1. 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.
HelioGenetics Inc

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. Size Distribution in a rae library
HelioGenetics Inc

9. 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

10. 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

11. Targeted Cells ExpRess Axl & Gas6
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 (2009)
HelioGenetics Inc

12. 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

13. 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

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

15. 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

16. 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