Expi293 Expression System Boosting Protein Expression to >1 G/L in a HEK 293 Mammalian Transient Protein Production System

Expi293 Expression System Boosting protein expression to >1 g/L in a HEK 293 mammalian transient protein production system

Boosting protein expression to >1g/L in the next generation mammalian transient protein production system: Expi293™

SanjaySanjay Vasu Va*, Chao*, Chao Yan Yan Liu† ,Liu Isabel+, Isabel Cisneros*, Cisneros*, Meredith Meredith Jones‡, SuzyJones Van++, Le*,Suzy Jon Van Zmuda Le*,‡ ,Jon Jeff ZmudaRogers*++ , Jeff Rogers*. *Thermo Fisher Scientific, Life Sciences Solutions, ++ † ‡ + 5781Thermo Van FisherAllen ScientifiWay, Carlsbad, c Corporation, CA 92008, Life Sciences USA. ThermoSolutions, Fisher *Carlsbad, Scientific, CA. Grand Life Island, Sciences NY. Frederick,Solutions, MD. 7305 Executive Way, Frederick, MD 21704,Expi B2 VLPExpi USA. B2 VLP FreeStyleThermo B2FreeStyle VLP B2 VLP Saturation BindingSaturation Curve Binding CurveSaturation BindingSaturation Curve Binding Curve with and withoutwith and100 withoutµM cold100withµM coldand withoutwith and100 withoutµM cold100 µM cold Fisher Scientific, Life Sciences Solutions, 3175 Staley Rd, Grand Island, NY 14072, USA. Propanolol,Propanolol, Propanolol,Propanolol, using 0.5 µgusing VLP/reaction 0.5 µg VLP/reactionusing 0.5 µgusing VLP/reaction 0.5 µg VLP/reaction

40 40 40 40

Expi B2 VLP FreeStyle B2 VLP AbstractAbstract ResultsResults Protein quality and30 functionality30 Saturation Binding 30Curve is maintainedTotal30 Bound SaturationTotal Bound Binding Curve Total Bound Total Bound with and without 100 µM cold with and without 100 µM cold Propanolol, Specific Bound SpecificPropanolol, Bound Specific BoundSpecific Bound using 0.5 µg VLP/reaction3 x Non-Specific BoundusingNon-Specific 0.5 µg VLP/reaction Bound Non-Specific BoundNon-Specific Bound Transient expression provides a rapid, fl exible, and >10x 20 20 20 20 40 yield Expi293™ 40 FreeStyle™ Transient expression provides a rapid, flexible, and UniqueUnique high-density High-Density medium MediumCell Cell line Lineoptimization Optimization Proteinyield Expi293™ quality andFreeStyle™ functionality are maintained 12 25 50 12 25 50 ng 30 Total Bound 30 Total Bound economical protein production alternative to the hIgG titer (passage-‐to-‐passage) 10 10 10 Specific Bound10 Specific Bound 16 60 Non-Specific Bound Non-Specific Bound 1200 100 1200 economical protein production alternative to the time- Day 5 20 20 75 d5 50 14 time-consuming and costly process of generating 0 0 10 0 0 10 50 1000 40 Radioactivity Bound (pmol/mg) Radioactivity Radioactivity Bound (pmol/mg) Radioactivity Radioactivity Bound (pmol/mg) Radioactivity 0 5 Bound (pmol/mg) Radioactivity 010 515 1020 15025 20305 25010 30515 1020 1525 2030 25 30 % Viability Day 6 >10x 12 25 d6 3x consuming and costly process of generating and ) Expi293 Media 25 16 6 yield 0 0 100 1,200 3yield Expi2933 3 FreeStyle3 Radioactivity Bound (pmol/mg) Radioactivity 0 0 5 10 15 20 25 30 Bound (pmol/mg) Radioactivity [ 0H] DHA5 10 (nM)[15 H]20 DHA25 (nM)30 and selecting stable, high-expressing cell lines. 2 3 4 5 6 7 8 Test Media 1 800 40 [ H] DHA (nM)[ H] DHA (nM) 40 10 1000 75 Day 7 3 3 14 Test Media 2 d7 Expi293 FreeStyle [ H] DHA (nM) [ H] DHA (nM) selecting stable, high-expressing cell lines. However, 50 1,000 30 Total bound 30 Total bound 8 Test Media 3 600 % Viability Specific bound Total Bound TotalSpecificSpecific Bound bound Bound 12 ■ Day 5 Total BoundTotal BoundTotalSpecificSpecific Bound Bound Bound Total Bound Specific Bound 25 ( µ g/mL) However, there is a growing demand for the higher ) 12 ng 25 ng 50 ng 12 ng 25 ng 50 ng hIgG (ug/ml) Bmax 31.41 30.08Nonspecific bound Bmax 30.01 Nonspecific29.14 bound

6 Expi293 medium Bmax 30.01Bmax 30.0129.14pmol/mg protein ■ Day 6 Bmax 20 31.41Bmax 31.4130.08pmol/mg protein 20 6 0 Kd 1.143 1.069 Kd 0.7543 1.057 there is a growing demand for the higher protein 2 3 4 5 6 7 8 800 Kd 0.7543Kd 0.75431.057nM 10 Test medium 1 400 ■ Day 7 Kd 1.143Kd 1.1431.069nM

