycoprotein 55. J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et Newcastle disease virus hemagglutinin-neuraminidase, al., 1993, Cancer Immunol. Immunother: 36: 373-380), gan Swine flu hemagglutinin, Swine flu neuraminidase, foot and glioside GM2 (Livingston et al., 1994, J. Clin. Oncol. mouth disease virus, hog cholera virus, Swine influenza virus, 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer African Swine fever virus, Mycoplasma hyopneumoniae, 45 Res. 53: 5244-5250), tumor-specific transplantation type of infectious bovine rhinotracheitis virus (e.g., infectious bovine cell-surface antigen (TSTA) such as virally-induced tumor rhinotracheitis virus glycoprotein E or glycoprotein G), or antigens including T-antigen DNA tumor viruses and Enve infectious laryngotracheitis virus (e.g., infectious laryngotra lope antigens of RNA tumor viruses, oncofetal antigen-alpha cheitis virus glycoprotein G or glycoprotein I), a glycoprotein fetoprotein such as CEA of colon, bladder tumor oncofetal of La Crosse virus (Gonzales-Scarano et al., 1982, Virology 50 antigen (Hellstrom et al., 1985, Cancer. Res. 45: 2210-2188), 120: 42), neonatal calf diarrhea virus (Matsuno and Inouye, differentiation antigen Such as human lung carcinoma antigen 1983, Infection and Immunity 39: 155), Venezuelan equine L6, L20 (Hellstromet al., 1986, Cancer Res. 46:3917-3923), encephalomyelitis virus (Mathews and Roehrig, 1982, J. antigens of fibrosarcoma, human leukemia T cell antigen Immunol. 129: 2763), punta toro virus (Dalrymple et al., Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of Immunospe 1981, in Replication of Negative Strand Viruses, Bishop and 55 cifically. 141: 1398-1403), neoglycoprotein, sphingolipids, Compans (eds.), Elsevier, NY. p. 167), murine leukemia virus breast cancer antigen such as EGFR (Epidermal growth factor (Steeves et al., 1974, J. Virol. 14:187), mouse mammary receptor), HER2 antigen (p85'), polymorphic epithelial tumor virus (Massey and Schochetman, 1981, Virology 115: mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 20), hepatitis B virus core protein and/or hepatitis B virus 17: 359), malignant human lymphocyte antigen-APO-1 Surface antigen or a fragment or derivative thereof (see, e.g., 60 (Bernhard et al., 1989, Science 245:301-304), differentiation U.K. Patent Publication No. GB 2034323A published Jun. 4, antigen (Feizi, 1985, Nature 314: 53-57) such as I antigen 1980; Ganem and Varmus, 1987, Ann. Rev. Biochem. 56:651 found in fetal erythrocytes, primary endoderm, I antigen 693: Tiolais et al., 1985, Nature 317:489-495), antigen of found in adult erythrocytes, preimplantation embryos, I (Ma) equine influenza virus or equine herpesvirus (e.g., equine found in gastric adenocarcinomas, M18, M39 found in breast influenza virus type A/Alaska 91 neuraminidase, equine 65 epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, influenza virus type A/Miami 63 neuraminidase, equine Myl, VIM-D5, D.56-22 found in colorectal cancer, TRA-1- influenza virus type A/Kentucky 81 neuraminidase equine 85 (blood group H). C14 found in colonic adenocarcinoma, US 8,715,677 B2 11 12 F3 found in lung adenocarcinoma, AH6 found in gastric rounds of replication within the host. When used as a vaccine, cancer, Y hapten, Le' found in embryonal carcinoma cells, the recombinant virus would go through limited replication TL5 (blood group A), EGF receptor found in A431 cells, E cycle(s) and induce a Sufficient level of immune response but series (blood group B) found in pancreatic cancer. FC10.2 it would not go further in the human host and cause disease. found in embryonal carcinoma cells, gastric adenocarcinoma Alternatively, inactivated (killed) virus may beformulated antigen, CO-514 (blood group Le') found in Adenocarci in accordance with the invention. Inactivated vaccine formu noma, NS-10 found in adenocarcinomas, CO-43 (blood lations may be prepared using conventional techniques to group Le”), G49 found in EGF receptor of A431 cells, MH2 “kill the chimeric viruses. Inactivated vaccines are "dead” in (blood group ALe”/Le’) found in colonic adenocarcinoma, the sense that their infectivity has been destroyed. Ideally, the 19.9 found in colon cancer, gastric cancer mucins, TsA, 10 infectivity of the virus is destroyed without affecting its found in myeloid cells, R found in melanoma, 4.2, G, immunogenicity. In order to prepare inactivated vaccines, the D1.1, OFA-1, G, OFA-2, G, and M1:22:25:8 found in chimeric virus may be grown in cell culture or in the allantois embryonal carcinoma cells, and SSEA-3 and SSEA-4 found of the chick embryo, purified by Zonal ultracentrifugation, in 4 to 8-cell stage embryos. In one embodiment, the antigen inactivated by formaldehyde or B-propiolactone, and pooled. is a T cell receptor derived peptide from a Cutaneous T cell 15 Completely foreign epitopes, including antigens derived Lymphoma (see, Edelson, 1998, The Cancer Journal 4: 62). from other viral or non-viral pathogens can also be engi In a preferred embodiment, the immunogenic composition neered into the virus for use in immunogenic compositions. used in methods of this invention is a cancer Vaccine. For example, antigens of non-related viruses Such as HIV Examples of cancer vaccines include, but are not limited to: (gp160, gp120, gp41) parasite antigens (e.g., malaria), bac antigen modified denritic cell (DC) vaccines such as, but not terial or fungal antigens or tumor antigens can be engineered limited to, Provenge, NeuVenge, Immunovex, Telomerase into the attenuated Strain. Typically such methods include vaccine, Uvidem, Collidem, DCVax-prostate, and DCVax inoculating embryonated eggs, harvesting the allantoic fluid, brain; peptide vaccines such as, but not limited to, Theratope, concentrating, purifying and separating the whole virus, L-BLP25. Oncophage (HSPPC-96), GTOPO-99, IGN-101, using for example Zonal centrifugation, ultracentrifugation, Favld, Panvac-VF, Prostvac-VF, Avicine, EP-2101, MyVax, 25 ultrafiltration, and chromatography in a variety of combina Biovaxid, Mitumomab (IMC-BEC2), IMG-GP75, HER-2 tions. DNA/Protein AutoVac, Zyc 300, and HER-2 protein Auto Virtually any heterologous gene sequence may be con Vac; whole tumor cell vaccines such as, but not limited to, structed into the chimeric viruses for use in immunogenic Canvaxin, Ony-P, Melacine, GVAX, GVAX and MDX-010, compositions. Preferably, heterologous gene sequences are and Oncovax, and viral vector vaccines Such as, but not lim 30 moieties and peptides that act as biological response modifi ited to, ALVAC-CEA/B&1, Allovectin-7, ALVAC, Lovaxin ers. Preferably, epitopes that induce a protective immune C, AdhTAP(OS-1), TroVax, and MVA-MUC1-IL2 response to any of a variety of pathogens, orantigens that bind (TG4010). Characteristics of these vaccines are summarized neutralizing antibodies may be expressed by or as part of the in Tables 1-4. chimeric viruses. For example, heterologous gene sequences Immunogens may comprise a virus, against which an 35 that can be constructed into the chimeric viruses include, but immune response is desired. In certain cases, the immuno are not limited to, influenza and parainfluenza hemagglutinin genic composition used in methods of this invention comprise neuraminidase and fusion glycoproteins such as the HN and F recombinant or chimeric viruses. In other cases, the immu genes of human PIV3. In addition, heterologous gene nogenic composition comprises a virus which is attenuated. sequences that can be engineered into the chimeric viruses Production of recombinant, chimeric and attenuated viruses 40 include those that encode proteins with immuno-modulating may be performed using standard methods known to one activities. Examples of immuno-modulating proteins include, skilled in the art. This invention also encompasses a live but are not limited to, cytokines, interferon type 1, gamma recombinant viral vaccine oran inactivated recombinant viral interferon, colony stimulating factors, interleukin-1, -2, -4. vaccine to be formulated in accordance with the invention. A -5, -6, -12, and antagonists of these agents. live vaccine may be preferred because multiplication in the 45 Other heterologous sequences may be derived from tumor host leads to a prolonged stimulus of similar kind and mag antigens, and the resulting chimeric viruses be used to gen nitude to that occurring in natural infections, and therefore, erate an immune response against the tumor cells leading to confers Substantial, long-lasting immunity. Production of tumor regression in vivo. In accordance with the present Such live recombinant virus vaccine formulations may be invention, recombinant viruses may be engineered to express accomplished using conventional methods involving propa 50 tumor-associated antigens (TAAS), including but not limited gation of the virus in cell culture or in the allantois of the chick to, human tumor antigens recognized by T cells (Robbins and embryo followed by purification. Kawakami, 1996, Curr. Opin. Immunol. 8:628-636, incorpo Recombinant virus may be non-pathogenic to the Subject rated herein by reference in its entirety); melanocyte lineage to which it is administered. In this regard, the use of geneti proteins, including gp100, MART-1/MelanA, TRP-1 (gp75) cally engineered viruses for vaccine purposes may require the 55 and tyrosinase; tumor-specific widely shared antigens, such presence of attenuation characteristics in these strains. The as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-1, introduction of appropriate mutations (e.g., deletions) into N-acetylglucosaminyltransferase-V and p15; tumor-specific the templates used for transfection may provide the novel mutated antigens, such as B-catenin, MUM-1 and CDK4: viruses with attenuation characteristics. For example, specific non-melanoma antigens for breast, ovarian, cervical and pan missense mutations which are associated with temperature 60 creatic carcinoma, HER-2/neu, human papillomavirus-E6. sensitivity or cold adaptation can be made into deletion muta -E7, MUC-1. tions. These mutations should be more stable than the point 5.1.2 Vaccines and Target Diseases mutations associated with cold or temperature sensitive A wide variety of vaccines may be used in connection with mutants and reversion frequencies should be extremely low. methods of this invention. A non-limiting list of vaccines that Alternatively, chimeric viruses with "suicide characteris 65 can be used in connection with this invention is provided in tics may be constructed for use in the immunogenic compo FIG.1. Target diseases for methods of the invention includes sitions. Such viruses would go through only one or a few cancer, other infectious or inflammatory diseases. US 8,715,677 B2 13 14 Methods of the invention can be used in the treatment of viruses, bunga viruses, phleboviruses and Nairo viruses); cancers, including, but not limited to, neoplasms, tumors, Arena viridae (e.g., hemorrhagic fever viruses); Reoviridae metastases, or any disease or disorder characterized by (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; uncontrolled cell growth. Specific examples of cancer Hepadnaviridae (Hepatitis B virus); Parvovirida (parvovi include, but are not limited to: cancers of the skin, Such as ruses); Papovaviridae (papilloma viruses, polyoma viruses); melanoma, lymph node, breast; cervix; uterus; gastrointesti Adenoviridae (most adenoviruses); Herpesviridae (herpes nal tract; lung; ovary; prostate; colon; rectum; mouth; brain; simplex virus (HSV) 1 and 2, varicella Zoster virus, cytome head and neck; throat; testes; kidney; pancreas; bone; spleen; galovirus (CMV), herpes virus); Poxyiridae (variola viruses, liver, bladder; larynx, nasal passages; and AIDS-related can vaccinia viruses, pox viruses); and Iridoviridae (e.g., African cers. Methods of the invention are particularly useful for 10 Swine fever virus); and unclassified viruses (e.g., the etiologi treating cancers of the blood and bone marrow, Such as mul cal agents of Spongiform encephalopathies, the agent of delta tiple myeloma and acute and chronic leukemias, for example, hepatitis (thought to be a defective satellite of hepatitis B lymphoblastic, myelogenous, lymphocytic, myelocytic leu virus), the agents of non-A, non-B hepatitis (class 1 inter kemias, and myelodysplastic syndromes including but not nally transmitted; class 2 