Generation of Novel Monoclonal Antibodies Binding to Cancer

Generation of Novel Monoclonal Antibodies Binding to Cancer Targets Using Diverse Display Libraries Michael Spruyt, Frank Buquicchio, Leigh-Ann Ricks & Neil Goldstein Helio Genetics Inc 46 De. Forrest Avenue East Hanover NJ 07936 Contact: Neil Goldstein, Ph. D. ngoldstein@heliogenetics. com 973 342 -7996 Helio. Genetics Inc

ABSTRACT Helio. Genetics has developed a rapid approach for generating and characterizing monoclonal Ig. G heavy chain molecules as an alternative to the widely used methods for identifying monoclonal antibodies. These molecules are derived from highly diverse DNA libraries consisting of randomized binding sites and include antigen binding sequences outside of the normal human repertoire. In-vitro selection identifies highly specific target binders and subsequent in-vitro functional screening allows for the selection of bio-active molecules. Overall, our technology can yield humanized antibodies which cannot be produced via traditional poly/monoclonal methodologies. Using this approach, we have developed antibodies to a number of cancer related targets including Axl, Mer. RT, Erb. B 3, EGFR, PD-L 1 and c-Met. One of our Axl “antibodies” has been further characterized and shown to bind to the appropriate Axlexpressing tumor cells lines and inhibit their growth in vitro. Additional studies are underway to further delineate the utility of this and other antibodies as candidates for further clinical development. Helio. Genetics Inc

INTRODUCTION The RAE system overcomes limitations of current methods for the isolation of human antibodies which bind to antigens of choice with high affinities and specificities. Human antibodies are required for many therapeutic applications. However, current methods for obtaining human antibodies with required bioactivities for therapeutic use are often unreliable. These methods include isolating antigen-specific hybridomas from human antibody -producing transgenic mice, and isolating antigen-specific human antibody genes from libraries displayed on bacteriophage, cells, or ribosomes by biopanning. The RAE system takes advantage of complete randomization of the antibody binding sites on the heavy and light chain of a human antibody to identify target specific binders with biological activity. The system allows “biology” (i. e. , biopanning of recombinant targets or tissue culture cells) to make a decision about which CDR sequence(s) is important for binding. A high level of diversity is obtained from both the random nature of the amino acid sequences of the antigen binding sites plus the potential for “repetitive length” within the sites themselves. Helio. Genetics Inc

BENEFITS OF THE RAE SYSTEM • RAE = Randomized Antibody Engineering • Ability to identify and perform initial biological characterization of target -specific binders within 5 weeks • Total randomization of antigen binding sites allowing “biology” to optimize target-specific sequence(s) • Additional diversity derives from “ repetitive length” within antibody binding site • Targets include recombinant proteins and proteins expressed in situ on tissue culture cells Helio. Genetics Inc

ANTIBODY CONSTRUCT IN M 13 PHAGE NH 2 - FLAG Antibody ETag gene III protein cloning site gene III Phagemid Phage Display on p. III: 3+3 Phagemid System M 13 ori p. UC ori Helio. Genetics Inc -COOH

BIOPANNING PROTOCOL Antibody Libraries Wash Bind to Target Repeat 3 -4 cycles H+ etc. Target specific Antibodys Elute Amplify A target is first coated on an immobilized surface (e. g. , microtiter plates). A phage library is added for several hr and non-binders removed by extensive washing. Target-specific phage are eluted with a low p. H buffer and amplified overnight at 37 o. C in combination with helper phage. Usually 3 -5 rounds are needed to identify target-specific binders and eliminate non-specific phage. Helio. Genetics Inc

TIME LINE TO ID TARGET-SPECIFIC ANTIBODIES Day Day 0: 1 -4: 5 -6: 6 -7: 7 -8: 9: 10 -13: Day 14 -18: Day 19: Day 20 -23: Day Day 24 -28: 28 -31: 32 -35: 36: Helio. Genetics Inc Coat target or cells on solid support Pan with high diversity antibody display library Plate and identify antibody expressing colonies Prepare “Master Plate” Rescue antibody expressing colonies ELISA vs. target or cells to identify binders PCR to identify clones; insert into expression vector; mini-preps for antibody expression studies Transient transfection in 293 cells ELISA for antibody expression Maxi-preps of clones positive for antibody expression Transient expression in 293 cells ELISA for antibody expression Biological assay(s) to determine antibody activity Go/No Go for stable expression and further biological and biochemical characterization