protein yields necessary to feed 800the increasing needs Viable Cells/mL (x10 4 Test medium 2 10 10 8 600 yields necessary to feed the increasing needs of Test medium 3 200 2 Figure 6. Antibodies produced in 0Figure 7. The GPCR,0 B2AR, produced in Radioactivity bound (pmol/mg) of pharmaceutical research. This study shows how 6 0 5 10 15 20 25 30 Radioactivity bound (pmol/mg) 0 5 10 15 20 25 30 400 3 3 0 0 FreeStyle™ and Expi293™ show Expi293™[ H] DHA (nM) and FreeStyle™,[ H] DHAdemonstrates (nM) 0 1 2 3 4 5 6 7 8 pharmaceutical research. This study shows how Viable cells/mL (x10 Protein (µg/mL) 4 600 Days in Culture Bank 1 Bank 2 High-‐yield Specific bound Specific bound redesigning the transient expression model enables 200 equivalent antigen reactivity. comparable and physiological antagonist 2 redesigning the transient expression model enabled B 30.08 pmol/mg protein B 29.14 pmol/mg protein Expi293™ adapted 293F cells Expi293F™ cells max max K 1.069 nM K 1.057 nM binding.d d 0 0 thethe superior superior protein protein yield yield and and reproducibility reproducibility typically typically 0 1 2 3 4 5 6 7 8 Figure 1. Expi293FDays in culture ™ cells were adapted into Bank 1 Bank 2 High-yield 400 Figure 2. A subpopulation of FreeStyle™ Figure 6. Antibodies Figure 7. The G protein–coupled obtained with stable cell lines while maintaining the various media over three passages. Cells Expi293-adapted 293-F cells Expi293F cells obtained with stable cell lines while maintaining the 293F cells adapted into Expi293™ were cultured for multiple passages in each produced in the Expi293 receptor (GPCR) B2AR demonstrates simplicitysimplicity and and ease ease of ofuse use associated associated with with current current A. Expi293™ B. 200 6 medium demonstrated greater viable cell and FreeStyle systems show comparable and physiologically Figuremedium 1. before Expi293F being cellsseeded were at 0.2adapted x 10 Figure 2. A subpopulation of Percent glycosylation Expi293™ transient expression systems. The combination of a densities, average diameter, and up to Expi293™ vs. FreeStyle™ 293 expressed human IgG transient expression systems. The combination of a intocells/ variousmL. Cell media density over and three viability passages. were Invitrogen™ FreeStyle™ 293-F Cells equivalent antigen reactivity. specifi c antagonist binding whether 50% higher protein expression capacity uniqueunique high-capacity high-capacity media, medium, a new a 293F293F cellcell variant,variant, monitored over 8 days. Expi293™ medium is produced in the Expi293 system or 0 Cells were cultured for multiple passages adaptedthan FreeStyle into Expi293™ 293F medium cells. andand the the ExpiFectamine Gibco™ ExpiFectamine 293™ transfection 293™ transfection reagent inunique each inmedium its support before of high being cell seededdensities demonstrated greater viable cell the FreeStyle system. ™ while still supporting6 transfections (see andreagent enhancer, and enhancertogether enable together the enable new Expi293™ the Gibco at 0.2 x 10 cells/mL. Cell density and densities, average diameter, and up A B below). Expi293FreeStyle™ systemExpi293 to achieve™ Expression expression System levels to achieve approaching expression and viability were monitored over 8 days. to 50% higher protein expression Gibco™ Expi293™ Expression Medium is capacity than FreeStyle 293-F cells. Expi293™ Percent glycosylation Human IgG expressed in the Expi293 system vs. FreeStyle 293 system exceedinglevels approaching 1 gram per and liter exceeding of protein within1 g/L of7 days.protein unique in its support of high cell densities 100% Wewithin present 7 days. data Wedemonstrating present data the demonstrating ability of the newthe 90% while still supporting transfections (see Antibodies produced in both expression systems show■ G0Fsimilar Figure 8. A. 80% Enhancers boost expression with ExpiFectamine™ ■ G1F 70% media to support the increased growth rate, patterns of glycosylation. ■ G2F ability of the medium to support the increased Figure 3). 