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Course: CHM 477, Fall 2008School: Lehigh
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Monoclonal Antibodies 101 - Presentation Overview Bill Strohl, Merck Vaccines and Biologics Research November 2, 2004 MAb structure MAb structure-function - overview Sources of MAb variability MAb Fc structure-function Generation and modification of Human MAbs Therapeutic MAb Cell Culture MAbs as Therapeutics Examples of Therapeutic MAbs Antibody Classes (a) IgG VL VH CL C1 T1/2 25d [12 mg/mL] Valency 2 (d) IgA...
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Monoclonal Antibodies 101 - Presentation Overview Bill Strohl, Merck Vaccines and Biologics Research November 2, 2004 MAb structure MAb structure-function - overview Sources of MAb variability MAb Fc structure-function Generation and modification of Human MAbs Therapeutic MAb Cell Culture MAbs as Therapeutics Examples of Therapeutic MAbs Antibody Classes (a) IgG VL VH CL C1 T1/2 25d [12 mg/mL] Valency 2 (d) IgA (dimer) VH C1 VL CL (e) IgM (pentamer) VL CL VH C 1 C 2 C 3 C 4 Hinge region C2 C3 (b) IgD VL VH CL C 1 C2 Hinge region Disulfide bond J chain C3 J chain T1/2 3d [0.03 mg/mL] Valency 2 (c) IgE C 2 C 3 VL L VH C C1 T1/2 2d [0.0003 mg/mL] C 2 C3 Valency 2 T1/2 6d [6 mg/mL] Valency 4 T1/2 5d [1 mg/mL] Valency 10 C 4 Therapeutic Antibodies: Focus on IgG Isotypes 1, 2, 4 IgG3 IgG1 IgG2 IgG4 Disulfide Bond IgG Flexibility IgG Structure Schematic View IgG Structure Heavy vs Light Chains (Reduction in presence of thiols) IgG Structure Proteolytic Fragmentation Proteolytic fragments Types of Monoclonal Antibodies Constructed Mouse V-regions VH CH1 CL CH2 CH3 VH VL Mouse Mab (Example: OKT3) Chimeric (~66% Human e.g., Rituxan) Mouse CDRs Human V-regions from Phage Display VH CH1 Humanized (~97% Human; e.g., Herceptin) VL CL CH2 CH3 Human (100%) VL scFv ca. 29 kDa T1/2 = < hr Antibody Structures and Functions Ligand-binding; Affinity maturation C1q binding (CMC) Glycosylation can affect both ADCC and T1/2 Fc binding (ADCC) FcRn binding (T1/2) Isotype (IgG1, IgG2, IgG4) Antibody Structures and Functions Ligand-binding; Affinity maturation C1q binding (CMC) Glycosylation can affect both ADCC and T1/2 Fc binding (ADCC) FcRn binding (T1/2) Isotype (IgG1, IgG2, IgG4) -Sheet structures of CH, CL, VH, and VL Domains Cavity binding: Enzymes and receptors Crystal structure of a monoclonal 2E8 Fab antibody fragment Nanobody Cavity binding: Enzymes and receptors Antibody::Ligand Interactions Four Processes to Generate Antibody Diversity 1. Different combinations of gene segments (VL, JL, VH, DH, JH) 2. Different pairing of VH and VL chains (including vs ) 3. Recombinant processes at joints 4. Somatic hypermutation ("affinity maturation" in secondary lymphoid organs) Germline Arrangement of Ig VDJ Chains Janeway et al Antibody V-Chain Structure and Diversity (VH; VDJ recombination) VH VH VH3-33 VH-CDR1 VH-CDR2 VH-CDR3 EVQLVESGA GVVQPGRSLR LSCAAS GFTFSNYGMH WVRNAP GKGLEWVA VIWYDGSNKY YAESVKG RSTISRDTSK KTLYLQLNSL RAEDTAIYYCAR EGDKNYWFYGMDV QVQLVESGG GVVQPGRSLR LSCAAS GFTFSSYGMH WVRQAP GKGLEWVA VIWYDGSNKY YADSVKG RFTISRDNSK NTLYLQMNSL RAEDTAVYYCAR YYYYYGMDV YWYFDL YFDY |--- CH1 WGQGTTVTVSS WGQGTTVTVSS (JH6) WGRGTLVTVSS (JH2) WGQGTLVTVSS (JH4) VH3-33 (51-65 VH germline families) (VL; VJ recombination) V Vk VK1-L4 VL-CDR1 VL-CDR2 VL-CDR3 AIQLTQSPTS LSASVADRVTITC RASQGISVSLA WYQQKPGKAPKLLIY DASSLESGVPS RFSGSGSGTD FTLTISCLQS EDFATYYC QQYYSFPPT AIQLTQSPSS LSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQFNSYP IT FT YT WT D (27) JH (6) |------CL (JK5) (JK5) (JK3) (JK2) (JK1) FGQGTRLEIK FGQGTRLEIK FGPGTKVDIK FGQGTKLEIK FGQGTKVEIK V1-L4 (40 V germline families; 30 V germline families) J (5J; 4J) - Hypermutated regions Variability of CDR Loop Structures VL VH Fab structure 3-D to Primary Sequence VH and CH1 Chains IGG1K 2H1P (MUR) |---------Signal sequence-----------| |----VH C VH-CDR1 MEFGLRWVFL VAILKDVQCD VQLVESGGGL VQPGGSLRLS --CAAS GFAYSSFWMH WVRQAP DVKLVESGGGL VKLGGSLKLS - - CAAS GFTFSSYFLS W VRQTP VH-CDR2 C96 GRGLVWVS RINPDGRITVYADAVKG RFTISRDNAK NTLYLQMNNL RAEDTAVYYCAR EKRLELVA TINSNGDKTYHPDTM G RFTI S RDNAK NTLYLQMS SL KSEDTALYYCAR K VH-CDR3 |--- CH1 starts here C144 GTRFLELTSRGQMDQ WGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP RDS- - - - SASLYFDY WGQGT TLTVSSAKTT PPSVYPLAPG SAAQTN SMVT LGCLVKGYFP (VH-C1 LINKER) C200 EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV EPVTVTW NSG SLSSGVHTFP AVLQS- DLYT LSSSVTVPSS TW PSETVTCN VAHPASSTKV DKKVEPK /// DKKI VPR / / / IGG1K 2H1P ( M R) U IGG1K 2 H1 P ( MUR) IGG1K 2H1P ( MUR) IGG1K 2 H1 P ( MUR) Kappa vs Lambda VL-CL Chains KAPPA1 1 KAPPA2 LAMBDA1 LAMBDA2 |------VL DIELTQSPAT LSLSPGERATLSC QIVSTQSPAI MSASPGEKVTMTC QSALTQPRSV -SGSPGQSVTISC ASVLTQPPSV -SGAPGQRVTISC VL-CDR1 RASQSV-----SSYLA SASSSR------SYMQ TGTSSDIGGY--NFVS TGSSSNIGAG--HNVK WYQQKPGQAPRLLIN WYQQKPGTSPKRWIY WYQQHPGKAPKLMIY WYQQLPGTAPKLLIF VL-CDR2 DASNRATGIPA DTSKLASGVPA DATKRPSGVPD H---NNAGVPD KAPPA1 61 KAPPA2 LAMBDA1 LAMBDA2 RFSGSGSGTD RFSGSGSGTS RFSGSKSGNT RFSVSKSGTS FTLTISSLEP YSLTISSMEA ASLTISGLQA ATLAITGLQA EDFVVYYC EDAATYYC EDEADYYC EDEADYYC VL-CDR3 QQRSNWP---LT HQRSS-----YT CSYAGDYTPGVV QSYDRSL---RV |----CL FGGGTKVEIKRT-V AAPSVFIFPP FGGGTKLEIKRT-V AAPSVFIFPP FGGGTKLTVLGQPK AAPSVTLFPP FGGGTKLTVLRQPK AAPSVTLFPP (VL-CL LINKER) KAPPA1 121 KAPPA2 LAMBDA1 LAMBDA2 KAPPA1 181 KAPPA2 LAMBDA1 LAMBDA2 SDEQLKSGTA SDEQLKSGTA SSEELQANKA SSEELQANKA LSKADYEKHK LSKADYEKHK PEQW--KSHR PEQW--KSHK SVVCLLNNFY SVVCLLNNFY TLVCLISDFY TLVCLISDFY VYACEVTHQG VYACEVTHQG SYSCQVTHEG SYSCQVTHEG PREAKVQWKV PREAKVQWKV PGAVTVAWKA PGAVTVAWKA LSSPVTKSFN LSSPVTKSFN STVEKTVAPT STVEKTVAPT DNALQSGNSQ DNALQSGNSQ DSSPVKAGVE DGSPVKAGVE RGEC* RGE-* ECS* ECS* ESVTEQDSKD ESVTEQDSKD TTTPSKQSNN TTTPSKQSNN STY-SLSSTLT STY-SLSSTLT KYAASSYLSLT KYAASSYLSLT Fc Structure::Function Analysis Therapeutic Antibodies: Focus on IgG Isotypes 1, 2, 4 IgG1 Normal serum T1/2 (days) Normal serum conc. (mg/mL) Activate classical complement ADCC Opsonization Cross placenta Diffusion extravascular Structural stability Bind FcRI (CD64; high affin 1e-9M) Bind FcRIIIa,b (CD16; low affin 5e-5M) Bind FcRIIa,b (CD32; low affin 2e-6M) 23 d 9 ++ +++ +++ ++ +++ +++ +++ ++ ++ IgG2 23 d 3 + +/+/+ +++ +++ + +/+/- IgG3 8d 1 +++ ++ ++ + +++ + +++ ++ ++ IgG4 23 d 0.5 +/+ + +++ +/+ +/+ Serum Half-life & Brambell Receptor (FcRn) 1960s - F.W.R. Brambell saturable binding IgG-specific pH-dependent protection in serum neonatal transport (FcRn) 1990s clone & crystalize FcRn FcRn KO mice low [IgG] short IgG half life FcRB/FcRn heavy chain 2 FcRn Neonatal Receptor Recycling of IgG by Vascular Endothelium pH 7.2 pH 5 proteolysis X rescue X X FcRn-Fc Structure binds FcR Fc FcRB Protein A (Bjorkman) Fc Sequence :: Function Analysis FcRn-binding sequences IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 -------Hinge region----| |--------CH2 P238 T250Q M252 C261 D265 D270 DKKAEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKTVERKCC- --VECPPCPA PP-VAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKRVESKYGP ---PCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP (Lonza=P) (Lower hinge) FcRn-bind B/C loop P331 N297* T307 C321 P329 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAPI EVQFNWYVDG MEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPI EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSSI C'E loop FcRn-bind F/G loop |----CH3 EKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY +++ AA=ADCC M428L H433 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK* FcRn-bind Therapeutic Antibodies: Focus on IgG Isotypes 1, 2, 4 IgG1 Normal serum T1/2 (days) Normal serum conc. (mg/mL) Activate classical complement ADCC Opsonization Cross placenta Diffusion extravascular Structural stability Bind FcRI (CD64; high affin 1e-9M) Bind FcRIIIa,b (CD16; low affin 5e-5M) Bind FcRIIa,b (CD32; low affin 2e-6M) 23 d 9 ++ +++ +++ ++ +++ +++ +++ ++ ++ IgG2 23 d 3 + +/+/+ +++ +++ + +/+/- IgG3 8d 1 +++ ++ ++ + +++ + +++ ++ ++ IgG4 23 d 0.5 +/+ + +++ +/+ +/+ Fc Sequence :: Function Analysis FcR-binding sequences IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 -------Hinge region----| |--------CH2 P238 T250Q M252 C261 D265 D270 DKKAEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKTVERKCC- --VECPPCPA PP-VAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKRVESKYGP ---PCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP (Lonza=P) (Lower hinge) FcRn-bind B/C loop P331 N297* T307 C321 P329 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAPI EVQFNWYVDG MEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPI EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSSI C'E loop FcRn-bind F/G loop |----CH3 EKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY +++ AA=ADCC M428L H433 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK* FcRn-bind Fc Sequence :: Function Analysis C1q-binding sequences IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 -------Hinge region----| |--------CH2 P238 T250Q M252 C261 D265 D270 DKKAEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKTVERKCC- --VECPPCPA PP-VAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKRVESKYGP ---PCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP (Lonza=P) (Lower hinge) FcRn-bind B/C loop P331 N297* T307 C321 P329 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAPI EVQFNWYVDG MEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPI EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSSI C'E loop FcRn-bind F/G loop |----CH3 EKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY +++ AA=ADCC M428L H433 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK* FcRn-bind Fc Sequence :: Function Analysis Composite functional sequences IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 IGG1 IGG2 IGG4 -------Hinge region----| |--------CH2 P238 T250Q M252 C261 D265 D270 DKKAEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKTVERKCC- --VECPPCPA PP-VAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP DKRVESKYGP ---PCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP (Lonza=P) (Lower hinge) FcRn-bind B/C loop P331 N297* T307 C321 P329 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAPI EVQFNWYVDG MEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPI EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSSI C'E loop FcRn-bind F/G loop |----CH3 EKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY EKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY +++ AA=ADCC M428L H433 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK* KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK* FcRn-bind