parenterally transmitted, e.g., limited to 5 q minus syndrome, or myelodysplastic Syn 15 Hepatitis C); Norwalk and related viruses, and astroviruses. dromes associated with other cytogenic abnormalities. The Retroviruses that results in infectious diseases in animals methods of the invention can be used for treating, preventing and humans include both simple retroviruses and complex or managing either primary or metastatic tumors. retroviruses. The simple retroviruses include the subgroups Other specific cancers include, but are not limited to, of B-type retroviruses, C-type retroviruses and D-type retro advanced malignancy, amyloidosis, neuroblastoma, menin viruses. An example of a B-type retrovirus is mouse mam gioma, hemangiopericytoma, multiple brain metastase, glio mary tumor virus (MMTV). The C-type retroviruses include blastoma multiforms, glioblastoma, brain stem glioma, poor Subgroups C-type group A (including Rous sarcoma virus prognosis malignant brain tumor, malignant glioma, recur (RSV), avian leukemia virus (ALV), and avian myeloblasto rent malignant glioma, anaplastic astrocytoma, anaplastic oli sis virus (AMV)) and C-type group B (including murine godendroglioma, neuroendocrine tumor, rectal adenocarci 25 leukemia virus (MLV), feline leukemia virus (FeLV), murine noma, Dukes C & D colorectal cancer, unresectable sarcoma virus (MSV), gibbon ape leukemia virus (GALV), colorectal carcinoma, metastatic hepatocellular carcinoma, spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) Kaposi's sarcoma, karotype acute myeloblastic leukemia, and simian sarcoma virus (SSV)). The D-type retroviruses Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, cutaneous include Mason-Pfizer monkey virus (MPMV) and simian T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large 30 retrovirus type 1 (SRV-1). The complex retroviruses include B-Cell lymphoma, low grade follicular lymphoma, meta the subgroups of lentiviruses, T-cell leukemia viruses and the static melanoma (localized melanoma, including, but not lim foamy viruses. Lentiviruses include HIV-1, but also include ited to, ocular melanoma), malignant mesothelioma, malig HIV-2, SIV. Visna virus, feline immunodeficiency virus nant pleural effusion mesothelioma Syndrome, peritoneal (FIV), and equine infectious anemia virus (EIAV). The T-cell carcinoma, papillary serous carcinoma, gynecologic sar 35 leukemia viruses include HTLV-1, HTLV-II, simian T-cell coma, Soft tissue sarcoma, Scelroderma, cutaneous vasculitis, leukemia virus (STLV), and bovine leukemia virus (BLV). Langerhans cell histiocytosis, leiomyosarcoma, fibrodyspla The foamy viruses include human foamy virus (HFV), simian sia ossificans progressive, hormone refractory prostate can foamy virus (SFV) and bovine foamy virus (BFV). cer, resected high-risk soft tissue sarcoma, unrescectable Examples of RNA viruses that are antigenic or immuno hepatocellular carcinoma, Waldenstrom's macroglobuline 40 genic in Vertebrate animals include, but are not limited to, the mia, Smoldering myeloma, indolent myeloma, fallopian tube following: members of the family Reoviridae, including the cancer, androgen independent prostate cancer, androgen genus Orthoreovirus (multiple serotypes of both mammalian dependent stage IV non-metastatic prostate cancer, hormone and avian retroviruses), the genus Orbivirus (Bluetongue insensitive prostate cancer, chemotherapy-insensitive pros virus, Eugenangee virus, Kemerovo virus, African horse sick tate cancer, papillary thyroid carcinoma, follicular thyroid 45 ness virus, and Colorado Tick Fever virus), the genus Rotavi carcinoma, medullary thyroid carcinoma, and leiomyoma. In rus (human rotavirus, Nebraska calf diarrhea virus, murine a specific embodiment, the cancer is metastatic. In another rotavirus, simian rotavirus, bovine or ovine rotavirus, avian embodiment, the cancer is refractory or resistance to chemo rotavirus); the family Picornaviridae, including the genus therapy or radiation. Enterovirus (poliovirus, Coxsackie virus A and B, enteric Infectious diseases are caused by infectious agents such as, 50 cytopathic human orphan (ECHO) viruses, hepatitis A virus, but not limited to, viruses, bacteria, fungi protozoa, helm Simian enteroviruses, Murine encephalomyelitis (ME) inths, and parasites. viruses, Poliovirus muris, Bovine enteroviruses, Porcine Examples of viruses that have been found in humans enteroviruses), the genus Cardiovirus (Encephalomyocardi include, but are not limited to, Retroviridae (e.g., human tis virus (EMC), Mengovirus), the genus Rhinovirus (Human immunodeficiency viruses, such as HIV-1 (also referred to as 55 rhinoviruses including at least 113 subtypes; other rhinovi HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other iso ruses), the genus Apthovirus (Foot and Mouth disease lates, such as HIV-LP); Picornaviridae (e.g., polio viruses, (FMDV); the family Calciviridae, including Vesicular exan hepatitis A virus; enteroviruses, human Coxsackie viruses, thema of swine virus, San Miguel sea lion virus, Feline picor rhinoviruses, echoviruses); Calciviridae (e.g., strains that navirus and Norwalk virus; the family Togaviridae, including cause gastroenteritis); Togaviridae (e.g., equine encephalitis 60 the genus Alphavirus (Eastern equine encephalitis virus, viruses, rubella viruses); Flaviridae (e.g., dengue viruses, Semliki forest virus, Sindbis virus, Chikungunya virus, encephalitis viruses, yellow fever viruses); Coronaviridae O'Nyong-Nyong virus, Ross river virus, Venezuelan equine (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomati encephalitis virus, Western equine encephalitis virus), the tis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); genus Flavirius (Mosquito borne yellow fever virus, Dengue Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, 65 virus, Japanese encephalitis virus, St. Louis encephalitis measles virus, respiratory syncytial virus); Orthomyxoviri virus, Murray Valley encephalitis virus, West Nile virus, Kun dae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan jin virus, Central European tick borne virus, Far Eastern tick US 8,715,677 B2 15 16 borne virus, Kyasanur forest virus, Louping III virus, Powas goatpox), the genus Suipoxvirus (Swinepox), the genus San virus, Omsk hemorrhagic fever virus), the genus Rubivi Parapoxvirus (contagious postular dermatitis virus, pseudo rus (Rubella virus), the genus Pestivirus (Mucosal disease cowpox, bovine papular stomatitis virus); the family Iri virus, Hog cholera virus, Border disease virus); the family doviridae (African swine fever virus, Frog viruses 2 and 3. Bunyaviridae, including the genus Bunyvirus (Bunyamwera Lymphocystis virus of fish); the family Herpesviridae, and related viruses, California encephalitis group viruses), including the alpha-Herpesviruses (Herpes Simplex Types 1 the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Val and 2, Varicella-Zoster, Equine abortion virus, Equine herpes ley fever virus), the genus Nairovirus (Crimean-Congo hem virus 2 and 3, pseudorabies virus, infectious bovine kerato orrhagic fever virus, Nairobi sheep disease virus), and the conjunctivitis virus, infectious bovine rhinotracheitis virus, genus Uuku virus (Uukuniemi and related viruses); the family 10 feline rhinotracheitis virus, infectious laryngotracheitis Orthomyxoviridae, including the genus Influenza virus (In virus), the Beta-herpesviruses (Human cytomegalovirus and fluenza virus type A (many human Subtypes), Swine influenza cytomegaloviruses of Swine, monkeys and rodents), the virus, and Avian and Equine Influenza viruses, influenza type gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's B (many human Subtypes), and influenza type C (possible disease virus, Herpes saimiri, Herpesvirus ateles, Herpesvi separate genus)); the family paramyxoviridae, including the 15 rus Sylvilagus, guinea pig herpes virus, Lucke tumor virus); genus Paramyxovirus (Parainfluenza virus type 1, Sendai the family Adenoviridae, including the genus Mastadenovi virus, Hemadsorption virus, Parainfluenza viruses types 2 to rus (Human Subgroups A, B, C, D, E and ungrouped; simian 5, Newcastle Disease Virus, Mumps virus), the genus Mor adenoviruses (at least 23 serotypes), infectious canine hepa billivirus (Measles virus, Subacute Sclerosing panencephalitis titis, and adenoviruses of cattle, pigs, sheep, frogs and many virus, distemper virus, Rinderpest virus), the genus Pneu other species), the genus Aviadenovirus (Avian adenovi movirus (respiratory syncytial virus (RSV), Bovine respira ruses), and non-cultivatable adenoviruses; the family tory syncytial virus and Pneumonia virus of mice); forest Papoviridae, including the genus Papillomavirus (Human virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong papilloma viruses, bovine papilloma viruses, Shope rabbit virus, Ross river virus, Venezuelan equine encephalitis virus, papilloma virus, and various pathogenic papilloma viruses of Western equine encephalitis virus), the genus Flavirius (Mos 25 other species), the genus Polyomavirus (polyomavirus, Sim quito borne yellow fever virus, Dengue virus, Japanese ian vacuolating agent (SV-40), Rabbit vacuolating agent encephalitis virus, St. Louisencephalitis virus, Murray Valley (RKV), K virus, BK virus, JC virus, and other primate encephalitis virus, West Nile virus, Kunjin virus, Central polyoma viruses such as Lymphotrophic papilloma virus); European tick borne virus, Far Eastern tick borne virus, the family Parvoviridae including the genus Adeno-associ Kyasanur forest virus, Louping III virus, Powassan virus, 30 ated viruses, the genus Parvovirus (Feline panleukopenia Omsk hemorrhagic fever virus), the genus Rubi virus (Rubella virus, bovine parvovirus, canine parvovirus, Aleutian mink virus), the genus Pestivirus (Mucosal disease virus, Hog chol disease virus, etc). Finally, DNA viruses may include viruses era virus, Border disease virus); the family Bunyaviridae, which do not fit into the above families such as Kuru and including the genus Bunyvirus (Bunyamwera and related Creutzfeldt-Jacob disease viruses and chronic infectious neu viruses, California encephalitis group viruses), the genus 35 ropathic agents. Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever Bacterial infections or diseases that can be treated by meth virus), the genus Nairovirus (Crimean-Congo hemorrhagic ods of the present invention are caused by bacteria including, fever virus, Nairobi sheep disease virus), and the genus Uuku but not limited to, bacteria that have an intracellular stage in virus (Uukuniemi and related viruses); the family Orthomyx its life cycle. Such as mycobacteria (e.g., Mycobacteria tuber oviridae, including the genus Influenza virus (Influenza virus 40 culosis, M. bovis, M. avium, M. leprae, or M. africanum), type A, many human Subtypes); Swine influenza virus, and rickettsia, mycoplasma, chlamydia, and legionella. Other Avian and Equine Influenza viruses; influenza type B (many examples of bacterial infections contemplated include, but human Subtypes), and influenza type C (possible separate are not limited to, infections caused by Grampositive bacillus genus); the family paramyxoviridae, including the genus (e.g., Listeria, Bacillus Such as Bacillus anthracis, Erysipelo Paramyxovirus (Parainfluenza virus type 1, Sendai virus, 45 thrix species), Gram negative bacillus (e.g., Bartonella, Bru Hemadsorption virus, Parainfluenza viruses types 2 to 5, cella, Campylobacter, Enterobacter, Escherichia, Fran Newcastle Disease Virus, Mumps virus), the genus Mor cisella, Hemophilus, Klebsiella, Morganella, Proteus, billivirus (Measles virus, Subacute Sclerosing panencephalitis Providencia, Pseudomonas, Salmonella, Serratia, Shigella, virus, distemper virus, Rinderpest virus), the genus Pneu Vibrio, and Yersinia species), spirochete bacteria (e.g., Bor movirus (respiratory syncytial virus (RSV), Bovine respira 50 relia species including Borrelia burgdorferi that causes Lyme tory syncytial virus and Pneumonia virus of mice); the family disease), anaerobic bacteria (e.g., Actinomyces and Rhabdoviridae, including the genus Vesiculovirus (VSV), Clostridium species), Gram positive and negative coccal bac Chandipura virus, Flanders-Hart Park virus), the genus Lys teria, Enterococcus species, Streptococcus species, Pneumo savirus (Rabies virus), fish Rhabdoviruses, and two probable coccus species, Staphylococcus species, Neisseria species. Rhabdoviruses (Marburg virus and Ebola virus); the family 55 Specific examples of infectious bacteria include, but are not Arenaviridae, including Lymphocytic choriomeningitis virus limited to: Helicobacter pyloris, Borelia burgdorferi, (LCM), Tacaribe virus complex, and Lassa virus; the family Legionella pneumophilia, Mycobacteria tuberculosis, M. Coronoaviridae, including Infectious Bronchitis Virus (IBV), avium, M. intracellulare, M. kansai, M. gordonae, Staphy Mouse Hepatitis virus, Human enteric corona virus, and lococcus aureus, Neisseria gonorrhoeae, Neisseria menin Feline infectious peritonitis (Feline coronavirus). 