60% ■ G0 50% A. B. C. B. The preparations are alsoFreeStyle similar in the extent to which each glycosylation■ M5 transfectiongrowth rate, efficiency, transfection and highereffi ciency, levels and of higher protein levels Transfection Efficiency Transfection Enhancers 40% (day 3 posttransfection) (added 18-24 hr posttransfection) moiety is present on the expressed protein.30% 4 20% ™ ™ Enhancers90 boost expression with ExpiFectamine 293 Reagent productivityof protein ofproductivity the new Expi293F™ of the Gibco cell Expi293F line. In some 10% cases, expression of both IgG and non-IgG proteins 80 0% cell line. In some cases, expression of both IgG A 70 Transfection efficiency B 3 Transfection enhancers C Protein yield (day 3 posttransfection ) (added 18—24 hr posttransfection ) Figure 8. (A) Antibodies produced in both expression systems show 60 200000 wereand increased non-IgG proteinsup to 12-fold was increasedover levels up obtained to 12-fold 90 Figure 9. EPO made in Expi293™ 50 4 2 700 similar patterns of glycosylation. (B) The preparations are also similar 80 with the FreeStyle™ 293 Expression System.™ ™ 40 and FreeStyle™ 293 systems was 150000 over levels obtained with the Invitrogen FreeStyle 600 in the extent to which each glycosylation moiety is present on the 70 Expi293 30 3 1 tested in a TF-1 cell proliferation Proteins produced in the Expi293™ system exhibit 500

60 % GFP-‐Posi*ve Cells Freestyle293

Fold Change In Expression expressed protein. 293 Expression System. Proteins produced in the 20 assay. Both preparations show 100000 50 400 biologically relevant target recognition, binding, and 10 2 0 equivalent stimulatory activity. Expi293 system exhibit biologically relevant target 40 200,000 0 Enh. 1 - + - + 300 Figure 9. EPO made in the 50000 native pharmacokinetic performance. The system is 30 30 mL 1 mL Neg Enh. 2 - - + + recognition, binding, and native pharmacokinetic (RLU) Units Luminescence Relative % GFP-positive cells 200 Expi293 and FreeStyle 293 1 20 150,000 0.0001 0.01 1 100 10000 Human IgG yield (mg/L) scalable and, as a result of the high yields, enables Relative expression level performance. The system is scalable and, as a result Figure10 3. A. ExpiFectamine™ 293 achieves high-efficiency100 transfection of high density-cultures systems was tested in a [Erythropoeitin] ng/mL Expi293 FreeStyle 293 the use of microplates for high-throughput 0 0 0 B. Enhancer combination boosts protein yield by almost 3-fold TF-1 cell proliferation assay. 100,000 of the high yields, enables the use of microplates 30 mL 1 mL Neg . Enhancers workEnh. specifically 1 - + with- ExpiFectamine+ T™ toA achieveE highF proteinPEI Expi293 yields applications. The system has been demonstrated to C Enh. 2 - - + + system Both preparations showed for high-throughput applications. The system has 50,000 maintain high yields with a wide variety of proteins Figure 3. (A) ExpiFectamine 293 Reagent achieves high-efﬁ ciency transfection of equivalent stimulatory activity. Relative luminescence units been demonstrated to maintain high yields with a and applications, including lentiviral and membrane high-density cultures. (B) A combination of enhancers boosts protein yield almost 0.0001 0.01 1 100 10,000 wide variety of proteins and applications, including Alternative applications for Expi293™Erythropoietin (ng/mL) protein production. By enabling the generation of 3-fold. (C) Enhancers work speciﬁ cally with ExpiFectamine 293 Reagent to achieve lentiviral and membrane protein production. By highUp protein to 12x yields. FreeStyle ™ Yields at >1 g protein/L A. Protein production in 96-well plates B. MembranePro™ substantially higher levels of protein in smaller culture FreeStyle™ 293 Expi293™ Alternative160 applications for the Expi293 system 140 KentaroExpi293™ enabling the generation of substantially higher levels 120 volumes, the Expi293™ system represents a h IgG Cripto B2AR VLPs Rb IgG FreeStyle A 100 B of protein in smaller culture volumes, the Expi293 Up to 12x yields compared to FreeStyle system, at >1 g protein/L Protein80 production in 96-well plates MembranePro VLPs powerful, economical, and unique expression 60 160 1200 1200 120 500 40 system represents a powerful, economical, and Expi293 FreeStyle 140 20 Expi293 technology for both research and production of 120 1000 1000 100 (mg/L) yield purification GST post 0 FreeStyle unique expression technology for both research and EPO Human IgG Cripto B2AR VLPs 400Rabbit IgG 100 therapeutic candidate proteins. 80 IOH2475 IOH1779 800 800 80 IOH21059 60 BC021983.1 production of therapeutic candidate proteins. 300 NM_005371.2NM_006521.3NM_022777.1NM_000854.2 NM_004728.2NM_005526.1 9 40 1,200600 1,200600 1,20060 1,200 20 Figure 10.