Monoclonal Antibody Glycosylation G0 SA-2-6-Gal-1-4-GlcNAc-1-2-Man-1 Fucose | 1 | 6 6 | Man-1-4-GlcNAc-1-4-GlcNAc-1-Asn297 3 SA-2-6-Gal-1-4-GlcNAc-1-2-Man-1 Note: Lack of glycosylation leads to lack of binding to FcRs and absence of ADCC, CMC, long T1/2, etc) Monoclonal Antibody Glycosylation SA-2-6-Gal-1-4-GlcNAc-1-2-Man-1 Fucose | 1 | 6 6 | Man-1-4-GlcNAc-1-4-GlcNAc-1-Asn297 3 SA-2-6-Gal-1-4-GlcNAc-1-2-Man-1 G0 Note: Lack of Fucose moiety leads to increased binding to FcRIII and increased ADCC FcR C1q FcRn Sources of Antibody Genes for Engineering Purposes (or... From Mouse to CaT ) Mouse - The old fashioned way... - Humanize using PDL technology Medarex - HuMAb mouse (mouse with human Ab genes) Abgenix - Xenomouse (hu Ab genes) + SLAM Biosite/Dyax - Phage display library of affinity matured mouse genes after immunization with desired target MorphoSys - Fab cys-based display of human antibody libraries Cambridge Antibody Technology (CaT) - scFv phage display library of human Ab genes Examples of Processes for Current Antibody Projects Example 1 Humanized mice Screened for activity Cloned and sequenced Converted to Fabs Affinity matured Converted to r-IgG4 Human Example 2 Human Library scFvs Panned and isolated Activity determined Affinity Matured Converted to r-IgG4 Example 3 Murine hybridomas Screened for activity Cloned and sequenced Humanized Converted to r-IgG1 Antibody Maturation A key Step in Development Affinity Maturation - Most antibodies from mice or libraries start at ~1 nM - Many targets require <100 pM (some <10 pM) affinity - Maturation can either be random or focus on CDRs Germlining - Most antibodies are 3-20 aa in variable framework regions away from germline sequence - Minimize changes from germline without sacrificing efficacy Choice of Isotype; Modification of Fc functionality - ADCC, CMC, phagocytic, "benign blocker" Random Mutagenesis x x xx x x xx x x x x VH N CDR1 CDR2 CDR3 linker CDR1 VL CDR2 CDR3 C (Random) Directed Mutagenesis x x x xx x x VH N CDR1 CDR2 CDR3 linker CDR1 VL CDR2 CDR3 C Two Options for ScFv Display antibody (scFv) mRNA PHAGE DNA (phagemid) RIBOSOME antibody (scFv) Phage display of scFv Ribosome display of scFv In vivo and in vitro steps In vitro steps only Phage vs Ribosome Display in Lead Optimisaton library size exceeds 109 amenable to cell surface selections proven track record Phage scFv library size exceeds 1011 simultaneous optimisation of many leads directed evolution Ribosome scFv The Ribosome Display Cycle Two predominant selectable and amplifiable expression systems Dihydrofolate reductase (DHFR) vector commonly used in CHO cells Amplify with methotrexate (MTX) Kauffman R. and Sharp P. JMB (1982) Glutamine synthetase (GS) vector used in NS0 cells Select in glutamine-free medium Amplify with methionine sulfoxamine Bebbington C. et al., Biotechnol. (1992) Robinson D. et al., Ann. NY Acad. Sci. (1994) Cell Line Development Antibody Production Commercialization of Antibodies Why and how do antibodies make good drugs? Or, Do They? Pharmacological Characteristics of a Therapeutic Monoclonal Antibody Protein IgG is 22 of ~150 kDa Ability to inhibit protein-protein interactions on cell-bound, extracellular, and serum targets (e.g., receptors, peptides, hormones, cytokines, exo-enzymes, etc) Avidity effects allow high affinity to targets (matured down to <10 pM) Long half-life in serum (23 days for IgG1, IgG2, IgG4) Ability to have: > Antibody-dependent cytotoxicity > Complement-mediated