60 gitidis, Listeria monocytogenes, Streptococcus pyogenes Illustrative DNA viruses that are antigenic or immuno (Group A Streptococcus), Streptococcus agalactiae (Group B genic in Vertebrate animals include, but are not limited to: the Streptococcus), Streptococcus viridans, Streptococcus faeca family Poxyiridae, including the genus Orthopoxvirus (Vari lis, Streptococcus bovis, Streptococcus pneumoniae, Haemo Ola major; Variola minor, Monkey pox Vaccinia, Cowpox, philus influenzae, Bacillus antracis, corynebacterium diph Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvi 65 theriae, Erysipelothrix rhusiopathiae, Clostridium rus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, perfingers, Clostridium tetani, Enterobacter aerogenes, other avian poxvirus), the genus Capripoxvirus (sheeppox, Klebsiella pneumoniae, Pasturella multocida, Fusobacte US 8,715,677 B2 17 18 rium nucleatum, Streptobacillus moniliformis, Treponema americana, Blattella germanica, Blatta Orientails, Tebanus pallidium, Treponema pertenue, Leptospira, Rickettsia, and spp., Musca domestica, Ephemeroptera spp., Culicidae sp., Actinomyces israeli. and Heterocera spp.; Fungal diseases that can be treated by methods of the epithelia, dander, hair and features such as, but not limited present invention include, but are not limited to, aspergillio to, Serinus Canaria, Felis catus (domesticus), Bos taurus, sis, crytococcosis, sporotrichosis, coccidioidomycosis, para Gallus gallus (domesticus), Canis familiaris, Anas platyrhyn coccidioidomycosis, histoplasmosis, blastomycosis, Zygo chos, Meriones unguiculatus, Capra hircus, Anser domesti mycosis, and candidiasis. cus, Cavia porcellus (cobaya), Mesocrietus auratus, Sus Parasitic diseases that can be treated by methods of the scrofa, Equus cabalus, Mus musculus, Psittacidae, Colum present invention include, but are not limited to, amebiasis, 10 bafasciata, Oryctolagus cuniculus, Rattus norvegicus, and malaria, leishmania, coccidia, giardiasis, cryptosporidiosis, Ovis aries, toxoplasmosis, and trypanosomiasis. Also encompassed are fungi such as, but not limited to, Cephalosporium acremo infections by various worms such as, but not limited to, nium, Alternaria tenuis, Aspergillus glaucus, Aspergillus fia ascariasis, ancylostomiasis, trichuriasis, strongyloidiasis, vus, Aspergillus filmigatus, Aspergillus nidulans, Aspergillus toXoccariasis, trichinosis, onchocerciasis, filaria, and dirofi 15 niger; Aspergillus terreus, Aspergillus versicolor, Aureoba lariasis. Also encompassed are infections by various flukes sidium pullulan (Pullularia pullulans), Drechslera Sorokini Such as, but not limited to, Schistosomiasis, paragonimiasis, ana, Helminthosporium sativum, Botrytis cinerea, Candida and clonorchiasis. Parasites that cause these diseases can be albicans, Chaetomium globosum, Cladosporium herbarum, classified based on whether they are intracellular or extracel Cladosporium sphaerospermum(Homodendrum hordei), lular. An "intracellular parasite, as used herein, is a parasite Drechslera spicifera (Curvularia spicifera), Epicoccum whose entire life cycle is intracellular. Examples of human nigrum (Epicoccum purpurascens), Epidermophyton flocco intracellular parasites include Leishmania spp., Plasmodium sum, Fusarium moniliforme, Fusarium Solani, Geotrichum spp., Trypanosoma Cruzi, Toxoplasma gondii, Babesia spp., candidum, Gliocladium viride, Helminthosporium Solani, and Trichinella spiralis. An "extracellular parasite.” as used Microsporum canis, Mucor circinelloides f. circinelloides, herein, is a parasite whose entire life cycle is extracellular. 25 Mucor circinelloidesf lusitanicus, Mucor plumbeus, Extracellular parasites capable of infecting humans include Mycogone perniciosa, Neurospora intermedia, Nigrospora Entamoeba histolytica, Giardia lamblia, Enterocytozoon Oryzae, Paecilomyces variotii, Penicillum brevi-compactum, bieneusi, Naegleria and Acanthamoeba as well as most hel Penicillum camembertii, Penicillum chrysogenium, Penicil minths. Yet another class of parasites is defined as being lum digitatum, Penicillum expansium, Penicillum notatum, mainly extracellular but with an obligate intracellular exist 30 Penicillum roquefortii, Phoma betae, Phoma herbarum, ence at a critical stage in their life cycles. Such parasites are Rhizopus Oryzae, Rhizopus stolonifer, Rhodotorula mucilagi referred to herein as "obligate intracellular parasites.” These nosa, Saccharomyces cerevisiae, Scopulariopsis brevicaulis, parasites may exist most of their lives or only a small portion Serpula lacrymans, Setosphaeria rostrata, Stemphyllium bot of their lives in an extracellular environment, but they all have rvosum, Stemphylium Solani, Trichoderma harzianum, Tri at least one obligate intracellular stage in their life cycles. 35 chophyton mentagrophytes, Trichophyton rubrum, and Tri This latter category of parasites includes Trypanosoma rhod chothecium roseum, esiense and Trypanosoma gambiense, Isospora spp., Smuts such as, but not limited to, Ustilago nuda, Ustilago Cryptosporidium spp., Eimeria spp., Neospora spp., Sarco cynodontis, Ustilago maydis, Sporisorium cruentum, Usti cystis spp., and Schistosoma spp. lago avenae, and Ustilago tritici; 5.2 Allergens 40 grasses such as, but not limited to, Paspalum notatum, This invention encompasses methods of reducing or inhib Cynodon dactylon, Poa compressa, Bromus inermis, Phalaris iting allergic reaction to an allergen in a Subject comprising arundinacea, Zea mays, Elytrigia repens (Agropyron repens), administering to the Subject an immunomodulatory com Sorghum haelpense, Poa pratensis, Festuca pratensis (ela pound of the invention prior to the Subject’s exposure to an tior), Avena sativa, Dactylis glomerata, Agrostis gigantea allergen. Optionally, in addition to the administration before 45 (alba), Secale cereale, Leymus (Elymus) condensatus, the exposure to an allergen, an immunomodulatory com Lolium perennessp. multiflorum, Lolium perenne, Anthoxian pound may be administered during and/or after the Subjects thum Odoratum, Phleum pratense, Holcus lanatus, Triticum exposure to an allergen. It is contemplated that any types of aestivum, and Elymus (Agropyron) Smithii; exposure to allergens including, but not limited to, the Sub weeds such as, but not limited to, Atriplex polycarpa, Bac jects exposure to naturally occurring allergens, exposure by 50 charis halinifolia, Baccharis sarothroides, Hymenoclea Sal the intake of food, and exposure through allergy vaccine sola, Amaranthus hybridus, Xanthium strumarium (com administration, are encompassed by methods of this inven mune), Rumex Crispus, Eupathium capillifolium, Solidago tion. spp., Amaranthus tuberculatus (Acnida tamariscina), Allen Examples of allergens (e.g., naturally occurring or those rolfea Occidentalis, Chenopodium botrys, Kochia scoparia, contained in allergy vaccines) include, but are not limited to, 55 Chenopodium album, Iva xanthifolia, Iva angustifolia, Che allergens from: nopodium ambrosioides, Artemisia vulgaris, Artemisia mites such as, but not limited to, Dermatophagoides fari ludoviciana, Urtica dioica, Amaranthus spinosus, Plantago nae, Dermatophagoides pteronyssinus, Acarus siro, Blomia lanceolata, Iva axillaris, Atriplex lentiformis, Ambrosia tropicalis, Chortoglyphus arcuatas, Euroglyphus may nei, dumosa, Ambrosia acanthicarpa, Ambrosia trifida, Ambrosia Lepidoglyphus destructor; Trophagus putrescentiae, and 60 artemisiifolia, Ambrosia confertiflora, Ambrosia bidentata, Glyphagus demesticus, Ambrosia psilostachya, Salsola kali (pestifer), Artemisia Venoms such as, but not limited to, Bombus spp., Vespa Californica, Artemisiafrigida, Artemisia tridentata, Atriplex crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Wrightii, Atriplex confertifolia, and Artemisia annua, Vespula spp., Dolichovespula maculata, and Dolichovespula trees such as, but not limited to, Acasia spp., Alnus gluti arenaria, 65 nosa, Alnus rubra, Alnus incana ssp. rugosa, Alnus rhombi insects such as, but not limited to, Camponotus pennsyl folia, Fraxinus velutina, Fraxinus pennsylvanica, Fraxinus vanicus, Solenopsis invicta, Solenopsis richteri, Periplaneta latifolia, Fraxinus americana, Populus tremuloides, Myrica US 8,715,677 B2 19 20 cerifera, Fagus grandifolia (americana), Casuarina equise animal foods such as, but not limited to, Bos taurus, Ovis tifolia, Betula lenta, Betula pendula, Betula nigra, Betula aries, and Sus scrofa, occudentalis (fontinalis), Betula populifolia, Acer negundo, poultry products such as, but not limited to, chicken (Gal Cryptomeria japonica, Juniperus ashei (Sabinoides), Junipe lus gallus) products and turkey (Meleagris gallopavO) prod rus virginiana, Tamarix gallica, Populus balsamifera ssp. ucts; trichocarpa, Populus deltoides, Populusfremontii, Populus dairy products Such as, but not limited to, bovine casein and wislizeni, Populus monilifera (sargentii), Cupressus ari bovine milk; Zonoca, Taxodium distichum, Cupressus sempervirens, nuts Such as, but not limited to, Bertholletia excelsa, Anac Ulmus americana, Ulmus crassifolia, Ulmus pumila, Euca ardium Occidentale, Cocos nucifera, Corylus americana, lyptus globulus, Celtis Occidentalis, Corylus americana, 10 Arachis hypogaea, Carya illinoensis, Juglans nigra, and Corylus avellana, Carya ovata, Carya laciniosa, Carya alba, Juglans regia, Juniferus monosperma, Juniperus princhotii, Juniperus miscellaneous allergens Such as, but not limited to, those Scopulorum, Juniperus Occidentalis, Robinia pseudoacacia, from Gossypium hirsutum, Linum usitatissimum, Acaia sene Mangifera indica, Acer macrophyllum, Acer rubrum, Acer 15 gal, Sterculia urens, Astragalus gummifer; Ceiba pentandra, saccharum, Melaleuca quinquenervia (leucadendron), Iris germanica var. florentina, Chrysanthemum cinerariifo Prosopis glandulosa (juliflora), Broussonetia papyrifera, lium, Bombyx mori, and Nicotiana tabacum, Morus rubra, Morus alba, Quercus gambelii, Ouercus velu dust Such as, but not limited to, barley grain dust, corn grain tina, Quercus macrocarpa, Quercus kelloggii, Ouercus agri dust, house dust, mattress dust, oat grain dust, wheat grain folia, Quercus lobata, Quercus ilex, Quercus stellata, Ouer dust, and upholstery dust. cus rubra, Quercus dumosa, Quercus virginiana, Quercus 5.3 Immunomodulatory Compounds nigra, Quercus garryana, Quercus alba, Olea europaea, As used herein and unless otherwise indicated, the terms Elaegnus angustifolia, Citrus sinensis, Arecastrum roman “immunomodulatory compounds of the invention' and Zofianum (Cocos plumosa), Carya illnoensis, Schinus mole, “IMiDs(R (Celgene Corporation) encompass certain small Schinus terebinthifolius, Pinus taeda, Pinus strobus, Pinus 25 organic molecules that inhibit LPS induced monocyte TNF palustris, Pinus ponderosa, Pinus elliottii, Pinus virginiana, C., IL-1 B, IL-12, IL-6, MIP-1C, MCP-1, GM-CSF, G-CSF, Pinus monticola, Pinus echinata, Populus migra, Populus and COX-2 production. Specific immunomodulatory com alba, Ligustrum vulgare, Liquidambar styraciflua, Platanus pounds are discussed below. occidentalis, Platanus Orientalis, Platanus racemosa, Plata TNF-C. is an inflammatory cytokine produced by macroph nus acerifolia, Juglans migra, Juglans Californica, Juglans 30 ages and monocytes during acute inflammation. TNF-C. is regia, Salix lasiolepsis, Salix nigra, and Salix discolor, responsible for a diverse range of signaling events within flowers such as, but not limited to, Chrysanthemum leu cells. Without being limited by a particular theory, one of the canthemum, Taraxacum officinale, and Helianthus annuus, biological effects exerted by the immunomodulatory com farm plants such as, but not limited to, Medicago sativa, pounds of