8 200 Post-GST purification yield (mg/L) 0 The yield and scalability of Expi293™ 1,000400 1,000400 1,00040 1,000 The system 7 Lentivirus Lentivirus (from pLenti6.3 production-‐EmGFP) produced in IOH2475 IOH1779 allows for high-throughput IOH21059 C. microplate (pLenti6.3-EmGFP) format using Expi293™ (A) 100 BC021983.1 The system protein yield (mg/L) NM_00854.2 NM_005526 protein yield (mg/L) protein yield (mg/L) 6 800200 800protein yield (mg/L) 200 80020 800 NM_005371.2NM_006521.3NM_022777.1 NM_004728.2 3.0E+07 production of human ORF clones in 5 600 0 600 0 600 0 6000 2.5E+07 96-well format, (B) generation of 4 2.0E+07 Figure 10. The yield and FS Expi FS Expi FS Expi 3 400 400 FS Expi 400 400 1.5E+07 MembranePro™ VLPs displaying C GFP TU/mL 1.0E+07 scalability of the Expi293 system Product Quantity Cat. No. Figure2 4. Expression levels of five proteins demonstrates a 3x to 12x increase in protein expression recombinant expressed GPCRs using 5.0E+06 Protein yield (mg/L) Protein yield (mg/L) Protein yield (mg/L) Protein yield (mg/L) Protein yield (mg/L) 200 200 200 200 3.0E+07 allows for (A) high-throughput Expi293 Expression Medium 1,000 mL A1435101 in1 Expi293™ vs. FreeStyle™ 293. Expression levels exceeded 1 g/L for a secreted antibody and a 0.0E+00 the Expi293™ system (see Figure 8 2.5E+07 0 0 0 0 0 6 well 12well 96well cell surface receptor. 2.0E+07 productionabove) of human and (C) ORF high clones titer production of Expi293 Expression Medium 6 x 1 L A1435102 plate format 1.5E+07 in 96-welllentivirus format,. (B) generation GFP TU/mL 1.0E+07 ExpiFectamine 293 Transfection Kit For 1 L culture A14524 ™ Figure 4. Expression levels of fi ve proteins demonstrate a 3–12x increase in protein 5.0E+06 of MembranePro virus-like ExpiFectamine 293 Transfection Kit For 10 L culture A14525 expression in the Expi293 system relative to the FreeStyle 293 system. Expression 0.0E+00 6 well 12 well 96 well particles (VLPs) displaying ExpiFectamine 293 Transfection Kit For 50 L culture A14526 levels exceeded 1 g/L for a secreted antibody (human IgG) and a cell surface receptor recombinant expressed GPCRs Figure 5. Protein yield per unit volume ConclusionsPlate format Expi293F Cells 1 mL A14527 (Cripto). using the Expi293 system (see Reproducibility and scalability remains constant from 1 mL (24-well The Expi293™ system defines the next generation of Expi293F Cells 6 x 1 mL A14528 Figure 7 above), and (C) high-titer Reproducibility and scalability plate) to 30 mL (flask) to 1000 mL (3 L protein production in mammalian cells, producing more Expi293 Expression System Kit 1 kit A14635 Grand Island Fernbach). Expression in 96-well format production of lentivirus. 250.00 Figure 5. Protein yield per unit protein in less culture volume than any other commercial Antibody-Expressing Positive Control Vector 1vial A14662 Frederick and preliminary work in 10 L WAVE™ Conclusions CarlsbadGrand Island volumebags also remains demonstrate constant excellent from 1 mL 200.00250 mammalianThe Expi293 system. system Bydefi doing nes the so, next it offers generation better economyof pcDNA 3.4 TOPO TA Cloning Kit 1 kit A14697 Frederick (24-wellscalability plate) (not shown).to 30 mL (fl ask) to Carlsbad in the use of equipment, tissue culture capacity, plasmid 150.00200 protein production in mammalian cells, producing 1,000 mL (3 L Fernbach). Expression DNA, and plastic cultureware. The high yield, linear inEPO 96-well was expressedformat and in preliminarythree culture more protein in less culture volume than any other 100.00150 mg/L EPO workformats in 10at labs L WAVE in Grand™ bags Island, also scalability,commercial and mammalian versatility system. of the system By doing enables so, it offersthe use Frederick, and Carlsbad (indicated by 50.00100 demonstrate excellent scalability of microplates for high-throughput applications and offers mg/L EPO blue, yellow, and green bars). Despite better economy in the use of equipment, tissue culture (not shown). a simple and high-efficiency method for the production of 0.0050 differences in operator handling and capacity, plasmid DNA, and plastic cultureware. FreeStyle Expi293 FreeStyle Expi293 FreeStyle Expi293 culture facilities, EPO yield