cytotoxicity > Antibody-mediated immune complex formation with clearance > Antibody-mediated phagocytosis of bacteria > Or, none of the above Lack of blood-brain barrier penetration (e.g., peripheral targets only) Ability to penetrate some tissues (Vss = ~blood volume) Relatively shallow peak-to-trough ratio Clearance mediated largely by liver of intact species (too large for kidney) Little or no off-target activity or drug-metabolism-related issues Little or no non-mechanism-mediated toxicity Monoclonal Antibody Mechanisms of Action Many Current Therapeutic Targets are Accessible to Biologics Therapeutic Target Classes Receptors (including GPCRs) Hormones, peptides and soluble factors Ion channels Enzymes* DNA Nuclear receptors Other % 45% 11% 5% 28% 2% 2% 7% Accessible to Biologics? yes yes yes some no no no 61% * Those present in serum, on cell surface, or extracellular spaces Source: CSFB; Science 17 Mar 2000 Challenges to Monoclonal Antibody Product Development Mode of administration IV (parenteral) Subcutaneous (limited to low doses) Local site of administration reactions Manufacturing Capital Investment ($300-400M for Manufacturing) Cost of goods Removal of all animal and human derived products in manufacturing Immunogenicity: all antibodies are unique In many cases, can't really tell until Phase II MAb Immunogenicity MAb OKT3 NRLu10 NRLu10 Remicade Rituxan Simulect ReoPro Dose/frequency Murine Ig 5 mg; 10-14x daily 10 mg; 1x Murine Fab 10 mg; 1x Chimeric Ig 350 mg; 3-9x 375 mg/m2; 4x 20 mg; 2x Chimeric Fab 20 mg; 1x 20 mg; 2x Humanized Ig 70 mg; 5x 210-140 mg; 30x Human Response >80% HAMA 71% HAMA 10% HAMA 13% HACA <1 % HACA <2% HACA 12-17% HACA 24% HACA 8% HAHA >1% HAHA Zenapax Herceptin Advantages to Development of Monoclonal Antibodies as Therapeutic Drugs Specificity of binding to target is extremely high Off target toxicity, drug metabolism, cytochrome P450, and other "drug issues" are minimal Uniform pharmacokinetics and long half-lives in bloodstream Compared with small molecule development, development programs can be faster Biology replaces medicinal chemistry; Molecular biology, cell culture and fermentation replace process chemistry Therapeutic Monoclonal Antibodies Success in Commercialization Scientific Advances Have Led to Increased Probability of Success 1998 10 9 8 7 6 Number 5 4 3 2 1 0 Murine 3% success rate Morrison Kohler & et al Milstein Chimeric or humanized 25% success rate* 1975 1980 1985 1990 1995 2000 * progress from Phase I to licensure Reichart, J. Nature Biotechnology (2001) Historic and Projected Sales of MAb Therapeutics (from 2000) $ in billions 16 14 12 10 8 6 4 2 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 Source: CSFB 2003 Actual = $5.5B Updated 2010 Prediction ~ $21B Non-mAb Biologics (e.g., Aranesp, Avonex, Epogen, Procrit, Humulin > $10B in 2002) Growth in MAb Sales Forecast to Exceed that of Therapeutic Proteins 60 Annual Sales (Billions $/year) 50 40 30 20 10 0 1 2001 Actual 2 2010 Estimated Therapeutic Proteins Monoclonal Antibodies MAb sales exceed erythropoeitins in 2010 Datamonitor Examples of Therapeutic Antibody Products on the Market Product Name (Manufacturer) ReoPro (Lilly/Centocor) Simulect (Novartis) Remicade (Centocor) Synagis (MedImmune) Rituxan (Biogen/Idec) Zenapax (Roche) Enbrel (Immunex) Herceptin (Genentech) Mylotarg (Wyeth) Campath (Berlex) Zevalin (Biogen/Idec) Xolair (Genentech) Raptiva (Genentech) Bexxar (Corixa) Humira (Abbott) Erbitux (ImClone/BMS) Avastin (Genentech) Launch Date 1994 1998...
