The Trianni Mouse: Best-In-Class Technology for Human Antibody Discovery Corporate Overview Co David Meininger, PhD, MBA Chief Business Officer, Trianni 1
Our Mission Trianni is a biotech company with the primary scientific mission of creating an optimized and highly versatile platform for isolating fully human monoclonal antibodies. 2
The Trianni Mouse TM Difference 3
Distinctive Features of Trianni s Technology Elimination of endogenous V, D and J gene segments Humanization of all three antibody loci H, K, L Designed immunoglobulin alleles Targeted insertion of arrays of synthetic antibody gene segments Optimizations throughout the arrays Chimeric gene segments (important re FTO) Mouse noncoding (regulatory) DNA Human coding DNA Platform flexibility Refinements can be generated quickly Alternative repertoire mice in development 4
Core Alleles Heavy (H) 44 x V H D H J H Eμ μ δ γ3 γ1 γ2b γ2c ε α Kappa (K) Lambda (L) 5 39 x V K J K C K 38 x V L J1C L J2C L J6C L J7C L J6C L J3C L Deletion of endogenous V, D and J gene segments Chimeric gene segments (human open reading frames paired with mouse regulatory regions) Chimeric antibodies (human Vs with mouse Cs) Other versions of the core alleles available or under development
In-House Platform Validation Antibody repertoire analysis All gene segments are used; CDR3 lengths and amino acid compositions as in humans Immunizations Ten model antigens tested (human extracellular protein domains) mabs similar to wild type mice in terms of affinity, specificity and epitope coverage 6
Trianni Mice: Replete With B Cells and Antibodies Control HHKK HHKKλλ 7
CD23 Igλ B220 Igλ Flow Cytometry of Spleen Cells from TRN H/ / Gated on: Total lymphocytes MZ B (CD21 Hi CD23 Lo ) CD19 Igκ Total B cells (CD19 + B220 + ) Fo B (CD21 + CD23 + ) 8 CD21 Igκ In gray, non-b cells in the same staining tube to illustrate the light chain staining backgrounds
A Highly Diverse Heavy Chain Repertoire 44/44 VHs used
H CDR3 Length Distribution IgM-Sorted Naïve B cells 10
Amino Acid Frequencies 0 20 40 60 80 100 Amino Acid Frequencies 0 20 40 60 80 100 Heavy Chain CDR3 Compositions Human Y W V T S R Q P N M L K I H G F E D C A CDR3s of 15aa in length 92 93 94 95 96 97 98 99 100 100A 100B 100C 101 102 103 Amino Acid Positions Trianni Y W V T S R Q P N M L K I H G F E D C A non-immune repertoire 92 93 94 95 96 97 98 99 100 100A 100B 100C 101 102 103 Amino Acid Positions 11
Five Recent External Evaluation Reports Three major pharmaceutical companies Multiple antigens Comparison to BALB/c and C57BL/6 mice A (clinical stage) biotechnology/pharmaceutical company 1 antigen A start-up small biotechnology company 1 antigen 12
Evaluation, Large Pharma #1 Target 2, two campaigns (v1 and v2): both WT and Trianni Mice mount strong and similarimmune responses 13
Anti-Target 2 Trianni Mouse mabs Demonstrate Superior Maximum and Average Potency than Wild-Type C57BL/6 Benchmark mabs 14
Evaluation, Large Pharma #2 - Two human cell surface proteins (Ag1 and Ag2) - A soluble human protein (Ag3) - Immunization: - Twice a week using Ribi adjuvant - A total of eight injections - Comparison to BALB/c mice - Three Phase Evaluation: - Immunization data - Immune response quality and diversity; fusion screening data - Detailed characterization of 20 mabs per target 15
Comparable Titers for All Three Antigens Trianni BALB/c 16
MAb Isolation Hybridoma generation Electrofusion of lymph node cells Primary screen Binding to human antigen Secondary screen Cross-reactivity to cynomolgus antigen Ligand-blocking activity Off-rates for all binders (using supernatants) Epitope competition (using supernatants) 17
Primary Screening Results Fusion parameters and primary binding similar to BALB/c 18
Secondary Screening Results Extended characterization of hu-ag+ supernatants similar to BALB/c Ligand blocking Cyno cross-reactivity Off-rates (majority of clones in 10-2 to 10-3 range using supernatants) Epitope coverage (Ag3 6/7 Trianni, 5/7 BALB/c) 19
Tertiary Analysis Subclone 20 hybridomas from each Tg animal/ag Determine productivity of hybridomas Characterize EC 50, IC 50 and K D for purified mabs Assess diversity of selected mabs 20
Data Summary Good quality antibodies were produced relative to control mice and benchmark antibodies Reasonable diversity seen with sequenced antibodies 21
TRIANNI Strongly Preferred in Side-by-Side Comparison Pharma #2 performed a side-by-side comparison with another in-vivo platform The Trianni Mouse performed much better the results were not even close. (Quote From Pharma #2) 22
Evaluation, Large Pharma #3 - Independent evaluation at two sites (US and Europe) - Multiple human antigens in both cases - Immunization: - RIMMS vs. conventional - Hybridomas and single B cell cloning - Antibody reformatting 23
Trianni Mouse-Derived Hybridoma Deliver Normal Production Yields and mab Quality 24
Purified Trianni MAbs Exhibit Sub-nM to nm Binding Affinities Apparent Affinity on Soluble Protein K d app (M) Affinity in subnm-nm range for both Trianni and BALB/c Mean ± SD (M) 4.8 x 10-10 ± 5.4 x10-10 Mean ± SD (M) 3.9 x 10-09 ± 9.2 x10-09 25
Trianni mabs Can Be Successfully Reformatted into Fully-Human IgG Anti CD38 Clones Purified Batch Productivity mg/l Purity SDS-PAGE Mass spectrometry MW (Da) ELISA on CD38 EC50 (nm) LC HC Reformatted Hybridoma 109HHKK-3 VA115073 135 98 % 23 301 51 486 0.23 0.46 820HHKK-3 VA115081 108 > 99 % 23 223 50 445 0.37 1.26 1195HHKK-3 VA115072 152 99 % 23 356 50 939 0.42 0.21 1370HHKK-3 VA115079 77 > 99 % 23 257 50 417 0.30 1.40 1883HHKK-3 VA115080 73 94 % 23 235 51 745 0.10 0.06 4487HHKK-5 VA115098 26 99 % 23 359 50 050 0.07 0.26 4527HHKK-5 VA115099 42 99 % 24 011 51 043 0.10 0.15 992HHKK-7 VA115100 62 99 % 23 390 50 443 0.10 0.05 from reformatted Balb/c 30-200 Sequence retrieved by RT-PCR from hybridoma Cloning VH and VL sequence into mammalian expression vectors Similar production yield and mab quality from reformatted mabs and Balb/c 26
Business Model 27
Our Partners Below is representative of our publically announced partnerships DISCOVERY PARTNERS STANFORD UAB JANSSEN CRO PARTNERS 28
TRIANNI Benefits Small company means: - easy access to our experts - lean processes (quicker time to use of our platform) - personal relationships 60 years combined industry experience Representation in key biotech locations - San Francisco, CA (West Coast, USA) - Research Triangle Park, NC (East Coast, USA) - Austria (Europe) Flexibility to create a custom partnership to match your needs Forward-thinking leadership Faster turn-around time Technical support, if needed An investment in your company. We value our partners and want you to succeed! 29
Presented at the 3 rd Biologics and Biosimilars Congress. To find out more, visit: www.global-engage.com 30