variability in lentivirus and MembranePro™ particles. As a complete 1ml 1ml 30ml 30ml 1000ml 1000ml EPO was expressed in three culture 0 Expi293™ was within 20% across all The high yield, linear scalability, and versatility of formats at labs in Grand Island, system that is simple and easy to use, Expi293™ offers FreeStyle Expi293 FreeStyle Expi293 FreeStyle Expi293 formats. the system enables the use of microplates for high- 1 mL 1 mL 30 mL 30 mL 1,000 mL 1,000 mL Frederick, and Carlsbad. Despite the leading solution for mammalian protein expression. throughput applications and offers a simple and high- differences in operator handling and culture facilities, EPO yield variability effi ciency method for the production of lentivirus and in the Expi293 system was within MembranePro particles. As a complete system that is CONFIDENTIAL. For Internal Use Only. 20% across all formats. simple and easy to use, the Expi293 system offers the Thermoleading Fisher Scientific solution • 5791 for Van mammalianAllen Way • Carlsbad, protein CA 92008 expression. • www.lifetechnologies.com

“In all my years working with transient expression systems, the Expi293 Expression System is the ﬁ rst one to achieve 2.3 g/L, beating every other HEK 293 transient expression system.” Jelte-Jan Reitsma, research associate

Thermo Fisher Scientiﬁ c | 81 Wyman Street | Waltham, MA 02451 | thermoﬁ sher.com Expi293 yield Expi293 yield Definition Gene symbol Protein Definition Gene symbol Protein (μg/mL) (μg/mL) Pleckstrin, mRNA (cDNA clone MGC:17111 IMAGE:4341823), complete cds PLEK AAH18549.1 5,610 Janus kinase 3 (JAK3), mRNA JAK3 NP_000206.2 16 Signal transducer and activator of transcription 3 STAT3 NP_644805.1 2,139 Tyrosine kinase 2, mRNA (cDNA clone MGC:20776 IMAGE:4591726), complete cds TYK2 AAH14243.1 15 (acute-phase response factor) (STAT3), transcript variant 1, mRNA BCL2-like 2 (BCL2L2), transcript variant 1, mRNA BCL2L2 NP_004041.1 15 BH3 interacting domain death agonist (BID), transcript variant 2, mRNA BID NP_001187.1 972 Keratin 5, mRNA (cDNA clone MGC:39301 IMAGE:5428482), complete cds KRT5 AAH24292.1 15 RAB38, member RAS oncogene family (RAB38), mRNA RAB38 NP_071732.1 702 Smoothened, frizzled family receptor (SMO), mRNA SMO NP_005622.1 15 S100 calcium binding protein A4 (S100A4), transcript variant 1, mRNA S100A4 NP_002952.1 609 Cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), mRNA CYP17A1 NP_000093.1 15 NAD(P)H dehydrogenase, quinone 1 (NQO1), transcript variant 1, mRNA NQO1 NP_000894.1 525 Metallothionein 3 (MT3), mRNA MT3 NP_005945.1 14 Prosaposin (PSAP), transcript variant 1, mRNA PSAP NP_002769.1 524 Nucleolin (NCL), mRNA NCL NP_005372.2 14 Transcription factor AP-2 alpha TFAP2A NP_001027451.1 478 (activating enhancer binding protein 2 alpha) (TFAP2A), transcript variant 2, mRNA Adenosylmethionine decarboxylase 1 (AMD1), transcript variant 1, mRNA AMD1 NP_001625.2 13 TH1-like (Drosophila), mRNA (cDNA clone MGC:22971 IMAGE:4860894), complete cds NELFCD AAH14952.1 411 Keratin 19, mRNA (cDNA clone MGC:15788 IMAGE:3504206), complete cds KRT19 AAH07628.1 13 Growth factor receptor-bound protein 2 (GRB2), transcript variant 1, mRNA GRB2 NP_002077.1 333 Lysosomal-associated membrane protein 1 (LAMP1), mRNA LAMP1 NP_005552.3 12 Inhibitor of DNA binding 1, Lamin B receptor (LBR), transcript variant 1, mRNA LBR NP_002287.2 12 ID1 NP_002156.2 295 dominant negative helix-loop-helix protein (ID1), transcript variant 1, mRNA Epoxide hydrolase 1, microsomal (xenobiotic) (EPHX1), transcript variant 1, mRNA EPHX1 NP_000111.1 12 S100 calcium binding protein A6 (S100A6), mRNA S100A6 NP_055439.1 246 G antigen 12I (GAGE12I), mRNA GAGE12I NP_001468.1 12 Epidermal growth factor receptor (EGFR), transcript variant 3, mRNA EGFR NP_958440.1 206 Prolactin receptor (PRLR), transcript variant 1, mRNA PRLR NP_000940.1 12 Integrin-linked kinase (ILK), transcript variant 2, mRNA ILK NP_001014794.1 199 Major histocompatibility complex, class I, A, HLA-A AAH08611.1 12 S100 calcium binding protein P (S100P), mRNA S100P NP_005971.1 197 mRNA (cDNA clone MGC:17191 IMAGE:4157200), complete cds Methylthioadenosine phosphorylase, Estrogen receptor 1 (ESR1), transcript variant 1, mRNA ESR1 NP_000116.2 