KnotBodiesTM: creating ion channel blocking antibodies by fusing Knottins into peripheral CDR loops Aneesh Karatt Vellatt Precision Medicine and Ion Channel Retreat 2017 Vancouver Confidential Antibody discovery at IONTAS Antibody drug discovery company founded in Oct 2012 Powerful track record – 24 antibody discovery projects with 14 European and US organisations to Q1 2017 100% record of success! Protein Lead Primary Lead Secondary Lead/back Target Selection production Isolation Screen Optimisation Screening up IgG Phage library 4 x 1010 clones High proportion of insert Bi-specific Mammalian display scFv KnotBodiesTM Fab Production Research Targeting ion channels with antibodies Plasma membrane VGSC Challenging target class for antibody generation Difficult to express and purify, low stability Limited epitope availability Dynamic molecules with multiple conformations Sea anemone ShK Kv1.3 blocker Velvet Tarantula ProTx-II Nav1.7 blocker Conus snail Ziconotide Cav2.2 blocker Black Mamba Mambalgin1 ASIC blocker Casewell et al Trends in Ecology and Evolution (2013) Knottins: nature’s ion channel inhibitor scaffold PcTx1 Huwentoxin-IV ω-conotoxin-MVIIA ASIC1 blocker Nav blocker : Cav blocker 30-40 amino acids, 3-4 disulfide bonds Forms a conserved (Inhibitory Cystine Knot) structural motif high sequence and functional diversity despite the structural conservation Also found in non venomous species and modulate wide variety of biological functions Knottins: therapeutic development Ziconotide (PRIALT®; Primary Alternative to Morphine) ω-conotoxin-MVIIA, blocks N-type calcium channels Approved for the treatment of neuropathic pain Dalazatide (ShK-186) Engineered ShK toxin, blocks Kv1.3 In phase II trials for the treatment of psoriasis Most naturally occurring knottins lack exquisite specificity Huwentoxin-IV block potently both Nav1.7 and Nav1.2 ShK (non-engineered) toxin equally blocks Kv1.3 and Kv1.1 Evolved to paralyse prey hence not specific and requires further engineering for therapeutic use Challenges in knottin engineering and therapeutic development Limited compatibility with robust library selection technologies that can sample large mutant libraries Rational design strategies are laborious and exert less control over the specificity of new binders Chemical synthesis can be complex and expensive Half life of minutes to hours: too short for a drug that is expensive to synthesise Scaffolds within scaffolds: Combining the benefits of knottins and antibodies Knottins Natural blockers of ion channels Lack specificity, short half life Difficult to engineer Antibodies Ecballium elaterium Large binding surface providing specificity (Jumping cucumber) Amenable to engineering using in vitro selection technologies Long half life KnotBodiesTM concept: Insert knottins into peripheral antibody CDR loops Engineer other CDR loops for improved potency EETI-II: Trypsin and selectivity using phage display technology inhibitor (as model knottin) Specific ion channel modulators with long half life! Inserting EETI-II knottin into VL CDRs Fv H chain VH L chain VL VH VL IgG Randomised linkers joining Variation in recipient VL EETI-II knottin to VL framework Knottin scaffold FR1 CDR1 FR2 FR3 CDR3 FR4 Selecting antibodies by phage display Genotype Genotype + Phenotype Select for phenotype i.e. antigen binding Antibody displayed Antibody on phage as pIII (in scFv format) fusion Promoter Sig P VH VL Gene III Gene encoding the displayed Phage display Vector antibody Immobilised antigen (Trypsin) McCafferty et al (1990) Nature 348 p552-4 Insertion into antibody CDRs make knottins amenable to phage display! 300000 EETI-II CDR2 Fusion 250000 EETI-II CDR1 Fusion 200000 150000 100000 EETI-II Trypsin binding (FU) binding Trypsin Direct phage display 50000 0 A B C Monoclonal ELISA of trypsin binding TM 250000 KnotBodies 200000 VL-CDR2 150000 2 rounds phage (42/94 binders) 100000 50000 Trypsin binding (FU) binding Trypsin 0 1 8 15 22 29 36 43 50 57 64 71 78 85 92 Clone number Confirm specificity with EETI-II loop 1 (trypsin binding loop) 250000 VL-CDR1 mutation 200000 2 rounds phage (18/94 binders) 150000 CPRILMRC 100000 CGAILMRC Trypsin binding (FU) binding Trypsin 50000 0 1 8 15 22 29 36 43 50 57 64 71 78 85 92 Clone number Correct linkers are important for function 100 90 80 70 60 50 40 30 20 Normalised tryspin binding Normalisedbinding tryspin (%) 10 0 "Selected Gly4Ser linker (Gly4Ser)2 "Selected Gly4Ser linker (Gly4Ser)2 linker" linker linker" linker KB_A07 KB_A12 Crystal structure of KnotBodiesTM KB_A12( 2.5Å) KB_A05 (1.95Å) Crystal structure of KnotBodiesTM Demonstrating the capabilities of the KnotBodyTM format (i) Improving the existing knottin binding by introducing additional