Antibody Selection from Immunoglobulin Libraries Expressed in Mammalian Cells

Antibody Selection From Immunoglobulin Libraries Expressed in Mammalian Cells

Ernest Smith, Ph.D. Senior Vice President, Research & CSO [email protected] 585-271-2884 www.vaccinex.com Vaccinex Corporate Summary

Headquarters: Rochester, NY

Employees: 51

History: Founded in 1997 Core technology invented by founders at the University of Rochester Exclusive world-wide license in all fields of use

Intellectual Property: 70+ U.S. and foreign patents and patent applications. Funding: Focus: Closed a $50M Round in 2009 Four antibodies in pre-clinical development $33M in previous venture funding Two INDs expected to be filed in Q4 2010 $10M in research grants Novel antibody discovery platform

2 Vaccinex Technology Summary: Discovering Antigens and Therapeutic Antibodies Vaccinia Virus Library Construction

Vaccinia Virus ____ Vaccinia virus infects most mammalian cells ____ Recombinant proteins expressed by vaccinia virus infected cells undergo normal post-translational modifications and trafficking ____ Very versatile mammalian expression vector, used for >20 years as a vector for basic scientific and vaccine research ____ The conventional recombination technology allows for the introduction of one defined gene at a time into vaccinia virus ____ This is sufficient for expression of a single gene product but does not enable functional cloning of unknown genes from a library

A New Technology was Needed to Enable More Efficient Creation of Recombinant Vaccinia Virus

4 Standard Homologous Recombination

Wt Vaccinia Virus

TK plasmid

Infect

plasmid Nucleus Ligate plasmid Transfect cDNA of interest

cDNA cDNA

There is no selection against wild type virus

Selection (ex: BUdR)

TK cDNA

99.9 % Wild type Virus 0.1% recombinant virus

5 Strategy to Generate 100% Recombinant Vaccinia Virus

V7.5/tk genomic DNA N A plasmid TK

Cut with N and A

Ligate Transfect arms + infect cDNA of interest defective helper virus

plasmid plasmid Transfect Nucleus

cDNA cDNA

Host cell containing ONLY recombinant vaccinia virus Nature Medicine 7:967-972 (2001)

6 Vaccinex Technology Summary

Vaccinex has developed proprietary technology that allows for the creation of large and diverse cDNA libraries in a vaccinia virus- based vector.

Millions of different vaccinia recombinants in each library. Nature Medicine 7:967-972 (2001)

Technology for functional cloning in mammalian cells. Efficient identification of immune target antigens Selection of fully human monoclonal antibodies

7 Antibody Libraries Expressed in Mammalian Cells

We have developed a large library-based antibody discovery technology that can efficiently express and select fully functional IgG antibodies in mammalian cells Directly expresses complete, bivalent antibodies

Separate heavy and light chain libraries 107 Ig-H X 107 Ig-L =1014 combinations rapid initial screening of 107 to 108 combinations Affinity improvement by sequential substitution of heavy and light chains Allows for efficient conversion of mouse MAbs to fully human

Expression of antibody libraries in secreted IgG format Ig-H gamma I (no TM domain) + Ig-L = secreted MAb Screen by ELISA (soluble antigen) or FMAT (cell surface antigen)

Expression of antibody libraries on the surface of mammalian cells Ig-H gamma I (with TM domain) + Ig-L = surface display Isolate specific antibodies by MACS/FACS Isolate specific antibodies following induction of apoptosis

8 Vaccinex Antibody Selection Platforms

1. Conversion of Mouse MAb to Human MAb

SSeeccrreetteedd A Annttibibooddyy P Plalattffoorrmm Membrane Antibody Platform: Selection by Cell Sorting

2. de novo Antibody Selection

3. Affinity Improvement of Human Antibodies

9 Conversion of Mouse MAb to Fully Human MAbs (Flow Chart)

Mouse Antibody

Clone V-genes, create vaccinia with chimeric Ig-H and Ig-K

Chimeric Ig-H

Use vaccinia with chimeric Ig-H to select replacement human Ig-K

Human Ig-K LIBRARY Use selected human Ig-Ks to select replacement human Ig-H

Human Ig-H LIBRARY

1st Generation Human MAbs

Optimize (If necessary)

Lead MAb

10 Conversion of Mouse Mab to Human Antibody

Perform ELISA or FMAT Screen

Fixed Ag-specific chain: complexity = 1

Irrelevant antigen

Plate out and amplify mini complementary Co-infect Fixed chain libraries from complex + mini-libraries parent libraries in HeLa cells Approximately 100 - Antigen of interest 1000 antibodies/well

Complexity = 100 - 1000 pfu Titer = 2x106/ml

100 replicate plates = 1x106 to 1x107 Immunoglobulin clones

Subclone specific chain from positive wells and rescreen

11 Use Mouse Ig-H to Select Human Ig-K: Round 1

12 Use Human Ig-K to Select Human Ig-H: Round 1

13 Expression and Characterization of Selected MAbs

The V genes in the clonal Ig-H chain and Ig-L chain vaccinia recombinants are cloned from vaccinia virus into mammalian expression plasmids.