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ECE 269VLSI System TestingKrish Chakrabarty Lecture 1: OverviewECE 269Krish Chakrabarty1Lecture 1 IntroductionClass website: www.ee.duke.edu/~krish/teaching/269.html VLSI realization process Verification and test Ideal and real t
Stanford - COSN - 1021
04/20/2002 03:45 AM ESTUNITED STATES DISTRICT COURT SOUTHERN DISTRICT OF NEW YORK IN RE INITIAL PUBLIC OFFERING SECURITIES LITIGATION X : : : : X : : : : : XCoSine Communications, Inc.Master File No. 21 MC 92 (SAS)01 Civ. 10105 (SAS) AMENDED
Duke - ECE - 269
ECE 269VLSI System TestingKrish Chakrabarty Lecture 13: Design for Testability (DFT): 1ECE 269Krish Chakrabarty1Outline Motivation and Goals Controllability and Observability Ad Hoc DFT Methods Control/Test Point Insertion Circuit res
Duke - ECE - 269
ECE 269VLSI System TestingKrish Chakrabarty Built-In Self-Test (BIST)ECE 269Krish Chakrabarty1BIST Motivation Useful for field test and diagnosis (less expensive than a local automatic test equipment) Software tests for field test and di
Duke - ECE - 269
Power Management for Wafer-Level Test-During-Burn-InSudarshan BahukudumbiIntel Corporation, Hillsboro, Oregon, USAKrishnendu ChakrabartyDepartment of Electrical and Computer Engineering Duke University Durham, NC, USAIntroduction Demand for d
Duke - ECE - 269
ECE 269VLSI System TestingKrish Chakrabarty Lecture 21: Testing for Small-Delay Defects"I choose a block of marble and chop off everything that I do not need." Francoise Auguste Rodin (on how he created his statues)1What Are We After (This Cl
Duke - ECE - 269
ECE 269VLSI System TestingKrish Chakrabarty System-on-Chip (SOC) TestingECE 269Krish Chakrabarty1Outline Motivation for modular testing of SOCs Wrapper design IEEE 1500 Standard Optimization Test access mechanism design and optimizat
Duke - ECE - 269
ECE 269VLSI System TestingKrish Chakrabarty Test Compression1Test Compression Why do we need test compression? Test data volume Test time Number of chip/tester pins Why can we compress test data? Large number of don't-cares (Xs) in scan
Duke - ECE - 269
TutorialSurvey of Test Vector Compression TechniquesNur A. ToubaUniversity of Texas at AustinTest data compression consists of test vector compression on the input side and response compaction on the output side. Test vector compression has been
Stanford - COSN - 1021
US District Court Civil Docket as of 6/28/2007 Retrieved from the court on Thursday, December 18, 2008U.S. District Court Southern District of New York (Foley Square) CIVIL DOCKET FOR CASE #: 1:01-cv-10105-SASCosine Comm. IPO, et al v. Cosine Comm
Stanford - COSN - 1021
JUDGE SCHEINDLINUNITED STATES DI STRICT COURT SOUTHERN DISTRICT OF NEW YORKft, IN RE INITIAL PUBLIC OFFERING SECURITIES LITIGATION 21 MC 92^4X LINC CORP., on Behalf of Itself and All Others Similarly Situated,Plaintiff,Index No.CLASS ACTION
Stanford - JDSU - 1023
1 2 3 4 5 6 7 8JORDAN ETH (BAR NO. 121617) TERRI GARLAND (BAR NO. 169563) PHILIP T. BESIROF (BAR NO. 185053) MORRISON & FOERSTER LLP 425 Market Street San Francisco, California 94105-2482 Telephone: (415) 268-7000 Facsimile: (415) 268-7522 pbesirof
Stanford - RBAKD - 1029
Robert S. Green (State Bar No. 136183) Robert A. Jigarjian (State Bar No. 171107) GREEN & JIGARJIAN LLP 235 Pine Street, 15th Floor San Francisco, CA 94104 Telephone: (415) 477-6700 Facsimile: (415) 477-6710 ABBEY GARDY, LLP Mark C. Gardy Charles H.