the invention is the reduction of myeloid cell Ricinus communis, Trifolium pratense, Brassica spp., and 35 TNF-C. production. Immunomodulatory compounds of the Beta vulgaris, invention may enhance the degradation of TNF-C. mRNA. plant food Such as, but not limited to, Prunus dulcis, Further, without being limited by theory, immunomodula Maluspunila, Prunus armeniaca, Musa paradisiaca (sapien tory compounds used in the invention may also be potent tum), Hordeum vulgare, Phaseolus lunatus, Phaseolus vul 40 co-stimulators of T cells and increase cell proliferation dra garis, Phaseolus sp., Phaseolus sp., Phaseolus vulgaris, matically in a dose dependent manner. Immunomodulatory Rubus allegheniensis, Vaccinium sp., Brassica oleracea var. compounds of the invention may also have a greater co botrytis, Fagopyrum esculentum, Brassica oleracea var. stimulatory effect on the CD8+ T cell subset than on the capitata, Theobroma cacao, Cucumis melo, Daucus Carota, CD4+ T cell subset. In addition, the compounds preferably Brassica oleracea var. botrytis, Apium graveolens var. dulce, 45 have anti-inflammatory properties against myeloid cell Prunus sp., Cinnamomum verum, Coffea arabic, Zea mays, responses, yet efficiently co-stimulate T cells to produce Vaccinium macrocarpon, Cucumis sativus, Allium sativum, greater amounts of IL-2, IFN-Y, and to enhance T cell prolif Zingiber officinale, Vitis sp., Citrus paradisi, Humulus lupu eration and CD8+ T cell cytotoxic activity. Further, without lus, Citrus limon, Lactuca sativa, Agaricus campestris, Bras being limited by a particular theory, immunomodulatory sica sp., Myristica fragrams, Avena sativa, Olea europaea, 50 compounds used in the invention may be capable of acting Allium cepa var. cepa, Citrus sinensis, Vigna unguiculata, both indirectly through cytokine activation and directly on Pisum sativum, Prunus persica, Pyrus communis, Piper Natural Killer (“NK) cells and Natural Killer T (“NKT) nigrum, Capsicum annuum var. annuum, Ananas comosus, cells, and increase the NK cells’ ability to produce beneficial Ipomoea batatas, Solanum tuberosum, Rubus idaeus var. cytokines such as, but not limited to, IFN-Y, and to enhance idaeus, Oryza sativa, Secale cereale, Sesamum Orientale (in 55 NK and NKT cell cytotoxic activity. dicum), Glycine max, Spinacia oleracea, Cucurbitapepo var. Specific examples of immunomodulatory compounds melopepo, Fragaria chiloensis, Lycopersicon esculentum (ly include cyano and carboxy derivatives of Substituted Styrenes copersicum), Brassica rapa var. rapa, Vanilla planifolia, Cit such as those disclosed in U.S. Pat. No. 5,929,117: 1-oxo-2- rullus lanatus var. lanatus, and Triticun aestivum, 60 (2,6-dioxo-3-fluoropiperidin-3-yl)isoindolines and 1,3-di fish and shellfish such as, but not limited to, Micropterus oxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl)isoindolines such sp., Ictalurus punctatus, Mercenaria mercenaria, Gadus as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476: morhua, Callinectes sapidus, Platichthys sp., Hippoglossus the tetra substituted 2-(2,6-dioxopiperdin-3-yl)-1-oxoisoin sp., Homarus americanus, Scomber scombrus, Crassostrea dolines described in U.S. Pat. No. 5,798,368: 1-oxo and 1,3- virginica, Sebastes marinus, Salmo salar; Clupeiformes, 65 dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines (e.g., 4-me Pecten magellanicus, Penaeus sp., Salvelinus sp., and Thun thyl derivatives of thalidomide), substituted 2-(2,6- nus sp., dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6- US 8,715,677 B2 21 dioxopiperidin-3-yl)-1-oxoisoindoles including, but not lim ited to, those disclosed in U.S. Pat. Nos. 5,635,517, 6,281, 230, 6,316,471, 6,403,613, 6,476,052 and 6,555,554: 1-oxo C NH and 1,3-dioxoisoindolines substituted in the 4- or 5-position of the indoline ring (e.g., 4-(4-amino-1,3-dioxoisoindoline 5 2-yl)-4-carbamoylbutanoic acid) described in U.S. Pat. No. 6,380.239; isoindoline-1-one and isolindoline-1,3-dione sub stituted in the 2-position with 2,6-dioxo-3-hydroxypiperidin NH2 5-yl (e.g., 2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)- 4-aminoisoindolin-1-one) described in U.S. Pat. No. 6,458, 10 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; 810; a class of non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579 and 5,877,200; and isolindole-imide compounds Such as those described in U.S. patent publication O O no. 2003/0045552 published on Mar. 6, 2003, U.S. patent 15 N publication no. 2003/0096841 published on May 22, 2003, N O and International Application No. PCT/US01/50401 (Inter M national Publication No. WO 02/059106). The entireties of each of the patents and patent applications identified herein w\, are incorporated herein by reference. Immunomodulatory NH2 compounds do not include thalidomide. Various immunomodulatory compounds of the invention 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and contain one or more chiral centers, and can exist as racemic mixtures of enantiomers or mixtures of diastereomers. This 25 invention encompasses the use of stereomerically pure forms O O of Such compounds, as well as the use of mixtures of those N N O forms. For example, mixtures comprising equal or unequal M 30 amounts of the enantiomers of a particular immunomodula w\, tory compounds of the invention may be used in methods and NH2 compositions of the invention. These isomers may be asym metrically synthesized or resolved using standard techniques 1,3-dioxo-2-(3-methyl-2,6-dioxopiperidin-3-yl)-4-ami 35 Such as chiral columns or chiral resolving agents. See, e.g., noisoindole, and optically pure isomers thereof. The com pounds can be obtained via Standard, synthetic methods (see Jacques, J., et al., Enantiomers, Racemates and Resolutions e.g., U.S. Pat. No. 5,635,517, incorporated herein by refer (Wiley-Interscience, New York, 1981); Wilen, S. H., et al., ence). The compounds are also available from Celgene Cor Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of 40 poration, Warren, N.J. As used herein, and unless otherwise indicated, the term Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. “optically pure” means a composition that comprises one H., Tables of Resolving Agents and Optical Resolutions p. 268 optical isomer of a compound and is substantially free of (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, other isomers of that compound. For example, an optically Ind., 1972). 45 pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the com Preferred immunomodulatory compounds of the invention pound. An optically pure composition of a compound having include, but are not limited to, 1-oxo- and 1.3dioxo-2-(2,6- two chiral centers will be substantially free of other diastere dioxopiperidin-3-yl)isoindolines substituted with amino in omers of the compound. A typical optically pure compound the benzo ring as described in U.S. Pat. No. 5,635,517 which 50 comprises greater than about 80% by weight of one enanti is incorporated herein by reference. These compounds have omer of the compound and less than about 20% by weight of the structure I: other enantiomers of the compound, more preferably greater than about 90% by weight of one enantiomer of the com pound and less than about 10% by weight of the other enan 55 tiomers of the compound, even more preferably greater than about 95% by weight of one enantiomer of the compound and less than about 5% by weight of the other enantiomers of the NH compound, more preferably greater than about 97% by weight of one enantiomer of the compound and less than 60 about 3% by weight of the other enantiomers of the com pound, and most preferably greater than about 99% by weight of one enantiomer of the compound and less than about 1% by weight of the other enantiomers of the compound. in which one of XandY is C—O, the other of X and Y is C—O Other specific immunomodulatory compounds of the or CH, and R is hydrogen or lower alkyl, in particular 65 invention belong to a class of Substituted 2-(2,6-dioxopiperi methyl. Specific immunomodulatory compounds include, but din-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin are not limited to: 3-yl)-1-oxoisoindoles, such as those described in U.S. Pat. US 8,715,677 B2 23 24 Nos. 6,281.230; 6,316,471; 6,335,349; and 6,476,052, and International Patent Application No. PCT/US97/13375 (In II ternational Publication No. WO98/03502), each of which is incorporated herein by reference. Representative compounds NH are of formula:

R O R2 X R NH 10 N N O and pharmaceutically acceptable salts, hydrates, Solvates, R3 Y clathrates, enantiomers, diastereomers, racemates, and mix R4 tures of stereoisomers thereof, wherein: 15 one of X and Y is C—O and the other is CH or C=O; R" is H. (C-Cs)alkyl, (C-C)cycloalkyl, (C-Cs)alkenyl, in which: (C-Cs)alkynyl, benzyl, aryl, (Co-Cal)alkyl-(C-C)heterocy cloalkyl, (Co-C)alkyl-(C-Cs)heteroaryl, C(O)R, C(S)R. one of X and Y is C—O and the other of X and Y is C—O C(O)OR, (C-C)alkyl-N(R), (C-C)alkyl-OR., (C-C) or CH: alkyl-C(O)OR, C(O)NHR, C(S)NHR, C(O)NR'R'', C(S) (i) each of R', R. R. and R', independently of the others, NR'R' or (C-C)alkyl-O(CO)R: is halo, alkyl of 1 to 4 carbonatoms, or alkoxy of 1 to 4 carbon R is H. F. benzyl, (C-Cs)alkyl, (C-Cs)alkenyl, or (C- atoms or (ii) one of R', R. R. and R is NHR and the Cs)alkynyl: remaining of R', R. R. and Rare hydrogen; 25 R and R are independently (C-Cs)alkyl, (C-C)cy R is hydrogen or alkyl of 1 to 8 carbon atoms; cloalkyl, (C-C)alkenyl, (C-Cs)alkynyl, benzyl, aryl, (Co R is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or C.)alkyl-(C-C)heterocycloalkyl, (Co-Cal)alkyl-(C-Cs)het eroaryl, (Co-Cs)alkyl-N(R), (C-C)alkyl-OR, (C-C) halo; alkyl-C(O)OR, (C-C)alkyl-O(CO)R, or C(O)OR: provided that R is other than hydrogen if X and Y are R" is (C-Cs)alkyl, (C-Cs)alkenyl, (C-Cs)alkynyl, (C- C=O and (i) each of R', R. R. and R is fluoro or (ii) one of C.)alkyl-OR, benzyl, aryl, (Co-C)alkyl-(C-C)heterocy R", R. R. or R is amino. cloalkyl, or (Co-Cal)alkyl-OC-Cs)heteroaryl; Compounds representative of this class are of the formulas: R is (C-Cs)alkyl, (C-Cs)alkenyl, (C-Cs)alkynyl, ben Zyl, aryl, or (C-Cs)heteroaryl; 35 each occurrence of R is independently H, (C-Cs)alkyl, O O (C-Cs)alkenyl, (C-Cs)alkynyl, benzyl, aryl, (C-Cs)het eroaryl, or (Co-Cs)alkyl-C(O)C R or the R groups can join to form a heterocycloalkyl group; N O, M 40 n is 0 or 1; and OCC * represents a chiral-carbon center. HN \, In specific compounds of formula II, when n is 0 then R' is (C-C)cycloalkyl, (C-Cs)alkenyl, (C-Cs)alkynyl, benzyl, aryl, (Co-Cal)alkyl-(C-C)heterocycloalkyl, (Co-C)alkyl and 45 (C-C)heteroaryl, C(O)R, C(O)OR, (C-C)alkyl-N(R), (C-C)alkyl-OR, (C-C)alkyl-C(O)OR, C(S)NHR, or (C-C)alkyl-O(CO)R: O O R’ is H or (C-Cs)alkyl; and 4. R1 NH R is (C-Cs)alkyl, (C-C,)cycloalkyl, (C-Cs)alkenyl, N O, (C-Cs)alkynyl, benzyl, aryl, (Co-Cal)alkyl-(C-C)heterocy M cloalkyl, (Co-Cal)alkyl-(C-Cs)heteroaryl, (C-C)alkyl-N OCC (R), (Co-Cs)alkyl-NH CO)OR: (C-C)alkyl-OR, (C- HN H Cs)alkyl-C(O)OR, (C-C)alkyl-O(CO)R, or C(O)OR: and the other variables have the same definitions. 