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 DeForrest Avenue East Hanover NJ 07936 Contact: Neil Goldstein, Ph.D. ngoldstein@heliogenetics.com 973 342-7996 HelioGenetics Inc

ABSTRACT HelioGenetics has developed a rapid approach for generating and characterizing monoclonal IgG 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, MerRT, ErbB3, EGFR, PD-L1 and c-Met.  One of our Axl “antibodies” has been further characterized and shown to bind to the appropriate Axl-expressing 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.    HelioGenetics 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. HelioGenetics 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 HelioGenetics Inc

ANTIBODY CONSTRUCT IN M13 PHAGE NH2- gene III protein -COOH Phage Display on pIII: 3+3 Phagemid System pUC ori M13 ori gene III Phagemid cloning site Antibody ETag FLAG HelioGenetics Inc

Target specific Antibodys BIOPANNING PROTOCOL Repeat 3-4 cycles Wash Bind to Target Target specific Antibodys Antibody Libraries H+ etc. Amplify Elute 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 pH buffer and amplified overnight at 37oC in combination with helper phage. Usually 3-5 rounds are needed to identify target-specific binders and eliminate non-specific phage. HelioGenetics Inc

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

Size Distribution in a rae library HelioGenetics 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 CDR3 Can be produced in 293 and CHO cells 2-step selection process has been used to select for binding only in absence of GAS6 Inhibits growth of cancer cells independent of added Gas6 HelioGenetics Inc

Axl ANTIBODY: Effects on Human Cancer Cells Control Ig + Gas6 Axl Antibody H1299 Control Ig Axl Antibody SW480 Control Ig Axl Antibody A549 Control Ig Axl Antibody H1299 No Gas6 Control Ig Axl Antibody SW480 Control Ig SW480 (colorectal carcinoma), H1299 (non-small cell lung cancer) and A549 (lung cancer) are plated in 96 well microtiter plates at 5000 cells per well in 100ul DMEM containing 1% FBS and incubated overnight at 37C. Axl antibody and controls are added to the appropriate wells in the presence or absense of Gas6 (200ng/ml) and incubated for 72 at 37C. Viability is determined using WST-8 (Sigma) and read at 450nM at various times. HelioGenetics Inc

Targeted Cells ExpRess Axl & Gas6 1 2 3 4 5 6 RT-PCR: SW480 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) HelioGenetics Inc

Viability Assay vs. SW480 Medium Control Control Antibody Axl Antibody Cl 163 Cl 36 Cl 8 Cl 243 Cl 247 SW480 cells (colorectal cancer) are plated in 96 well microtiter plates at 5000 cells per well in 100ul DMEM containing 1% FBS and incubated overnight at 37C. Samples and appropriate controls (100ul/well) are added and the plates incubated for 72 hours. 10ul 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 450nM. Antibodies: Control antibody, anti-IL-8; anti-Axl antibody (vs. recombinant Axl); cl 163, anti-MerRT; cl 36, anti-Axl; cl 8, anti-ErbB3; cl 243 and 247, anti-Axl-Gas6 complex with negative pan. HelioGenetics Inc

Biological Effects OF aNTIBODIES on SW480 Medium Control Cl 8 Cl 36 Cl 163 Cl 243 Phase Stained Control Antibody Axl Cl 247 Antibodies: Control antibody, anti-IL-8; anti-Axl antibody (vs. recombinant Axl); cl 163, anti-MerRT; cl 36, anti-Axl; cl 8, anti-ErbB3; cl 243 and 247, anti-Axl-Gas6 complex with negative pan. HelioGenetics Inc

Size Distribution of Anti-Axl & Anti-Mer Antibodies Anti-Mer RT HelioGenetics Inc

Targets Panned Cancer Cells Pancreatic Breast Melanoma NSCLC Ovarian Recombinant Targets Axl receptor Mer receptor PD-L1 EGFR ErbB3 PDGFR FGFR Cancer Cells Pancreatic Breast Melanoma NSCLC Ovarian HelioGenetics Inc

Overview Rapid generation and characterization of target-specific antibodies Targets can be purified recombinant proteins or those expressed in situ on tissue culture cells 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 HelioGenetics Inc