Stanford - RBAKD - 1029
Duke - EE - 164
Task List Group management plan Background studies Link budget: optical/electrical Build, test learning Rx board continued Order components for transceiver (TxRx) module Design, circuit schematic, layout of full duplex TxRx module (Run 1) Buil
Duke - ECE - 4006
Group 1 7 3 4 6 2 7 1 5 2 3 2 4 4 6 2 7 3 5 6 4 5 3 1 6 7 7 4 6 2 1 5 1 5First Ragad Shilo Tiffany James Theron Richard Jed Michael Melanie Curtis Ming Joshua Elisa Jinesh Gautam Christopher Dhairya Michelle Barry Vikas Bishan Nihar John Mustansir
Duke - ECE - 4006
Task List Group management plan Background studies Link budget: optical/electrical Build, test learning Rx board continued Order components for transceiver (TxRx) module Design, circuit schematic, layout of full duplex TxRx module (Run 1) Buil
Duke - ECE - 261
Combination LockECE261: VLSI December 11, 2007Chris Gregory Archana Ramamoorthy Viresh Thusu Andrew WangFeaturesMultiple users with different password protection for each user. User friendly indicators using LEDs. Time out for incorrect entrie
Princeton - COS - 116
COS 116 The Computational Universe Homework 61. The notion of exponential growth is mentioned in each of the following excerpts from a magazine or newspaper. Which of them use the notion correctly and which don't? Justify each answer in a line. Note
Princeton - COS - 116
COS 116 The Computational Universe Homework 2 Due 3/9/2006Q1) Why do supermarket chains and drugstores issue "preferred customer" cards? Q2) Convert 45 from decimal to binary. Convert 110101 from binary to decimal. Q3) In class we saw how to model t
Princeton - COS - 116
COS 116 The Computational Universe Writing and Blogging AssignmentDiscussion for Tuesday, May 2: Searle's brief but controversial article. Class discussion will focus on the following topics. Topic 1: Try to find passages in Searle's article that su
Princeton - COS - 116
COS 116: The Computational UniverseAdam Finkelstein COS116: 2/5/07COS 116: The Computational Universe Instructor: Adam Finkelstein Head TA: UmarSyedPreceptors: ForresterCole Ari Feldman Labs will be held in (Friend 005) Tues7-10
Princeton - COS - 116
COS 116 The Computational Universe More about selfreproducing programsThis handout gives more details on the construction of selfreproducing programs, which was sketched in Lecture 10. In this construction we assume that the machine has a separate
Duke - ECE - 3040
ECE 3040 d,e Microelectronic Devices and CircuitsFall, 2000 Class Meetings: M 1:05 1:55 pm (d lecture) OR M 2:05 2:55 pm (e lecture) AND T Th 12:05 1:25 pm (d lecture) OR TTh 1:35 2:55 pm (e lecture) Instructors: Prof. Nan Marie Jokerst and Prof
Duke - ECE - 3040
GEORGIA INSTITUTE OF TECHNOLOGY School of Electrical and Computer Engineering ECE 3040 D,E Course Outline Fall 2000 Text References P=Pierret J = Jaeger Web Notes J 1.2 P 1.1 P 1.2 P 2.2 P 2.3 P 2.4 P 2.5 P 3.1 P 3.2 P 3.3 P 3.4 P 4.1 P 5.1-5.2 P 6.1
Duke - ECE - 3040
4. The diode circuit in Figure 1 is a rectifier circuit. The voltage across the load R (vC) is to be a DC voltage of 5 V with a maximum ripple of +- 50 mV (100 mV peak to peak). Use the information given below to determine the smallest capacitance C,
Duke - ECE - 3040
dtime 0IS 1.00E-13Nk T 1 1.38E-23q 300 1.60E-19f 60C 3.00E-04time 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0vS 0 0.06 0.13 0.19 0.25 0.31 0.38 0.44 0.5 0.56 0.63 0.69 0.75 0.81 0.88
Duke - ECE - 269
ECE 269: VLSI System TestingSpring 2009 Krish Chakrabarty Homework 1 Assigned: January 22, 2009 Due: February 3, 2009 Instructions: You are required to work on the homework on your own. If you think a question has several interpretations, make reaso
Duke - ECE - 269
ECE 269: VLSI System TestingSpring 2009 Krish Chakrabarty Homework 2 Assigned: February 3, 2009 Due: February 17, 2009 (start of class) Instructions: You are required to work on the homework on your own. If you think a question has several interpret
Duke - ECE - 269
Problem 4 There are some parameters to be chosen when running the Fastscan to generate stuck-at test patterns. 1. N-detection Students can choose different n-detection patterns in the DOFILE using the following command. For an ordinary stuck-at test
Duke - ECE - 269