11 MTAP AAH26106.1 182 mRNA (cDNA clone MGC:33067 IMAGE:4820938), complete cds Twist basic helix-loop-helix transcription factor 1 (TWIST1), mRNA TWIST1 NP_000465.1 10 Ornithine aminotransferase (OAT), ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 OAT NP_000265.1 179 ELAVL2 NP_001164666.1 10 nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA (Hu antigen B) (ELAVL2), transcript variant 2, mRNA Chromosome 12 open reading frame 57 (C12orf57), mRNA C12orf57 NP_612434.1 175 Ring finger protein 14 (RNF14), transcript variant 2, mRNA RNF14 NP_899645.1 10 Tumor suppressor candidate 4, Synovial sarcoma translocation, chromosome 18, NPRL2 AAH21984.1 175 SS18 AAH96222.1 10 mRNA (cDNA clone MGC:22898 IMAGE:4068981), complete cds mRNA (cDNA clone MGC:116875 IMAGE:40004909), complete cds N-myc downstream regulated 1 (NDRG1), transcript variant 2, mRNA NDRG1 NP_006087.2 162 Carcinoembryonic antigen-related cell adhesion molecule 1 MutS homolog 2, colon cancer, nonpolyposis type 1 CEACAM1 NP_001192273.1 9 MSH2 NP_000242.1 159 (biliary glycoprotein) (CEACAM1), transcript variant 6, mRNA (E. coli) (MSH2), transcript variant 1, mRNA Surfactant protein C, mRNA (cDNA clone MGC:14509 IMAGE:4043169), complete cds SFTPC AAH05913.1 9 Calreticulin (CALR), mRNA CALR NP_004334.1 148 Mucosa associated lymphoid tissue MALT1 NP_776216.1 9 Major vault protein (MVP), transcript variant 2, mRNA MVP NP_005106.2 139 lymphoma translocation gene 1 (MALT1), transcript variant 2, mRNA Mitogen-activated protein kinase kinase 4, Cancer/testis antigen family 45, member A1 (CT45A1), mRNA CT45A1 NP_001017417.1 9 MAP2K4 AAH36032.1 138 mRNA (cDNA clone MGC:33126 IMAGE:5272439), complete cds Fatty acid synthase, mRNA (cDNA clone IMAGE:4299348), complete cds FASN AAH07909 9 X-ray repair complementing defective repair in Chinese hamster cells 3, Oxidized low density lipoprotein (lectin-like) receptor 1 (OLR1), XRCC3 AAH11725.1 138 OLR1 NP_002534.1 8 mRNA (cDNA clone MGC:19630 IMAGE:4138588), complete cds transcript variant 1, mRNA Budding uninhibited by benzimidazoles 1 homolog beta (yeast), BUB1B AAH18739.1 135 Fas ligand (TNF superfamily, member 6) (FASLG), mRNA FASLG NP_000630.1 8 mRNA (cDNA clone MGC:31884 IMAGE:4649881), complete cds GNAS complex locus (GNAS), transcript variant 3, mRNA GNAS NP_536351.1 125 Growth differentiation factor 15 (GDF15), mRNA GDF15 NP_004855.2 8 BCL2-like 1 (BCL2L1), nuclear gene encoding CD4 molecule (CD4), transcript variant 1, mRNA CD4 NP_000607.1 108 BCL2L1 NP_612815.1 8 mitochondrial protein, transcript variant 1, mRNA Tumor protein p53, mRNA (cDNA clone MGC:646 IMAGE:3544714), complete cds TP53 AAH03596.1 100 Caveolin 1, caveolae protein, 22kDa (CAV1), transcript variant 1, mRNA CAV1 NP_001744.2 8 HNF1 homeobox A (HNF1A), mRNA HNF1A NP_000536.5 84 Proprotein convertase subtilisin/kexin type 7, Sulfotransferase family 1E, estrogen-preferring, member 1, PCSK7 AAH06357.1 8 SULT1E1 AAH27956.1 79 mRNA (cDNA clone MGC:12699 IMAGE:4125594), complete cds mRNA (cDNA clone MGC:34459 IMAGE:5210178), complete cds FYN oncogene related to SRC, FGR, YES (FYN), transcript variant 1, mRNA FYN NP_002028.1 7 Chitinase 3-like 1 (cartilage glycoprotein-39), CHI3L1 AAH38354.1 76 Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), mRNA EIF4EBP1 NP_004086.1 7 mRNA (cDNA clone MGC:17199 IMAGE:4212419), complete cds C-src tyrosine kinase (CSK), transcript variant 1, mRNA CSK NP_004374.1 72 Interleukin 10 (IL10), mRNA IL10 NP_000563.1 7 Thrombomodulin, mRNA (cDNA clone MGC:45302 IMAGE:5176531), complete cds THBD AAH35602.2 71 Retinoic acid receptor, beta (RARB), transcript variant 1, mRNA RARB NP_000956.2 6 Ras-related C3 botulinum toxin substrate 1 (rho family, Janus kinase 2 (JAK2), mRNA JAK2 NP_004963.1 69 RAC1 NP_008839.2 6 small GTP binding protein Rac1) (RAC1), transcript variant Rac1, mRNA Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3B AAH09113.1 67 Small nuclear ribonucleoprotein polypeptide E (SNRPE), mRNA SNRPE NP_003085.1 6 (semaphorin) 3B, mRNA (cDNA clone MGC:18122 IMAGE:4153377), complete cds HORMA domain containing 1 (HORMAD1), transcript variant 1, mRNA HORMAD1 NP_115508.2 66 Nescient helix loop helix 1 (NHLH1), mRNA NHLH1 NP_005589.1 6 Transmembrane