Monoclonal antibody is expressed as full length IgG1/Kappa by transfection of the plasmids into CHO cells. IgG is purified using Protein A, and each selected MAb is tested for specificity, affinity and function.

As shown on the following slide, purified IgG was tested against an antigen of interest as well as a panel of control antigens.

14 Specificity of Antigen-Specific MAb

The V Genes From Selected MAbs are Transferred into Mammalian Expression Vectors and expressed as soluble IgG

15 Affinity of Antigen-Specific Antibodies

A large panel of human antigen-specific MAbs were selected including the two leads MAb 2071 and MAb 2090:

Biacore MAb affinity (nM) Chimeric 0.08 MAb 2071 0.03 MAb 2090 0.03

Selected Antibodies compete with the mouse antibody for binding to the antigen of interest and demonstrate functional activity that is superior to that of the chimeric antibody

16 Vaccinex Antibody Selection Platforms

1. Conversion of Mouse MAb to Human MAb

Secreted Antibody Platform Membrane Antibody Platform: Selection by Cell Sorting

2. de novo Antibody Selection

3. Affinity Improvement of Human Antibodies

17 Vaccinex Antibody Platform Transformation of Mouse MAb to Human MAb

Add labeled antigen then anti-IgG Single antigen-specific Magnetic chimeric heavy chain in beads / FACS vaccinia

Human Ig light chain n o library in vaccinia i s s e r p x E g Repeat infection I for additional Use specific selection cycle(s) Ag binding light chain to Collect positive identify human cells heavy chain Extract virus Analyze and analyze positive Amplify select best light pool by flow virus chain clones cytometry

18 Transformation of Mouse MAb into Human MAbs

Wild Type Chimeric Ig-H + Chimeric Ig-L r r e e t t t t a a c c S S d d w w F F

Ag Binding Ag Binding Chimeric Ig-H +Human Ig-L Human Ig-H + Human Ig-L r r e e t t t t a a c c S S d d w w F F

Ag Binding Ag binding

19 Relative affinity of fully human antibodies and chimeric MAb

The Ig-H and Ig-L chain genes are cloned from recombinant vaccinia virus into mammalian expression plasmids. Antibody is produced as full length soluble IgG1/Kappa by transfection of the plasmids into CHO cells.

IgG is purified using Protein A, and each selected MAb is tested for specificity, affinity and function.

Biacore MAb affinity (nM) chimeric 0.05 Human Mab 416 0.27 All MAbs compete for binding to Human Mab 926 0.15 antigen with the original mouse MAb Human Mab 1156 0.09 and demonstrate strong functional activity Human Mab 1338 0.07

Human Mab 1339 0.05 Human Mab 1259 0.09

20 Advantages of

For the conversion of mouse MAbs into human MAbs, our library technology allows for:

The selection of multiple candidate lead antibodies derived from distinct VH and VL germ line genes Multiple distinct Leads and backups Conservation of epitope specificity Affinity improvement from the original MAb Built in selection for good expression levels in mammalian cells

21 Screening for Human MAbs in Secreted IgG Format

Ig-H chain library: complexity = 100 clones/well Titer = 2x106/ml Irrelevant antigen

1 Co-infect Ig-H + Ig-L mini- libraries in HeLa cells Complexity = 1000 2 antibodies/well Plate out and amplify mini vv Ig libraries from Perform ELISA 3 complex parent libraries

Ig-L chain library: Isolate antigen-specific Antigen of interest complexity = 10 clones/well Ig-L and Ig-H by limiting Titer = 2x106/ml dilution cloning and re-screening 4

22 Affinity Improvement

Perform ELISA or FMAT Screen

Fixed Ag-specific chain: complexity = 1

Irrelevant antigen

Plate out and amplify mini complementary Co-infect Fixed chain libraries from complex + mini-libraries parent libraries In HeLa cells Approximately 100 - Antigen of interest 1000 antibodies/well

Complexity = 100 - 1000 pfu Titer = 2x106/ml

Subclone specific chain from positive wells and rescreen

23 Affinity Improvement of Human MAbs by V-Gene Replacement

The V genes from positive clones are isolated and cloned into mammalian expression plasmids. Antibody is produced as full length soluble IgG1/Kappa by transfection of the plasmids into CHO cells. IgG is purified using Protein A, and each selected MAb is tested for specificity, affinity and function