ECE 269: VLSI System TestingSpring 2009 Krish Chakrabarty Homework 3 Assigned: February 24, 2009 Due: March 17, 2009 (start of class) Instructions: You are required to work on the homework on your own. If you think a question has several interpretat
Duke - ECE - 269
ECE 269: VLSI System TestingSpring 2009 Krish Chakrabarty Solutions to Homework 3 Problem 1 Note: the solution of problem 1 is prepared by Yang Zhao. (a) Testing time for a full-scan circuit with n flip-flops, m scan chains, and p test patterns. The
Duke - ECE - 269
ECE 269: VLSI System TestingSpring 2009 Krish Chakrabarty Homework 4 Assigned: March 31, 2009 Due: April 14, 2009 (start of class) Instructions: You are required to work on the homework on your own. If you think a question has several interpretation
Stanford - EE - 102
Stanford University Spring 20082009ModulationThe modulation theorem tells us how to place a message signal m(t) on a carrier cos(ct). How do we recover m(t) from the modulated signal? What are the problems?Signal Processing and Linear Systems I
Duke - ECE - 4006
VCSEL Parameters Max Eyesafe Power Pout(max),VCSEL (mW) Fiber Attenuation (dB) Fiber Attenuation (Linear) Extinction Ratio (dB) Min Power Output Pout(min), VCSEL (mW) Imod (mA) VCSEL Manufacturer Honeywell Honeywell Optek Est Values based on Typical
Duke - ECE - 4006
Part Part # Units Unit Price (US$) Total(US$) Phone Cost of 1 Cost of 1000 Laser Driver Maxim 3287 1 6.59 6.59 9.84 6.59 TIA Maxim 3266 1 5.37 5.37 8 5.37 Limiting Amp Maxim 3264 1 6.39 6.39 4087377600 9.84 6.39 VCSEL Honeywell HFE4384-522 1 12 12 18
Duke - EE - 3270
#%!PS-Adobe-3.0 %Creator: Windows PSCRIPT %Title: Microsoft Word - NIVAMOD.DOC %BoundingBox: 14 9 597 784 %DocumentNeededResources: (atend) %DocumentSuppliedResources: (atend) %Pages: (atend) %BeginResource: procset Win35Dict 3 1 /Win35Dict 290 dict
Duke - EE - 3270
Duke - EE - 164
GIGABIT ETHERNET DESIGN ECE164.02 - Spring 2004Presentation # 6Week Progress Summary Obtainedand Built Boards of our Design Tested Our Boards Verified Operation with Eye Diagrams Updated GANTT Chart Began Photodiode TestingGantt ChartPr
Elon - CIS - 211
Small-group discussion A consulting firm requires that all consultants use software that records their interactions with applications and files in order to help automate the client billing process.What are some of the security, privacy, and ethical
Duke - ECE - 135
Project GabE ECE 135 Spring 2005Final PresentationOmar Al-Jadda Patrick Crosby Adam Durity Rahmin SarabiProgress Report Builtfinal board design and verified Tx and verified Rx Testing Tested Tested LoopbackGantt ChartID 1 2 3 4 5 6 7
Duke - ECE - 4006
ECE4006 Group 6 Presentation 1Division of Labor Vikas Parekh Group management, project schedule manager, background studies. Gautum Khanna Analog design and testing for the receiver board. Akil Khamisi Sutton Part management, link
Duke - ECE - 4006
ECE4006 Group 6 Presentation 2 Overview This Week's AccomplishmentsNext Week's GoalsRevised Gantt Chart Optical Link Budget Website Built and Tested Evaluation Board Revised Gantt Chart Updated Optical Link BudgetSources: Tab
Duke - ECE - 4006
ECE 4006 Group 6Presentation 4 February 13, 2003Accomplishments & Goals Whats been done. Optical Link Budget Verified Schematic Verified Contacted Vendors and Ordered Parts Initial Transceiver Layout What needs to be done. Verify Layou
Duke - ECE - 4006
ECE4006 Group 6 Presentation 7 Overview Last WeekThis WeekAssembled First Fabrication Baseline Tests on Transmitter Completed Paper Continued Testing Anticipated PD Arrival First Run Layout First Run Soldered Board Pe
Duke - ECE - 4006
ECE4006 Final PresentationGroup 6, Spring 2003 Gigabit EthernetVikas ParekhGroup Management Plan Vikas Parekh Group manager, background research, scheduling and budgets. Theron Davis Webmaster, chief editor and solder expert. Domin
Duke - ECE - 4006
Gigabit Ethernet Design ECE 4006C Spring 2003Group 7 Presentation #2 Jed EatonJanuary 23rd, 2003Progress Report First Lab Session: Decipher Rx layout. Familiarized with lab tools. Important safety tips. Built and TestedReceiver Board Rea
Duke - ECE - 4006
Gigabit Ethernet Design ECE 4006C Spring 2003Group 7 Presentation #3 Pranjal ShahJanuary 30th, 2003Progress Report Optical Link Budget: VCSEL options narrowed to 2. Block diagram, started to fill in the numbers. Familiarization withoverall