protein 37, Alpha-methylacyl-CoA racemase, TMEM37 AAH46362.1 62 AMACR AAH09471.1 6 mRNA (cDNA clone MGC:50757 IMAGE:5221396), complete cds mRNA (cDNA clone MGC:3743 IMAGE:2958112), complete cds Actin-like 8, mRNA (cDNA clone MGC:24924 IMAGE:4933032), complete cds ACTL8 AAH28909.1 56 Surfactant protein D, mRNA (cDNA clone MGC:22626 IMAGE:4696173), complete cds SFTPD AAH22318.1 6 Kallikrein-related peptidase 10, Intercellular adhesion molecule 1 (ICAM1), mRNA ICAM1 NP_000192.2 54 KLK10 AAH02710.1 5 mRNA (cDNA clone MGC:3667 IMAGE:3632557), complete cds Dihydrofolate reductase, DHFR AAH70280.1 47 mRNA (cDNA clone MGC:88273 IMAGE:6714220), complete cds Jun proto-oncogene (JUN), mRNA JUN NP_002219.1 5 FEZ family zinc finger 2, mRNA (cDNA clone MGC:26179 IMAGE:4794121), FEV (ETS oncogene family) (FEV), mRNA FEV NP_059991.1 47 FEZF2 AAH22464.1 5 complete cds Chromosome 8 open reading frame 4 (C8orf4), mRNA C8orf4 NP_064515.1 46 Interleukin 6 (interferon, beta 2) (IL6), mRNA IL6 NP_000591.1 5 Fatty acid binding protein 1, liver (FABP1), mRNA FABP1 NP_001434.1 43 Lymphoid enhancer-binding factor 1 (LEF1), transcript variant 1, mRNA LEF1 NP_057353.1 5 Matrix metallopeptidase 10 (stromelysin 2) (MMP10), mRNA MMP10 NP_002416.1 41 Cholinergic receptor, nicotinic, alpha 3 (neuronal) (CHRNA3), Protein tyrosine phosphatase, non-receptor type 11 (PTPN11), CHRNA3 NP_000734.2 5 PTPN11 NP_542168.1 40 transcript variant 1, mRNA transcript variant 2, mRNA Transforming growth factor, beta receptor II TGFBR2 NP_003233.4 4 Olfactory receptor, family 51, subfamily E, member 2 (OR51E2), mRNA OR51E2 NP_110401.1 39 (70/80kDa) (TGFBR2), transcript variant 2, mRNA F-box and WD repeat domain containing 7, FBXW7 NP_060785.2 39 Cell division cycle 25B (CDC25B), transcript variant 1, mRNA CDC25B NP_068659.1 4 E3 ubiquitin protein ligase (FBXW7), transcript variant 2, mRNA Carboxypeptidase M (CPM), transcript variant 3, mRNA CPM NP_001005502.1 4 SMAD family member 4 (SMAD4), mRNA SMAD4 NP_005350.1 35 Cytochrome P450, family 3, subfamily A, Echinoderm microtubule associated protein like 4, CYP3A5 NP_000768.1 4 EML4 AAH08685 33 polypeptide 5 (CYP3A5), transcript variant 1, mRNA mRNA (cDNA clone IMAGE:3868340), complete cds Trefoil factor 1 (TFF1), mRNA TFF1 NP_003216.1 4 Synovial sarcoma, X breakpoint 2 (SSX2), transcript variant 2, mRNA SSX2 NP_783629.1 32 E74-like factor 3 (ets domain transcription factor, epithelial-specific) V-myc myelocytomatosis viral related oncogene, ELF3 NP_001107781.1 4 MYCN NP_005369.2 32 (ELF3), transcript variant 2, mRNA neuroblastoma derived (avian) (MYCN), mRNA ATP-binding cassette, sub-family C (CFTR/MRP), Estrogen receptor 2 (ER beta), ABCC5 NP_001018881.1 4 ESR2 AAH24181.1 32 member 5 (ABCC5), transcript variant 2, mRNA mRNA (cDNA clone MGC:26496 IMAGE:4825937), complete cds Keratin 18 (KRT18), transcript variant 1, mRNA KRT18 NP_000215.1 4 Cancer susceptibility candidate 1 (CASC1), transcript variant 3, mRNA CASC1 NP_001076441.1 31 Ewing sarcoma breakpoint region 1 (EWSR1), transcript variant 2, mRNA EWSR1 NP_005234.1 4 Thioredoxin (TXN), transcript variant 1, mRNA TXN NP_003320.2 30 24-dehydrocholesterol reductase (DHCR24), mRNA DHCR24 NP_055577.1 4 Glutathione S-transferase mu 1 (GSTM1), transcript variant 2, mRNA GSTM1 NP_666533.1 30 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA5 AAH08915.1 4 Fanconi anemia, complementation group C (FANCC), transcript variant 1, mRNA FANCC NP_000127.2 29 member 5, mRNA (cDNA clone MGC:16086 IMAGE:3618167), complete cds Cytochrome P450, family 2, subfamily A, polypeptide 6, CYP2A6 AAH96255.1 29 Siah E3 ubiquitin protein ligase 1 (SIAH1), transcript variant 2, mRNA SIAH1 NP_001006611.1 3 mRNA (cDNA clone MGC:116920 IMAGE:40006064), complete cds Pleiotrophin (PTN), mRNA PTN NP_002816.1 3 Tumor necrosis factor receptor superfamily, member 10b, TNFRSF10B AAH01281.1 28 mRNA (cDNA clone MGC:5144 IMAGE:3458466), complete cds Frizzled-related protein (FRZB), mRNA FRZB NP_001454.2 3 Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), Ribosomal protein S6 (RPS6), mRNA RPS6 NP_001001.2 3 PIN1 NP_006212.1 28 transcript variant 1, mRNA Folate hydrolase (prostate-specific membrane antigen) FOLH1 NP_001014986.1 3 