55 In other specific compounds of formula II, R is H or wherein R' is hydrogen or methyl. In a separate embodiment, (C-C)alkyl. the invention encompasses the use of enantiomerically pure In other specific compounds of formula II, R is (C-Cs) forms (e.g. optically pure (R) or (S) enantiomers) of these alkyl or benzyl. compounds. In other specific compounds of formula II, R is H. (C- Still other specific immunomodulatory compounds of the 60 Cs)alkyl, benzyl, CHOCH, CHCHOCH or invention belong to a class of isoindole-imides disclosed in U.S. Patent Application Publication Nos. US 2003/0096841 and US 2003/0045552, and International Application No. oci-Cy PCT/US01/50401 (International Publication No. WO 65 O 02/059106), each of which are incorporated herein by refer ence. Representative compounds are of formula II: US 8,715,677 B2 25 In another embodiment of the compounds of formula II, R' is III R O , 5 R2 Y N N N O --C).O well-C)S O M R7 R7 R3 X R6 R4 well-( ) 10 Yo and pharmaceutically acceptable salts, hydrates, Solvates, clathrates, enantiomers, diastereomers, racemates, and mix tures of stereoisomers thereof, wherein: wherein Q is O or S, and each occurrence of R is indepen 15 dently H. (C-C)alkyl, (C-C7)cycloalkyl, (C-C)alkenyl, one of X and Y is C—O and the other is CH or C=O; (C-Cs)alkynyl, benzyl, aryl, halogen, (Co-Cal)alkyl-(C-C) R is H or CHOCOR'; heterocycloalkyl, (Co-Cal)alkyl-(C-Cs)heteroaryl, (Co-Cs) alkyl-N(R), (C-Cs)alkyl-OR, (C-Cs)alkyl-C(O)CR, (i) each of R. R. R., or R', independently of the others, is (C-Cs)alkyl-O(CO)R, or C(O)OR, or adjacent occur halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon rences of R7 can be taken together to form a bicyclic alkyl or atoms or (ii) one of R', R. R. or R is nitro or -NHR and aryl ring. the remaining of R. R. R. or R are hydrogen; In other specific compounds of formula II, R is C(O)R. R is hydrogen or alkyl of 1 to 8 carbons In other specific compounds of formula II, R is (CO-C4) Rhydrogen, alkyl of 1 to 8 carbonatoms, benzo, chloro, or alkyl-C2-C5)heteroaryl, (C1-C8)alkyl, aryl, or (Co-Cal)alkyl 25 OR. fluoro; In other specific compounds of formula II, heteroaryl is R" is R7 CHR0 N(RR): pyridyl, furyl, or thienyl. R’ is m-phenylene or p-phenylene or -(CH2)— in In other specific compounds of formula II, R' is C(O)OR". which n has a value of 0 to 4; In other specific compounds of formula II, the H of C(O) 30 NHC(O) can be replaced with (C-C)alkyl, aryl, or benzyl. each of R and R taken independently of the other is Further examples of the compounds in this class include, hydrogen or alkyl of 1 to 8 carbon atoms, or RandR taken but are not limited to: 2-(2,6-dioxo-piperidin-3-yl)-1,3-di together are tetramethylene, pentamethylene, hexamethyl oxo-2,3-dihydro-1H-isoindol-4-ylmethyl-amide: (2-(2,6- ene, or —CH2CH2XCH-CH - in which X is —O—, dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4- 35 —S—, or —NH-; ylmethyl)-carbamic acid tert-butyl ester;4-(aminomethyl)-2- R" is hydrogen, alkyl of to 8 carbonatoms, or phenyl; and (2,6-dioxo(3-piperidyl)-isoindoline-1,3-dione; N-(2-(2,6- dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4- * represents a chiral-carbon center. ylmethyl)-acetamide: N-(2-(2,6-dioxo(3-piperidyl)-1,3- Other representative compounds are of formula: dioxoisoindolin-4-yl)methylcyclopropyl-carboxamide: 40 2-chloro-N-(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindo lin-4-yl)methyl)acetamide: N-(2-(2,6-dioxo(3-piperidyl))- 1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide: 3-1-oxo R1 O O R10 R8 4-(benzylamino)isoindolin-2-yl)piperidine-2,6-dione: 2-(2, 2 / R X N-CH-O-C-R7-C-N 6-dioxo(3-piperidyl)-4-(benzylamino)isoindoline-1,3- 45 N V N O R9 dione; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin M 4-yl)methylpropanamide: N-(2-(2,6-dioxo(3-piperidyl))- R3 Y R6 1,3-dioxoisoindolin-4-yl)methyl-3-pyridylcarboxamide: N-(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl) R4 methylheptanamide: N-(2-(2,6-dioxo(3-piperidyl)-1,3- 50 dioxoisoindolin-4-yl)methyl)-2-furylcarboxamide: {N-(2- (2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl) wherein: carbamoylmethyl acetate; N-(2-(2,6-dioxo(3-piperidyl)-1, one of X and Y is C—O and the other of X and Y is C—O 3-dioxoisoindolin-4-yl)pentanamide: N-(2-(2,6-dioxo(3- or CH: piperidyl))-1,3-dioxoisoindolin-4-yl)-2- 55 thienylcarboxamide: N-(2-(2,6-dioxo(3-piperidyl)-1,3- (i) each of R. R. R., or R', independently of the others, is dioxoisoindolin-4-yl)methyl (butylamino)carboxamide: halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon N-2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl) atoms or (ii) one of R', R. R. and R is NHR and the methyl(octylamino)carboxamide; and N-(2-(2,6-dioxo(3- remaining of R. R. R. and Rare hydrogen; piperidyl))-1,3-dioxoisoindolin-4-yl)methyl (benzylamino) 60 R is hydrogen or alkyl of 1 to 8 carbon atoms: carboxamide. Rishydrogen, alkyl of 1 to 8 carbonatoms, benzo, chloro, Still other specific immunomodulatory compounds of the or fluoro; invention belong to a class of isoindole-imides disclosed in U.S. Patent Application Publication Nos. US 2002/0045643, R’ is m-phenylene or p-phenylene or -(CH2)— in International Publication No. WO 98/54170, and U.S. Pat. 65 which n has a value of 0 to 4; No. 6,395,754, each of which is incorporated herein by ref each of R and R taken independently of the other is erence. Representative compounds are of formula III: hydrogen or alkyl of 1 to 8 carbon atoms, or RandR taken US 8,715,677 B2 27 28 together are tetramethylene, pentamethylene, hexamethyl of the other is hydrogen or alkyl of 1 to 8 carbonatoms, or R ene, or —CH2CH2XCHCH in which X* is —O , and R taken together are tetramethylene, pentamethylene, —S-, or —NH-; and hexamethylene, or —CH2CH2XCH-CH in which X* is R" is hydrogen, alkyl of to 8 carbon atoms, or phenyl. —O— —S— or —NH-; and Other representative compounds are of formula: R" is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl. Other specific immunomodulatory compounds of the invention include, but are not limited to, 1-oxo-2-(2,6-dioxo R O 3-fluoropiperidin-3-yl)isoindolines and 1,3-dioxo-2-(2,6-di oxo-3-fluoropiperidine-3-yl)isoindolines such as those R2 X NH 10 N described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of N O M which is incorporated herein by reference. Representative R3 Y R6 compounds are of formula: R4 15 in which NH one of X and Y is C—O and the other of X and Y is C—O or CH: each of R', R. R., and R', independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R', R. R. and R is nitro or protected amino and the remaining of R. R. R. and Rare hydrogen; and wherein: R is hydrogen, alkyl of 1 to 8 carbonatoms, benzo, chloro, 25 Y is oxygen or H and or fluoro. each of R', R. R. and R', independently of the others, is Other representative compounds are of formula: hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino. 30 Other specific immunomodulatory compounds of the R O invention include, but are not limited to, the tetra substituted R2 X NH 2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in N N O U.S. Pat. No. 5,798.368, which is incorporated herein by M reference. Representative compounds are of formula: R3 Y R6 35 R4 R / O R2 / NH in which: N one of X and Y is C—O and the other of X and Y is C—O 40 N O or CH: / (i) each of R', R. R. and R', independently of the others, R3 H is halo, alkyl of 1 to 4 carbonatoms, or alkoxy of 1 to 4 carbon R4 atoms or (ii) one of R', R. R. and R is NHR and the remaining of R. R. R. and Rare hydrogen; R is hydrogen, alkyl of 1 to 8 carbonatoms, or CO R” – 45 wherein each of R', R. R. and R, independently of the CH(R')NR'R' in which each of R7, R. R. and R' is as others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to herein defined; and 4 carbon atoms. R is alkyl of 1 to 8 carbonatoms, benzo, chloro, or fluoro. Other specific immunomodulatory compounds of the Specific examples of the compounds are of formula: invention include, but are not limited to, 1-oxo and 1,3-dioxo 50 2-(2,6-dioxopiperidin-3-yl)isoindolines disclosed in U.S. Pat. No. 6,403,613, which is incorporated herein by refer ence. Representative compounds are of formula:

55 R1 O O

N N O M C 60 V in which: R2 Y one of X and Y is C—O and the other of X and Y is C—O or CH: R is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, in which chloro, or fluoro; 65 Y is oxygen or H2, R’ is m-phenylene, p-phenylene or -(CH2)—in which a first of R' and R is halo, alkyl, alkoxy, alkylamino, n has a value of 0 to 4: each of RandR taken independently dialkylamino, cyano, or carbamoyl, the second of R' and R. US 8,715,677 B2 29 30 independently of the first, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl, and O O R is hydrogen, alkyl, or benzyl. 4. Y-R O M is / | Specific examples of the compounds are of formula: N-C-(CH2)-C-R 2 / Y. X V XI O

NH 10 in which the carbon atom designated C constitutes a center of chirality (when n is not zero and R' is not the same as R); one of X and X is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X" or X is hydrogen; each of R' and R independent of the other, is hydroxy or NH Z: R is 15 hydrogen, alkyl of one to six carbons, halo, or haloalkyl, Z is hydrogen, aryl, alkyl of one to six carbons, formyl, or acyl of wherein one to six carbons; and n has a value of 0, 1, or 2; provided that a first of R' and R is halo, alkyl of from 1 to 4 carbon if X is amino, and n is 1 or 2, then R' and Rare not both atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in hydroxy; and the salts thereof. which each alkyl is of from 1 to 4 carbon atoms, cyano, or Further representative compounds are of formula: carbamoyl; the second of R' and R, independently of the first, is O O hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of 4. Y-s: O from 1 to 4 carbon atoms, alkylamino in which alkyl is of 25 N M | from 1 to 4 carbonatoms, dialkylamino in which each alkyl is N- (CH)-C-R of from 1 to 4 carbon atoms, cyano, or carbamoyl; and X2 C R3 R is hydrogen, alkyl of from 1 to 4 carbon atoms, or XI benzyl. Specific examples include, but are not limited to, 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline. 30 in which the carbon atom designated C constitutes a center Other representative compounds are of formula: of chirality when n is not zero and R is not R; one of X and X is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X" or X is hydrogen; each of R' and Rindepen 35 dent of the other, is hydroxy or NH Z: R is alkyl of one to six carbons, halo, or hydrogen, Z is hydrogen, aryl or an alkyl or acyl of one to six carbons; and n has a value of 0, 1, or 2. Specific examples include, but are not limited to, 2-(4- NH amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-bu 40 tyric acid and 4-(4-amino-1'-oxo-1,3-dihydro-isoindol-2-yl)- 4-carbamoyl-butyric acid, which have the following structures, respectively, and pharmaceutically acceptable salts, Solvates, prodrugs, and stereoisomers thereof:

45 O O wherein: OH a first of R' and R is halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in N which each alkyl is of from 1 to 4 carbon atoms, cyano, or 50 carbamoyl; NH2 NH2 and the second of R' and R, independently of the first, is O hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino in which alkyl is of 55 from 1 to 4 carbonatoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl; and R is hydrogen, alkyl of from 1 to 4 carbon atoms, or O O benzyl. 60 NH2 Other specific immunomodulatory compounds of the N invention include, but are not limited to, 1-oxo and 1,3-diox oisoindolines substituted in the 4- or 5-position of the indo line ring described in U.S. Pat. No. 6,380.239 and co-pending NH2 OH. U.S. application Ser. No. 10/900.270, filed Jul. 28, 2004, 65 O which are incorporated herein by reference. Representative compounds are of formula: US 8,715,677 B2 31 32 Other representative compounds are of formula: -continued O O OH. / 'VY-R: O M is / | N-C-(CH2)-C-R N X2 CV R3 / OH O XI O NH O 10 O in which the carbon atom designated C constitutes a center of chirality when n is not zero and R' is not R; one of X" and Other specific examples of the compounds are of formula: X is amino, nitro, alkyl of one to six carbons, or NH Z, and the other of X" or X is hydrogen; each of R' and Rindepen 15 dent of the other, is hydroxy or NH Z: R is alkyl of one to O O six carbons, halo, or hydrogen, Z is hydrogen, aryl, oran alkyl 4. Y-s: O M is / | oracyl of one to six carbons; and n has a value of 0, 1, or 2; and N-C-(CH)-C-R the salts thereof. 2 / Y. Specific examples include, but are not limited to, 4-car X \, bamoyl-4-4-(furan-2-yl-methyl)-aminol-1,3-dioxo-1,3- X dihydro-isoindol-2-yl)-butyric acid, 4-carbamoyl-2-4- (furan-2-yl-methyl)-amino-1,3-dioxo-1,3-dihydro isoindol-2-yl)-butyric acid, 2-4-(furan-2-yl-methyl)- 25 wherein: amino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-4- one of X" and X is nitro, or NH-Z, and the other of X" or phenylcarbamoyl-butyric acid, and 2-4-(furan-2-yl X is hydrogen; methyl)-amino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)- each of R' and R, independent of the other, is hydroxy or pentanedioic acid, which have the following structures, respectively, and pharmaceutically acceptable salts, Solvate, NH Z: prodrugs, and stereoisomers thereof. R is alkyl of one to six carbons, halo, or hydrogen; Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of one to six carbons; and O 35 n has a value of 0, 1, or 2; and O OH, if COR and -(CH),COR" are different, the carbon atom designated C constitutes a center of chirality. N Other representative compounds are of formula: / NH2 40 NH O O O O O 4. \ -R2 O M is / 45 N-C-(CH-)-C-R1 O X2 / Y.