SIZE DISTRIBUTION IN A RAE LIBRARY Helio. Genetics Inc

ATTRIBUTES OF AN ANTI-AXL ANTIBODY • Derived using RAE Technology vs. recombinant Axl • Binds to purified Axl and Axl-expressing cancer cells • Novel amino acid sequence in CDR 3 • Can be produced in 293 and CHO cells • 2 -step selection process has been used to select for binding only in absence of GAS 6 • Inhibits growth of cancer cells independent of added Gas 6 Helio. Genetics Inc

AXL ANTIBODY: EFFECTS ON HUMAN CANCER CELLS A 549 Axl Antibody Control Ig H 1299 Axl Antibody Control Ig SW 480 Axl Antibody Control Ig 10 9 8 7 6 5 4 3 2 1 + Gas 6 0 A 549 Axl Antibody 10 Control Ig 9 5 10 15 20 8 7 H 1299 Axl Antibody 6 Control Ig 5 No Gas 6 4 3 SW 480 Axl Antibody 2 Control Ig 1 0 5 10 15 20 SW 480 (colorectal carcinoma), H 1299 (non-small cell lung cancer) and A 549 (lung cancer) are plated in 96 well microtiter plates at 5000 cells per well in 100 ul DMEM containing 1% FBS and incubated overnight at 37 C. Axl antibody and controls are added to the appropriate wells in the presence or absense of Gas 6 (200 ng/ml) and incubated for 72 at 37 C. Viability is determined using WST-8 (Sigma) and read at 450 n. M at various times. Helio. Genetics Inc

TARGETED CELLS EXPRESS AXL & GAS 6 1 2 3 4 5 6 RT-PCR: SW 480 Lane 1 Marker Lane 2 Actin Control Lane 3 Axl Lane 4 Gas Lane 5 EGFR Lane 6 IGFR Western Blot Phosphorylation ELISA From Li et al. , Oncogene 28 3442 (2009) Helio. Genetics Inc

VIABILITY ASSAY VS. SW 480 Medium Control Antibody Axl Antibody Cl 163 Cl 36 Cl 8 Cl 243 Cl 247 0 0. 5 1 1. 5 2 OD 450 SW 480 cells (colorectal cancer) are plated in 96 well microtiter plates at 5000 cells per well in 100 ul DMEM containing 1% FBS and incubated overnight at 37 C. Samples and appropriate controls (100 ul/well) are added and the plates incubated for 72 hours. 10 ul of WST-8 (Sigma) are added to each well and the plates read at 1, 2, 3 and 4 hours in an ELISA reader at 450 n. M. Antibodies: Control antibody, anti-IL-8; anti-Axl antibody (vs. recombinant Axl); cl 163, anti-Mer. RT; cl 36, anti-Axl; cl 8, anti-Erb. B 3; cl 243 and 247, anti-Axl-Gas 6 complex with negative pan. Helio. Genetics Inc

BIOLOGICAL EFFECTS OF ANTIBODIES ON SW 480 Phase Stained Medium Control Cl 243 Control Antibody Cl 163 Axl Cl 247 Stained Cl 36 Cl 8 Antibodies: Control antibody, anti-IL-8; anti-Axl antibody (vs. recombinant Axl); cl 163, anti-Mer. RT; cl 36, anti. Axl; cl 8, anti-Erb. B 3; cl 243 and 247, anti-Axl-Gas 6 complex with negative pan. Helio. Genetics Inc

SIZE DISTRIBUTION OF ANTI-AXL & ANTIMER ANTIBODIES Anti-Axl Helio. Genetics Inc Anti-Mer RT

TARGETS PANNED Recombinant Targets • • Helio. Genetics Inc Axl receptor Mer receptor PD-L 1 EGFR Erb. B 3 PDGFR FGFR Cancer Cells • Pancreatic • Breast • Melanoma • NSCLC • Ovarian

OVERVIEW • Rapid generation antibodies and characterization • Targets can be purified recombinant expressed in situ on tissue culture cells of target-specific proteins or those • Secondary libraries can be generated to improve affinity, potency and selectivity • Antibodies are biologically active • Novel composition of matter for IP protection • Available for research collaborations and partnerships Helio. Genetics Inc