Placenta-specific 1 (PLAC1), mRNA PLAC1 NP_068568.1 28 1 (FOLH1), transcript variant 2, mRNA Melanoma antigen family C, 2, Cyclin-dependent kinase 4 (CDK4), mRNA CDK4 NP_000066.1 28 MAGEC2 AAH13318.1 3 CUB domain containing protein 1 (CDCP1), transcript variant 2, mRNA CDCP1 NP_835488.1 27 mRNA (cDNA clone MGC:13377 IMAGE:4133159), complete cds Dihydrolipoamide dehydrogenase (DLD), Synovial sarcoma, X breakpoint 1 (SSX1), mRNA SSX1 NP_005626.1 3 DLD NP_000099.2 27 Wingless-type MMTV integration site family, nuclear gene encoding mitochondrial protein, mRNA WNT5A NP_003383.2 2 X antigen family, member 1A (XAGE1A), transcript variant a, mRNA XAGE1A NP_001091061.2 27 member 5A (WNT5A), transcript variant 1, mRNA Cholecystokinin (CCK), transcript variant 1, mRNA CCK NP_000720.1 2 Signal transducer and activator of transcription 5A (STAT5A), mRNA STAT5A NP_003143.2 26 Vimentin (VIM), mRNA VIM NP_003371.2 2 Ras homolog family member C (RHOC), transcript variant 1, mRNA RHOC NP_786886.1 25 Xeroderma pigmentosum, complementation group A (XPA), transcript variant 1, mRNA XPA NP_000371.1 2 Neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), mRNA NRAS NP_002515.1 25 Neuregulin 1, mRNA (cDNA clone IMAGE:3161700), complete cds NRG1 AAH07675 2 Apolipoprotein D (APOD), mRNA APOD NP_001638.1 24 Homeobox B13 (HOXB13), mRNA HOXB13 NP_006352.2 2 Alpha-fetoprotein (AFP), mRNA AFP NP_001125.1 23 Desmin (DES), mRNA DES NP_001918.3 2 BCL2-associated agonist of cell death (BAD), transcript variant 1, mRNA BAD NP_004313.1 22 Kallikrein-related peptidase 4, BCL2-antagonist/killer 1 (BAK1), mRNA BAK1 NP_001179.1 2 KLK4 AAH69429.1 22 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 4 (Hu antigen D), mRNA (cDNA clone MGC:96926 IMAGE:7262135), complete cds ELAVL4 AAH36071.1 2 Transmembrane protease, serine 2 (TMPRSS2), transcript variant 2, mRNA TMPRSS2 NP_005647.3 19 mRNA (cDNA clone MGC:33705 IMAGE:5286347), complete cds CLPTM1-like (CLPTM1L), mRNA CLPTM1L NP_110409.2 2 V-rel reticuloendotheliosis viral oncogene homolog B (RELB), mRNA RELB NP_006500.2 19 Lymphocyte-specific protein tyrosine kinase, LCK AAH13200.1 1 PDZ and LIM domain 5 (PDLIM5), transcript variant 5, mRNA PDLIM5 NP_001011516.1 18 mRNA (cDNA clone MGC:17196 IMAGE:4341278), complete cds Diablo, IAP-binding mitochondrial protein (DIABLO), DIABLO NP_063940.1 18 Prolactin (PRL), transcript variant 1, mRNA PRL NP_000939.1 1 nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA Matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), mRNA MMP3 NP_002413.1 1 Chromosome X open reading frame 48 (CXorf48), transcript variant 2, mRNA CXorf48 NP_060333.1 18 TIMP metallopeptidase inhibitor 3 (TIMP3), mRNA TIMP3 NP_000353.1 1 Cysteine-rich, angiogenic inducer, 61 (CYR61), mRNA CYR61 NP_001545.2 17 Major histocompatibility complex, class I, E, Dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) HLA-E AAH02578.1 17 DLST NP_001924.2 1 mRNA (cDNA clone MGC:1408 IMAGE:3140835), complete cds (DLST), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA Insulin-like growth factor binding protein 3, High mobility group AT-hook 1 (HMGA1), transcript variant 2, mRNA HMGA1 NP_002122.1 17 IGFBP3 AAH00013.1 1 mRNA (cDNA clone MGC:2305 IMAGE:3506666), complete cds Ret proto-oncogene, mRNA (cDNA clone IMAGE:3507569), complete cds RET AAH03072 17 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase), MMP9 AAH06093.1 1 Angiotensin II receptor, type 2 (AGTR2), mRNA AGTR2 NP_000677.2 17 mRNA (cDNA clone MGC:12688 IMAGE:4054882), complete cds Insulin-like growth factor binding protein 1, Parathyroid hormone (PTH), mRNA PTH NP_000306.1 17 IGFBP1 AAH35263.2 1 Transforming growth factor, beta 1, mRNA (cDNA clone MGC:30041 IMAGE:4800940), complete cds TGFB1 AAH01180.1 16 mRNA (cDNA clone MGC:2323 IMAGE:3356605), complete cds PAS domain containing 1 (PASD1), mRNA PASD1 NP_775764.2 1 Insulin-like growth factor binding protein 2, 36kDa (IGFBP2), mRNA IGFBP2 NP_000588.2 16 Interleukin 8 (IL8), mRNA IL8 NP_000575.1 1 Kallikrein-related peptidase 3 (KLK3), transcript variant 1, mRNA KLK3 NP_001639.1 16 Melanoma inhibitory activity (MIA), transcript variant 1, mRNA MIA NP_006524.1 1

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