O NH2, X \,

50 N

/ OH wherein: O NH O O one of X" and X is alkyl of one to six carbons; 55 each of R' and R, independent of the other, is hydroxy or NH Z: R is alkyl of one to six carbons, halo, or hydrogen; O Z is hydrogen, phenyl, an acyl of one to six carbons, or an O NH 60 alkyl of one to six carbons; and n has a value of 0, 1, or 2; and N y s and if COR and -(CH2)COR" are different, the carbon OH atom designated C constitutes a center of chirality. O 65 Still other specific immunomodulatory compounds of the NH invention include, but are not limited to, isoindoline-1-one and isoindoline-1,3-dione substituted in the 2-position with US 8,715,677 B2 33 34 2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S. Pat. In one embodiment, this invention encompasses treatment No. 6,458.810, which is incorporated herein by reference. or prevention of cancer. Examples of cancer that can be Representative compounds are of formula: treated or prevented using methods of the invention include those described in Section 5.1.2, above. In some embodi ments, cancers to be treated or prevented using methods of the / O invention are metastatic. In other embodiment, specific can / NH cers that can be treated or prevented using methods of the invention are sarcoma, carcinoma, melanoma, lymphoma and N O M leukemia. X R2 : In another embodiment, this invention encompasses meth ods of vaccinating against cancer by reducing the inhibition R1 OH of anti-tumor immune response in a subject (e.g., a human) comprising administering to the Subject an immunomodula wherein: tory compound of the invention prior to the administration of 15 a cancer vaccine. This invention also encompasses methods the carbon atoms designated constitute centers of chiral of enhancing immune response to a cancer vaccine in a Sub ity; ject comprising administering to the Subject an immuno X is —C(O)— or —CH2—, modulatory compound of the invention prior to the adminis R" is alkyl of 1 to 8 carbon atoms or - NHR: tration of a cancer vaccine. Examples of cancer vaccines that R is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen; can be used in connection with methods of the invention and include those listed in Tables 1-4. In specific embodiment, R is hydrogen, cancers against which vaccination is performed are sarcoma, alkyl of 1 to 8 carbon atoms, unsubstituted or substituted carcinoma, melanoma, lymphoma and leukemia. In another with alkoxy of 1 to 8 carbon atoms, halo, amino, or alky specific embodiment, the cancer vaccine is an antigen modi lamino of 1 to 4 carbon atoms, 25 fied denritic cell vaccine, a peptide vaccine, a whole tumor cycloalkyl of 3 to 18 carbon atoms, cell vaccine, or a viral vector vaccine. phenyl, unsubstituted or substituted with alkyl of 1 to 8 In another embodiment, this invention also encompasses carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or treatment or prevention of an infectious disease. Examples of alkylamino of 1 to 4 carbon atoms, infectious diseases that can be treated or prevented using benzyl, unsubstituted or substituted with alkyl of 1 to 8 30 methods of the invention are described in Section 5.1.2, carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or above. In some embodiments, infectious diseases that can be alkylamino of 1 to 4 carbon atoms, or -COR in which treated or prevented using methods of the invention include R is hydrogen, those caused by viruses, bacteria, fungi, and parasites. alkyl of 1 to 8 carbon atoms, unsubstituted or substituted In another embodiment, this invention encompasses meth with alkoxy of 1 to 8 carbon atoms, halo, amino, or alky 35 ods of vaccinating against an infectious disease by reducing lamino of 1 to 4 carbon atoms, the inhibition of immune response in a subject (e.g., a human) cycloalkyl of 3 to 18 carbon atoms, comprising administering to the Subject an immunomodula phenyl, unsubstituted or substituted with alkyl of 1 to 8 tory compound of the invention prior to the administration of carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or a vaccine against an infectious disease. This invention also alkylamino of 1 to 4 carbon atoms, or 40 encompasses methods of enhancing immune response to a benzyl, unsubstituted or substituted with alkyl of 1 to 8 vaccine against an infectious disease in a subject comprising carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or administering to the Subject an immunomodulatory com alkylamino of 1 to 4 carbon atoms. pound of the invention prior to the administration of the All of the compounds described can either be commer vaccine. Examples of infectious diseases against which a cially purchased or prepared according to the methods 45 Subject can be vaccinated according to methods of the inven described in the patents or patent publications disclosed tion are described in Section 5.1.1, above. In a specific herein. Further, optically pure compounds can be asymmetri embodiment, infectious diseases are those caused by viruses, cally synthesized or resolved using known resolving agents or bacteria, fungi, and parasites. In a specific embodiment, the chiral columns as well as other standard synthetic organic vaccine against an infectious disease is hepatitis B vaccine. chemistry techniques. 50 5.5 Methods of Administration Compounds used in the invention may be Small organic Methods encompassed by this invention comprise admin molecules having a molecular weight less than about 1,000 istering one or more immunomodulatory compounds, or a g/mol, and are not proteins, peptides, oligonucleotides, oli pharmaceutically acceptable salt, Solvate, Stereoisomer, or gosaccharides or other macromolecules. prodrug thereof, to a Subject (e.g., a human) prior to the It should be noted that if there is a discrepancy between a 55 exposure to or administration of an immunogen or an aller depicted structure and a name given that structure, the gen. depicted structure is to be accorded more weight. In addition, Any route of administration may be used. For example, an if the stereochemistry of a structure or a portion of a structure immunomodulatory compound can be orally, parenterally, is not indicated with, for example, bold or dashed lines, the transdermally, rectally, Sublingually, mucosally, or nasally structure or portion of the structure is to be interpreted as 60 administered. In addition, an immunomodulatory com encompassing all stereoisomers of it. pounds can be administered in a form of pharmaceutical 5.4 Methods of Treatment and Prevention composition and/or unit dosage form. Suitable dosage forms This invention encompasses methods of treating and/or include, but are not limited to, capsules, tablets (including preventing (e.g., prophylactic treatment such as vaccination) rapid dissolving and delayed release tablets), powder, syrups, of various disorders using the dosing regimen involving an 65 oral Suspensions and Solutions for parenteral administration. immunomodulatory compounds of the invention as described Pharmaceutical compositions may contain one of more phar herein. maceutically acceptable excipients. See, e.g., Rowe et al., US 8,715,677 B2 35 36 Handbook of Pharmaceutical Excipients, 4" Ed. (2003), and 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperi entirety of which is incorporated herein by reference. In addi dine-2,6-dione (lenalidomide), but not thalidomide, inhibits tion, an immunomodulatory compound of the invention may the suppressive function of these cells. The inhibition of T be included in a kit, which may comprise an immunogen oran regulatory cell function and production by these compounds allergen, one or more other active ingredients, and devices 5 was shown to be not due to any cytotoxic or apoptotic effects and directions for administration. Other ingredients (e.g., of the IMiDs on the cells, but the inhibition of function was immunogen, allergen, and other active ingredients) may be associated with a decrease in FOXP3 expression in CTLA4+ included in the same formulation with the immunomodula CD25'8"CD4+ cells. tory compound of the invention, or in separate formulations. 6.1.1 Effects on T. Function The specific amount of the agent will depend on the spe 10 Regulatory T cells were isolated by the Dynal Tregulatory cific agent used, the type of disease or disorder being treated cell isolation kit, and treated for 24 hours with varying con or managed, and the amount(s) of an immunomodulatory centrations of an immunomodulatory compound (ActimidTM compound of the invention and any optional additional agents or lenalidomide) or DMSO. The cells were washed and incu concurrently administered to the patient. Typical dosage bated at a 1:2 ratio with CD25CD4" cells, which were also forms of the invention comprise an immunomodulatory com 15 isolated by the Dynal T regulatory cell isolation kit. Results pound of the invention or a pharmaceutically acceptable salt, were expressed as the mean '% change in proliferation com Solvate, Stereoisomer, or prodrug thereof in an amount of pared to the cpms obtained from DMSO treated CD25" cells from about 0.001 to about 150 mg. In particular, dosage forms incubated with CD25 cells. As shown in FIG. 2, pre-treat comprise an immunomodulatory compound of the invention ment of CD25"CD4" cells with the IMiDs tested significantly or a pharmaceutically acceptable salt, Solvate, Stereoisomer, increased the proliferation of CD25 cells in the presence of or prodrug thereof in an amount of about 0.001, 0.01, 0.1. 1, CD25"CD4" cells as compared to the DMSO treated CD4" 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200 mg. In CD25" cells. Thalidomide showed little effect under these a particular embodiment, a dosage form comprises assay conditions. The results suggest that the IMiDS tested 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione reduce or inhibit the suppressive activity of regulatory T cells. in an amount of about 0.001, 0.01, 0.1. 1, 2, 5, 10, 25 or 50 mg. 25 6.1.2 Effects on Foxp3 Expression In some embodiments, this invention encompasses admin CD4"CD25" cells were incubated for 24 hours with vary istration of racemic mixture, optically pure (R)-isomer, or ing concentrations of DMSO, ActimidTM, lenalidomide, or optically pure (S)-isomer of 4-(amino)-2-(2,6-dioxo(3-pip thalidomide and then washed twice with RPMI medium. eridyl))-isoindoline-1,3-dione. In one specific embodiment, Cells were stained with CD152-PE, CD4-PERCP, and CD25 the racemic 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindo 30 APC. Intracellular Foxp3 staining and CD152 staining were line-1,3-dione is administered at an amount of 1, 2, 5, 10, or carried out after permeabilizing the CD4" CD25" cells. 25 mg per day. As (S)-isomer of 4-(amino)-2-(2,6-dioxo(3- Results were expressed as percentage of expression of Foxp3 piperidyl))-isoindoline-1,3-dione is reported to have a higher in the CD4"CD25" population or the CD4"CD25 popula potency than the racemic mixture, a lower dose can be given tion. As shown in FIG. 3, cells pre-treated with the IMiDs when (S)-isomer is used. For examples, (S)-4-(amino)-2-(2, 35 showed inhibition of Foxp3 expression, while DMSO and 6-dioxo(3-piperidyl))-isoindoline-1,3-dione can be adminis thalidomide showed little effects. The results show that the tered at an amount of 0.01, 0.1, 1.2.5, 5, or 10 mg per day. inhibition of T cells by the IMiDs tested may be associated (R)-isomer of 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindo with the compounds’ ability to inhibit Foxp3 expression. line-1,3-dione can be administered at an amount comparable 6.13 Effects on Level of T. Cells to the racemic mixture. 