Biacore Antibody affinity (nM) Parental 40 Replacement #1 27 Replacement #2 6 Replacement #3 4

24 Summary of Selected Projects

Approximate number of Project Primary MAbs selected Selection type A 10 De novo B 60 mouse to human X 10 mouse to human I 20 De novo P 20 De novo IL6 60 mouse to human C35 90 De novo and mouse to human VX5 60 mouse to human VX70 20 mouse to human VX90 30 mouse to human

25 Development of New Antibody Selection Strategies Chimeric Receptor Based Selection Strategies

Because of their size, the throughput of MACS/FACS with mammalian cells is still lower than what can be achieved with yeast or Phage ______Employ hybrid receptors whose signaling results in the induction of apoptosis and loss of cell adherence (recombinant virus can be recovered from floating apoptotic cells) Candidate: Fas Anti-Fas antibody induce apoptosis Create Ig-Fas Chimeric molecules (ECD = Ig; ICD = Fas DD): Infect Hela in monolayer Add Ag Harvest apoptotic cells (floaters) after overnight incubation ______Advantages: Selection system No FACS/Beads needed, so throughput is very large

27 Selection of Human MAbs using Hybrid Ig-Fas Receptor Libraries

Add Antigen Ag binding Heavy Chain Ig-Fas induces apoptosis: Library in vaccinia cells detach

Human Ig light chain library in vaccinia

Repeat Harvest Floating Cells infection to enrich

Amplify Extract virus virus Analyze clones analyze positive pool by flow cytometry

28 Test Cell Detachment with Ig-Fas Recombinant Vaccinia Virus

Confluent monolayers of HeLa cells were infected at moi = 1 each of Ig-Fas and Ig-L Antigen specific and control Ig-Fas used ______6 hours after infection, Antigen is added ______Cells allowed to detach for 24 hours ______Supernatant was harvested, the wells were washed two times with PBS ______Remaining cells were stained with crystal Violet Take Pictures Solubilize with Acetic Acid and read OD570

29 Ig-Fas Recombinant Vaccinia Virus Mediates Antigen-Dependent Cell Detachment

Antigen Specific Ig-Fas

Cells remaining after detachment

Control Ig-Fas

30 Test Enrichment with Ig-Fas Recombinant Vaccinia Virus

Confluent monolayers of HeLa cells were infected at moi = 1 with antigen specific Ig-Fas and co-infected with admixtures of Ig-L virus 1.0% Antigen specific Ig-L + 99% Control Ig-L 0.1% Antigen specific Ig-L + 99.9% Control Ig-L 0.01% Antigen specific Ig-L + 99.99% Control Ig-L ______6 hours after infection, Antigen was added ______Cells allowed to detach for 24 hours ______Harvest Floating cells, extract and amplify virus ______Use this virus for a second round of enrichment. ______After the second round, take the virus and test for enrichment by staining for Antigen specific binders by flow cytometry.

31 Enrichment for Antigen specific Ig-L

1.0% 0.1% 0.01%

Starting Sample

After Enrichment

32 Transformation of Mouse MAb to Human MAb Using Ig-Fas Hybrid Libraries

Add Antigen Single antigen Ag binding specific Ig-Fas induces apoptosis: vaccinia cells detach

Human Ig light chain library in vaccinia

Use specific light chain to Harvest Floating Cells identify human heavy chain

Extract virus Amplify Analyze and Sort for Ag repeat for 2nd select best light specific cycle of chain clones binders after 2nd cycle enrichment

33 Transformation of Mouse MAb into Human MAbs

Control Ig-Fas Antigen Specific Control Ig-Fas n n o o i i s s s s e e r r p p x x E E g g I I

Ag Binding Ag Binding Clone 6 Clone 11 n n o i o i s s s s e e r r p p x x E E g I g I

Ag Binding Ag binding

34 Competitive Advantages

Challenge Vaccinex Technology Advantage

Conversion of Non-human Selection of antibodies with conserved epitope specificity Antibodies to Fully Human and similar or even improved affinity and functional activity. and Affinity Improvement of Selection of multiple antibodies derived from distinct VH and Existing Human Antibodies VL germ line genes with different biochemical properties.

Intrinsic selection for high expression in mammalian cell lines, Manufacturing easily adaptable to manufacturing.

De novo Antibody Broader target range than mouse-based platforms Selection Very Large Throughput

Re-engineering Vaccinex expresses complete MAbs that do not require re- engineering into IgG format.