VH contacts (ii) Create a bispecific molecule by introducing a VH that binds to different target (iii) Alter the specificity of the original knottin scaffold by loop diversification (iv) Generate ion channel blocking KnotBodies Change in SPR Trypsin binding (FU) of KnotBody affinity Improved 100000 150000 200000 250000 300000 350000 400000 50000 0 B06 binding Trypsin Trypsin A10 B07 C01 Trypsin dissociation dissociation Trypsin E06 D08 F11 VH selectionpartner D09 C06 ELISA ranking using Affinity Off E08 F02 Time (S) Time - rate analysis SPR analysis using rate H07 A07 D01 Clone ID Clone H05 F07 C02 H01 E07 C10 G03 B08 D10 E10 B03 B11 KB_A12 (parent clone) (parent KB_A12 KB_A07 (Parent clone) (Parent KB_A07 clone improved Affinity F05 TM KB_A12 E12 D12 G12 KB_A07 through through B12 A03 Bispecific binding through binding Bispecific through VH VL VL selection Parent KnotBody cMET bi-specfic KnotBody 30000 ) ) 360000 U U F F ( ( 20000 240000 g g n n i i d 10000 d 120000 n n i i B B 0 0 T 6 4 n T 6 4 n E s R i E s R i a F s a F s M G p M G p c G ry VH c G ry F T F T partner VH Gas6 bi-specific KnotBody FGFR4 bi-specific KnotBody ) ) 360000 360000 U U F F ( ( 240000 240000 g g n n i i d VH d n 120000 n 120000 i i B B 0 0 6 4 T 6 4 n T n VL VL i i E s R s E s R s M a F p M a F p c G G y c G G y r r F T F T Altering specificity of knottin “donor” KB_A12 KnotBody Randomise L1 loop of EETI-II donor-> 4 x 109 library (loop lengths= 6, 8, 9, 10 using VNS codons) Altering specificity of knottin “donor” 200000 600000 180000 c-Met 550000 b-galactosidase 500000 160000 450000 140000 400000 120000 350000 100000 300000 80000 250000 200000 60000 150000 40000 100000 20000 50000 Antigen binding (FU) binding Antigen 0 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 Clone number Clone number -CPRILMRC- 23/47 unique L1 loop 11/47 unique Role of potassium channels in T effector cell signaling ShK mutant Central Activated CM memory Naïve Naïve effector pore Effector Activated EM memory Kv1.3 Model from: Murray et al. (2015) J. Med Chem. Autoreactive TEM cells depend heavily on Kv1.3, while naïve and central memory T cells depends on KCa3.1 Autoreactive TEM cells can be eliminated via selective inhibition of Kv1.3 Measuring Kv1.3 ion channel currents using automated patch clamp control, t = 0 min 100 ms 0.5 nM, t = 4 min A n 5 5 nM, t = 8 min . 0 50 nM, t = 12 min 500 nM, t = 16 min +30 mV, every 10 s -80 mV Sophion QPatch Functional inhibition of Kv1.3 by KnotBodiesTM ShK KnotBody Kaliotoxin KnotBody 100 100 100 g g g n n n i i i 75 75 75 n n n i i i a a a m m m e e e r r r KB_HsTX1 KB_ShK KB_KalioTx 50 50 50 t t t IC = 3.9 nM IC = 8.6 nM IC = 430 nM n n n 50 50 50 e e e r r r r r r u u u c c c 25 25 25 % % % 0 0 0 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 Log [M] Log [M] Log [M] Functional inhibition of Kv1.3 by KnotBodiesTM 100 100 100 g g g n n n i i i 75 75 75 n n n i i i a a a m m m e e e r r r KB_HsTX1 KB_ShK KB_KalioTx 50 50 50 t t t IC = 3.9 nM IC = 8.6 nM IC = 430 nM n n n 50 50 50 e e e r r r r r r u u u c c c 25 25 25 % % % 0 0 0 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 Log [M] Log [M] Log [M] ShK KnotBody inhibited cytokine and Granzyme B secretion by activated PBMCs Functional inhibition of ASIC1a by KnotBodiesTM Therapeutic areas: neuropathic pain, neurological disorders PcTX1 (Psalmotoxin) - ASIC1a toxin blocker KnotBody A12_PcTX1 KnotBody A07_PcTX1 100 100 g g n n i i 75 75 n n i i a a m m e e r r 50 IC = 98 nM 50 IC50 = 68 nM t 50 t n n e e r r r r u u c 25 c 25 % % 0 0 -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 Log [M] Log [M] Rapid generation of cell lines for specificity screening pINT_ IC Vectors CHO-S cells pIONTAS IC Vectors MaxCyte STX Electroporation Suspension cells Transient expression High transfection efficiency and scalability Easy to culture Polyclonal stable expression using transposase system High cell viability No dissociation required Expression from a specific Efficient multi plasmid co- locus using nuclease mediated transfection Direct use in APC integration Comparison with monoclonal stable cell line Test Kv1.3-CHO Kv1.3 Transient Kv1.3 + Transposase Parameters Stable Cell Line MaxCyte MaxCyte (CRL) (24 h after EP) (2 weeks after EP) Cell Viability (%) ≈ 99 ≈ 99 ≈ 98 Mean IK+ (nA) 1.5 ± 1.3 4.7 ± 3.1 2.8 ± 3.4 % IK+ >0.5nA 91 88 87 % seal >1 G 89 83 88 I-V V1/2 (mV) -1.2 2.5 1.1 I-V slope (mV) 26.7 21.7 20.4 Data obtained using Sophion QPatch, Single hole mode Evaluating the pharmacology of Kaliotoxin Kaliotoxin Kv1.3-CHO Kv1.3 Transient Kv1.3 + Kv1.1 Transient stable Transposase IC50 (nM) 0.78 0.68 0.56 2.5 Kaliotoxin Kaliotoxin g 100 g 100 n n i i n n i i a 75 a 75 m m e e r r t 50 Kv1.3 Stable t 50