40 PBMCs were treated with 150 UAml of IL-2. Some of the In a specific embodiment, a dosage form comprises 3-(4- cultures were also treated with Actimid TM or lenalidomide. amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-di Cells were stained with CD25-FITC/CD152-PE/CD4 one in an amount of about 0.001, 0.01, 0.1, 1, 5, 10, 25 or 50 PerCP/NKG2D-APC and analyzed using a FACSCalibur. As mg. Typical dosage forms comprise the second active ingre shown in FIG. 4, levels of CD4, CD25high, CD52high dient in an amount of 1 g to about 1000 mg, from about 0.01 45 expressing cells are reduced in groups pre-treated with an to about 500 mg. from about 0.1 to about 350 mg. or from IMiD as compared to the untreated group. The results suggest about 1 to about 200 mg. This invention also encompasses the that the IMiDs of the invention also decrease the levels of use of racemic mixture, (S)-isomer, and (R)-isomer of 3-(4- regulatory T cells or inhibits the proliferation of such cells. amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-di 6.2 Effects on Acquired Antibody Resistance one. Typically, racemic 3-(4-amino-1-oxo-1,3-dihydro 50 Rituximab-resistant cell lines (RRCL) were generated by isoindol-2-yl)-piperidine-2,6-dione can be administered at an chronic exposure of Rajicells to escalating doses of rituximab amount of 1, 5, 10, 15, 25, or 50 mg per day. Optical isomers alone (2R) or along with human complement (4RH). Func also can be administered at an amount comparable to racemic tional assays including antibody-dependant cellular cytotox mixture. Doses can be adjusted depending on the type of icity (ADCC) and complement-mediated cytotoxicity disease or disorder being treated, prevented or managed, and 55 (CMC) were performed to demonstrate resistance to ritux the amount(s) of an immunomodulatory compound of the imab. To study the effects of lenalidomide-priming of invention and any optional additional agents concurrently PBMC's against RRCL, peripheral blood mononuclear cells administered to the patient, which are all within the skill of from healthy donors were cultured with either DMSO or the art. lenalidomide (at final concentrations of 10 or 20 ug/ml), with 60 or without IL-2 (20 IU/ml), over a 5-day period at 37°C.,5% 6. EXAMPLES CO. Parentral Raji, and RRCL (2R and 4RH) were labeled with 'Cr and exposed to either rituximab or trastuzumab 6.1 Effects of IMiDs on Regulatory T Cells (Isotype control at 20 ug/ml) in the presence of an IMiD or An assay in which the ability of isolated T to suppress control stimulated-PBMCs (Effector:Target ratio of 40:1). anti-CD3 mAb activated CD4+CD25-cells was performed. 65 'Cr release was measured and the percentage of lysis was Results showed that pre-incubation of T with 4-(amino)-2- calculated. Statistical differences were analyzed by chi (2,6-dioxo(3-piperidyl)-isoindoline-1,3-dione (ActimidTM) square test. US 8,715,677 B2 37 38 In vitro exposure of PBMC to IMiD+/-IL-2 improved two key transcription factors by the IMiD favors Th1 differ rituximab-associated ADCC in RRCL. Exposure of PBMC to entiation of human naive CD4 T cells. Enhancement of T-bet IMiD+/-IL-2 for 5 days led to a statistically significant by the IMiD results in increased tyrosine phosphorylation of increase in rituximab-mediated ADCC in 2R cells IMiD T-bet, increased expression of IL-12RB2, and increased mean % lysis 26.9+/- 1.18% IMiD+IL-2 mean % lysis IFN-Y production, compared to treatment with anti-CD3 38.4+/-4.14% when compared to control-stimulated alone. PBMC's mean % lysis 17.6+/-5.6%). Similar effects were A similar effect of the IMiD on T-betand GATA-3 was also observed in 4RH cells. The mean % of lysis by ADCC for observed in differentiated human Th2 cells in vitro under Th2 combination IMiD/IL-2 exposed PBMC's on 4RH cells was polarizing condition. The intracellular cytokine staining of found to be highest at 38.4+/-4.1%, as compared to IMiD 10 IL-4 and IFN-Y on re-stimulated Th2 cells showed that the (mean % lysis 26.5+/- 1.83%) or vehicle exposed PBMC's IMiD reduced the number of IL-4 producing cells and (mean % lysis 17.6+/-5.69%) (P=0.01). These results sug increased the number of IFN-Y producing cells in the presence gest that modulation (e.g., PBMC-priming) of the immune of plate bound anti-CD3 antibody. The effect of the IMiD on system by the IMiD of the invention (+/-IL-2) improves polarized Th2 cells includes reversal of Th2 cell differentia rituximab anti-tumor activity and may partially overcome 15 tion and enforcement of IFN-Y expression in IL-4 positive rituximab resistance in RRCL via augmentation of ADCC. cells, which is greatly enhanced by addition of exogenous 6.3 Effects on Growth Arrest and Apoptosis IL-12. These results suggest that the IMiDs of the invention Direct effects of IMiDs on NHL tumor cells were tested by not only preferentially induce Th1 immune response by treating Raji cells with IMiDs alone, or in combination with enhancing T-bet, but also inhibit Th2 lineage commitment by anti CD20 antibodies B1 or rituxan. IMiD 1 alone caused up reducing GATA-3 expression. to 40% inhibition of proliferation at 10 uM in Raji cells, 6.6 Effects on T Cell Activation which corresponded to G1 arrest. In combination with B1, The Gab proteins, including Gab1, Gab2 and Gab3 com ActimidTM showed a small additive effect at 10 uM, while prise a growing family of phospho-tyrosine regulated Scaf lenalidomide effects were minimal up to 10 uM. In combina folding molecules involved in RTKsignal transduction. Phos tion with rituxan. ActimidTM showed a slight additive effectat 25 phorylation of Gab1 in B cells is associated with P13-kinase 10 uM, and lenalidomide showed the same at 50 uM. activity and cell proliferation. While Gab1 is expressed in B A co-culture assay of PBMC and NHL tumor cells were cells, only Gab2 is expressed in T cells. Although Gab2 is developed as an in vitro model of tumor-host immune system tyrosine phosphorylated upon TCR activation by ZAP-70, it interaction, to further explore the anti-tumor potential of functions as a negative regulator of TCR signaling via a Shp-2 IMiDs in NHL cells. This assay is non-radioactive and flow 30 dependent mechanism. Overexpression of Gab2 in T cells cytometry based. Using Raji and PBMC, it was shown that results in the inhibition of IL-2 production (Yamasaki et al., J. pre-treatment of PMBC with an IMiD can enhance the PBMC Biol. Chem., 2001). The effectoflenalidomide on Gab2 phos activity in inducing Raji cell apoptosis in a dose dependent phorylation and activation in anti-CD3/CD28 stimulated Jur manner. In addition, it was shown that pre-treatment of Raji kat T cells was examined. Lenalidomide inhibited Gab2 cells with an IMiD can further enhance the apoptosis induced 35 phosphorylation dose-dependently (with approximately 50% by PBMC pre-treated with an IMiD. These results suggest inhibition at about 1 uM) in a manner that correlated with T that the IMiDs of the invention directly induce NHL tumor cell costimulation and enhancement of IL-2 production. The cell growth arrest and effectively enhance tumor cell apopto results show that the mechanism of action of lenalidomide is sis induced by PBMC. therefore consistent with inhibition of phosphorylation of 6.4 Effects on HSC Expansion 40 Gab2 in anti-CD3/CD28-stimulated T cells. The ability of IMiDs to enhance the expansion of hemato 6.7 Effects on Y&T Cells poietic stem cells (HSC) ex vivo in combination with growth 6.7.1 Materials and Methods factors were tested. It was shown that the IMiDs of the inven Phenotyping of PBMC Preparations Stimulated with IL-2 tion dramatically enhance the expansion of CD34+ cells in a and IPPIMDS: serum-free system, achieving up to 100-fold expansion after 45 PBMC preparations were obtained and treated weekly with 14 days in culture. In addition, the IMiDs of the invention IL-2 and IPP (150 units/ml and 10 uM respectively). Expres enabled a preferential expansion of CD34+CD38-cells, a sion of Öy TCR and NKG2D were measured by FACS overa more immature phenotype. period of three weeks. IMiDs showed similar activities on HSC from all sources Generation of Y&T Cells: tested: bone marrow, cord blood and peripheral blood 50 PBMC preparations were treated with IL-2 (150 units/ml) (steady-state or G-CSF-mobilized). It was also shown that and IPP (25 uM) weekly. Cultures were split and replenished IMiDs can efficiently expand CD34+ cells isolated from fro weekly with fresh IL-2 and IPP and % yö TCR+ve cells Zen cord blood units. determined by FACS. After 3-4 weeks lyö T cells were purified Global gene expression (Affymetrix) analysis of IMiDs by negative magnetic separation using CD4+ and CD8+ expanded CD34+ cells revealed that the IMiDs of the inven 55 Dynalbeads and maintained in IL-2. tion modulate several genes involved in cell differentiation, Measurement of Cytokine Production in Purified Yö T cell adhesion and cell self-renewal. The IMiDs of the inven Cells and Fresh Yö Cells in PBMC Preparations: tion also upregulated many genes involved in immune Purified Yö T cells were stimulated with IPP-IMiDs (10 responses and antigen presentation. g/mL) or with the MM cell line RPMI-8226 (+IMiDs (10 6.5 Effects on T Cell Differentiation 60 g/mL)) in 24 well plates and were incubated 8-72 hours. Effects of IMiDs on T cell differentiation were investigated Cell-free supernatants were collected and stored at -70° C. using various methods. It was demonstrated that, in combi until assayed by ELISA. IFN-y, TNF-C. and IL-2 were mea nation with anti-CD3 stimulation, the IMiD of the invention sured by ELISA (BD pharmingen). For fresh Öy preps, directly increases expression of Th1 transcription factor T-bet PBMCs were stimulated with plate bound anti-CD3 (1.25 via enhanced T-bet RNA transcription at 4 hours after stimu 65 ug/ml) for 48 hours and the expression of TNF-C. IFNY, IL-2 lation. A concomitant decrease in expression of Th2 tran and IL-4 were measured by intracellular FACS on cells scription factor GATA-3 was also observed. The regulation of stained for yö TCR. US 8,715,677 B2 39 40 Measurement of Apoptosis in Öy Cells: with increasing tumor to YöT cells ratio. The results show that Gamma delta T cells were treated with a single dose of 25 immunomodulatory compounds of the invention enhance the uMIPP and weekly with 150 U/ml of IL-2 for 4 weeks and 3 production of IFN-y by Yö T cells, and the effects increase in days. Cells were then either left untreated or treated with response to increasing tumor to Yö T cells ratio. ActimidTM, IPP or ActimidTM and IPP. Apoptosis was 5 6.7.6 Effects on Cytotoxicity of Y&T Cells assessed by staining of cells with annexin V PE and 7-AAD at Gamma delta cells were treated with a single dose of 25uM various time points and analysis using a FACSCalibur. IPP and weekly with 150 U/ml of IL-2 for 22 days. RPMI Cytotoxicity Assays: 8226 target cells were incubated overnight with 50 uMpam Gamma delta T cells were treated with a single dose of 25 idronate, then treated with 3 MBq 51 Cr. Target and effector uMIPP and weekly with 150 U/ml of IL-2 for 3 weeks and 1 10 cells were incubated at various ratios with fresh Actimid TM day. RPMI-8226 target cells were incubated overnight with and chromium release assayed after 4 hours. ActimidTM was 50 uMpamidronate then treated with 3MBq 51Cr. Target, and also added to some wells for the 22 day pretreatment with effector cells were incubated at different ratios and chromium IL-2 and IPP (FIG.9A) or just for the 4 hr chromium release release was assayed after 4 hours. To determine the effects of assay (FIG.9B). ActimidTM, the compound was either included in the 22 day 15 As shown in FIG. 9, the addition of Actimid TM during preincubation before the assay and in the chromium release either the pretreatment or the chromium release assay step, or was included during the chromium release assay. enhanced the cytotoxicity of Yö T cells toward RPMI-8226 6.7.2 Effects on the Expression of Y&T Cells and NKG2D MM cell lines, although a better effect was observed with the PBMCs were treated with a single dose of 25 uMIPP and addition of ActimidTM during the pretreatment of period. The then weekly with 150U/ml of IL-2. In addition, some cultures results suggest that immunomodulatory compounds of the were treated with 10 uM ActimidTM or lenalidomide. IL-2 invention enhance the cytotoxicity of Yö T cells toward tumor treated cells were stained with CD25 FITC/CD4 PE/CD3 cells, and the effects may be improved by pretreating the PerCP/NKG2D APC, and IL-2 plus IPP treated cells were tumor cells with the compounds of the invention. stained with Öy TCR FITC/alpha beta TCR PE/CD3 PerCP/ 6.8 Effects on Invariant NKT Cells NKG2D APC and analysed using a FACSCalibur. 25 The establishment of highly purified primary invariant As shown in FIG. 5, cells treated with an immunomodula NKT (iNKT) cell lines from health donors and multiple tory compound of the invention exhibited higher yö T cells myeloma (MM) patients has been tested, and the effects of and NKG2D expression. The results show that immuno IMiD 2 on iNKT cells were further explored. iNKT cells modulatory compounds of the invention enhance the expres derived from peripheral blood or bone marrow mononuclear sion of yöT cells and NKG2D in PMBCs activated with IL-2 30 cells were enriched with anti-TCRVC. 24 mAb or anti-6B11 and IPP mAb and further expanded by several rounds of stimulation 6.7.3 Effects of Apoptosis of y& T Cells with C-GalCer-loaded dendritic cells. Phenotype analysis Gamma delta T cells were treated with a single dose of 25 confirmed 95% purity in expanded iNKT cell lines. No sig uMIPP and weekly with 150U/ml of IL-2 for 31 days. Cells nificant phenotypic difference was observed in iNKT cells were then either left untreated or treated with ActimidTM, IPP 35 between healthy donors and MM patients. or ActimidTM and IPP in combination. Apoptosis was Majority of iNKT cells expressed CD161 and CD28, assessed by staining of cells with annexin V PE and 7-AAD at whereas CD56 expression was at very low level. Following the stated time points and analysis using a FACSCalibur. anti-CD3 or C-GalCer-loaded dendritic cells stimulation, Annexin V PE negative/7-AAD negative cells are designated iNKT cells showed strong proliferative activity as measured live, annexin V PE positive/7-AAD negative early apoptotic, 40 by H-TdRincorporation assay and production of IFN-Y mea annexin V PE positive/7-AAD positive late apoptotic and sured by ELISA. annexin V PE negative/7-AAD positive dead. Next, the effects of IMiD 2, which is known to enhance T As shown in FIG. 6, ActimidTM offered protection against cell costimulation and NK cell activity, on iNKT cells were apotosis in Yö T cells with or without IPP. The results suggest evaluated. From the tests, it was observed that IMiD 2 that immunomodulatory compounds of the invention protect 45 enhances anti-CD3 mediated proliferation of expanded iNKT against apotosis of Yö T cells. cells by 1.4 fold, and the enhanced expression and fluorescent 6.7.4 Effects on Cytokine Production by Yö T Cells intensity of CD25 (MFI 68.6 versus 28.5) on iNKT cells The effects of Actimid TM on IFN-y, TNF-C., and IL-4 were treated with IMiD 2 compare to untreated iNKT cells. Addi examined in freshly prepared Yö T cells and Yö T cell lines tionally, compared to the control group stimulated with stimulated with IPP. As shown in FIG. 7A, ActimidTM 50 C-GalCer-loaded dendritic cells alone, IMiD 2 plus C-Gal enhanced the production of both IFN-Y and TNF-C. in TCRYö Cer-loaded DC also enhanced the production of IL-2. These cells from within a freshly prepared PMBC population. In results provide the preclinical feasibility and rationale to addition, as shown in FIG. 7B, ActimidTM enhanced the pro clinically evaluate the efficacy of adoptive transfer of iNKT duction of IFN-Y, but not IL-4, in Yö T cells stimulated with cells in MM. Additionally, the results demonstrate the ability IPP. The results show that immunomodulatory compounds of 55 of the IMiDs of the invention to augment the immunoreactiv the invention stimulate the production of IFN-Y and TNF-C. ity of iNKT cells, suggestive of their use in enhancing iNKT but not IL-4. cell mediated immunotherapy in myeloma. 6.7.5 Effects on IFN-Y Production in Response to Varying 6.9 Use with Hepatitis B Vaccine Tumor to Y& T Cells Ratio A two-center, randomized, double-blind, placebo-con Tumor cells pre-incubated with (FIG. 8B) or without (FIG. 60 trolled trial is designed. A single dose of Hepatitis B vaccine 8A) pamidronate were incubated with Öy T cells at different is administered to subjects. An IMiD or placebo is adminis tumor (RPMI-8226 MM) to Y& T cells ratios as indicated in tered to 64 patients for 7 days prior to and 7 days after the FIG.8. Some of the cells were further treated by Actimid TM. vaccine. Collection of blood samples for immune analysis is Intracellular IFN-gamma production was measured by flow performed prior to the initiation of the IMiD administration, cytometry. 65 at the time of vaccination, and 7, 14, and 28 days after vacci As shown in FIGS. 8A and 8B, ActimidTM augmented nation. Safety assessments is performed at day 14, the last day IFN-Y production by Yö T cells. IFN-Y production increased of study drug. US 8,715,677 B2 41 42 Subjects may opt for 2" and 3" doses of vaccine in order compounds, materials, and procedures. All Such equivalents to complete the usual course of hepatitis B vaccination. Opt are considered to be within the scope of the invention and are ing for additional vaccinations is not a requirement of this encompassed by the appended claims. study. Patients opting to receive the second (day 28) and 3" (6 month) vaccination may have their blood samples collected What is claimed is: prior to the 2" and 3" and 1 month after 3" vaccination. The 1. A method of eliciting an enhanced immune response 28 day blood draw serves as the blood draw prior to the 2" from an immunogen in a Subject comprising administering to dose of vaccine. The blood draws one month after the 2" and the Subject an immunomodulatory compound prior to initial 3 dose are not required for subjects wishing to receive the 2" introduction of the immunogen as a vaccine to the Subject, and 3" dose of vaccine. 10 wherein the immunomodulatory compound is a compound of The effect of the IMiD on the response to hepatitis B formula I, or a pharmaceutically acceptable salt or stereoiso vaccine in Subjects with plasma cell dyscrasias, as measured mer thereof: by change in antibody titer against hepatitis B surface antigen (HbSAg), can be determined following the above procedures. In addition, serum and blood cells can be collected to: a) 15 O assess the development of T cell responses against HbSAg following vaccination; b) identify phenotypic changes in X R. NH peripheral blood cells following the IMiD administration N O M especially with regard to CD3, CD4, CD8 T cells, and NKand OCY NKT cells; and c) determine changes in gene expression HN profile of immune cells before and after the treatment of the IMiD using micro array protocols. 6.10 T. Cell Phenotyping and Functional Analyses from wherein: Patients Undergoing Lenalidomide Treatment one of X and Y is C—O, the other of X and Y is C=O or Patients with any malignancy which are selected for lena 25 CH: lidomide treatment in are asked to participate in this study. R’ is hydrogen or lower alkyl. The cycle of dosing for the patients selected for lenalidomide 2. The method of claim 1, wherein the immunomodulatory treatment is 3 weeks of dosing with 25 mg lenalidomide daily, compound is administered from about 10 days to about 12 followed by 1 week without dosing, followed by three more hours prior to the introduction of an immunogen. weeks of dosing, in repeated cycles. Forty ml samples of 30 3. The method of claim 2, wherein the immunomodulatory blood are collected into heparin tubes and 5 mls into serum compound is administered from about 7 days to about 12 tubes at time points from 1 hour to 24 hrs before the first hours prior to the introduction of an immunogen. administration of lenalidomide (25 mg/dose) and at 21 days 4. The method of claim3, wherein the immunomodulatory and 49 days after dosing. compound is administered from about 5 days to about 1 day The blood in the heparin tubes is layered onto histopaque 35 prior to the introduction of an immunogen. and spun for 25 minutes at 600 g to separate the buffy coat 5. The method of claim 4, wherein the immunomodulatory layer. The buffy coat containing the peripheral blood mono compound is administered from about 3 days to about 1 day nuclear cells and malignant haematogical cells is isolated. prior to the introduction of an immunogen. The cells isolated are subjected to the following procedures: 6. The method of claim 1, which further comprises a sec 6.10.1 Phenotype Analysis Using a FACscalibur Machine 40 ond administration of an immunomodulatory compound after Dominant phenotypes of the PBMCs freshly isolated from the introduction of an immunogen. each patient are analyzed, and the percentage of cells in the 7. The method of claim 6, wherein the immunomodulatory patients that are of a regulatory T cell phenotype (CD4" compound is administered from about 12 hours to about 10 CD25" positive cells, staining positive also for FOXP3 and days after the introduction of an immunogen. CTLA-4) is measured. 45 8. The method of claim 7, wherein the immunomodulatory 6.10.2 Isolation of CD4"CD25 Cells from the Patients compound is administered from about 12 hours to about 7 PBMCS days after the introduction of an immunogen. CD4"CD25" cells and CD4"CD25 cells are isolated from 9. The method of claim 8, wherein the immunomodulatory the patients' PBMCs using standard magnetic bead kits (In compound is administered from about 1 day to about 5 days vitrogen). The ability in-vitro of the CD4"CD25" cells to 50 after the introduction of an immunogen. inhibit the proliferation of CD4"CD25 cells, upon stimula 10. The method of claim 9, wherein the immunomodula tion with anti-CD3, is assessed. tory compound is administered from about 1 day to about 3 6.10.3 Analysis of Serum days after the introduction of an immunogen. 11. The method of claim 1, wherein the immunogen com Serums are analysed for TGF-beta, IL-10, IL-4, IL-6, prises a tumor antigen. IFN-Y and TNF-C. concentrations, using methods described 55 herein as well as those well-known in the art. 12. The method of claim 1, wherein the immunomodula All of the references cited herein are incorporated by ref tory compound is 4-(amino)-2-(2,6-dioxo(3-piperidyl)- isolindoline-1,3-dione. erence in their entirety. While the invention has been 13. The method of claim 12, wherein the immunomodula described with respect to the particular embodiments, it will tory compound is enantiomerically pure. be apparent to those skilled in the art that various changes and 60 modifications may be made without departing from the spirit 14. The method of claim 1, wherein the immunomodula and scope of the invention as recited by the appended claims. tory compound is 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2- The embodiments of the invention described above are yl)-piperidine-2,6-dione. 15. The method of claim 14, wherein the immunomodula intended to be merely exemplary, and those skilled in the art tory compound is enantiomerically pure. will recognize, or will be able to ascertain using no more than 65 routine experimentation, numerous equivalents of specific k k k k k