VECTOR OPTIMIZATION FOR NON-VIRAL ANTIBODY GENE TRANSFER AND EXPRESSION OF ANTI- SARS-COV2, HUMAN MONOCLONAL ANTIBODIES IN MICE
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Vector optimization for non-viral antibody gene transfer and expression of anti- SARS-CoV2, human monoclonal antibodies in mice Nathaniel Silver, Sid Jindal, Nicholas Parsonnet, Luke Hamm, Matt Manganiello, Henry Sun, Debra Klatte, Phillip Samayoa, Doug Kerr Generation Bio, 301 Binney Street, 4th Floor, Cambridge, MA 02142 Since their initial approval over 30 years ago, monoclonal antibodies (mAbs) have seen great success as a therapeutic class. However, their high cost of production as well as the need for frequent administration has limited their widespread use in areas outside of oncology and autoimmune diseases. Antibody gene transfer (AGT) provides an alternative means of delivering specific mAbs, wherein an antibody is vectorized and produced in vivo. This may enable patients to produce their own biotherapeutic for an extended period. AAV-based delivery of vectorized antibodies has demonstrated a capacity to produce efficacious levels of antibodies in a variety of pre-clinical, disease models. However, there remain significant limitations to AAV that have limited its use for AGT in the clinic. In particular, its genetic cargo capacity of ~4.7kb limits encoding of multiple polypeptides, immunogenicity of the viral capsid restricts dosing to a single administration, and costly, scale-limited manufacturing processes prevent use in wide-spread diseases like HIV, Influenza, or COVID19, particularly in the prophylactic setting where they might be the most impactful. Non-viral gene therapy is an attractive solution to address these limitations and potentially enable prophylactic use of AGT on a much broader scale than is possible with passive administration of recombinant mAb or AAV based delivery. Generation Bio has developed a non-viral gene therapy platform to deliver and durably express therapeutic proteins systemically in vivo. It is comprised of ceDNA, an engineered, double-stranded, linear, covalently closed-ended DNA construct, formulated in a lipid nanoparticle delivery system, ctLNP. The ctLNP delivery system has been designed for hepatocyte-specific delivery by using a biological targeting ligand and does not contain any component of the viral capsid, allowing repeated administration. Studies in immunocompetent mice have demonstrated that systemic administration of a single dose of ctLNP-formulated ceDNA results in delivery to hepatocytes and durable transgene expression. Administration of a second dose further increased expression. Here we present the use of our non-viral gene therapy platform to express a monoclonal, SARS-CoV2 neutralizing antibody (Vir Biotechnology) as well as our vector optimization approach to maximize expression. We compare bicistronic, furin/2A-peptide based expression cassettes to paired heavy and light chain vectors as well as multi-promoter, scarless designs. While larger in size, we find that the latter two vector formats, in particular bidirectional promoter cassettes, significantly improve expression of antibodies in vivo relative to the bicistronic designs commonly used in AAV vectors to encode multiple peptides. Further optimization of regulatory as well as heavy and light chain sequences enhanced ceDNA- based expression, achieving therapeutically relevant levels of systemic mAb expression in mice. These data demonstrate early capabilities of our non-viral AGT platform and provide a path for non-viral delivery of antibodies for disease prevention and treatment. 1
Forward-looking statements Any statements in this presentation about future expectations, plans and prospects for the company, including statements about our strategic plans or objectives, our technology platforms, our research and clinical development plans, and other statements containing the words “believes,” “anticipates,” “plans,” “expects,” and similar expressions, constitute forward-looking statements within the meaning of The Private Securities Litigation Reform Act of 1995. Actual results may differ materially from those indicated by such forward-looking statements as a result of various important factors, including: uncertainties inherent in the identification and development of product candidates, including the conduct of research activities, the initiation and completion of preclinical studies and clinical trials and clinical development of the company’s product candidates; uncertainties as to the availability and timing of results from preclinical studies and clinical trials; whether results from preclinical studies will be predictive of the results of later preclinical studies and clinical trials; expectations for regulatory approvals to conduct trials or to market products; challenges in the manufacture of genetic medicine products; whether the company’s cash resources are sufficient to fund the company’s operating expenses and capital expenditure requirements for the period anticipated; the impact of the COVID-19 pandemic on the company’s business and operations; as well as the other risks and uncertainties set forth in the “Risk Factors” section of our most recent annual report on Form 10-K, which is on file with the Securities and Exchange Commission, and in subsequent filings the company may make with the Securities and Exchange Commission. In addition, the forward-looking statements included in this presentation represent the company’s views as of the date hereof. The company anticipates that subsequent events and developments will cause the company’s views to change. However, while the company may elect to update these forward-looking statements at some point in the future, the company specifically disclaims any obligation to do so. These forward-looking statements should not be relied upon as representing the company’s views as of any date subsequent to the date on which they were made. 2
ceDNA is a closed-ended, linear, duplex DNA vector whose structure imparts key
features for non-viral gene therapy
GFP expression in non-dividing cells GFP expression in dividing cells
4000 150
Dividing cells (293T)
Non-dividing cells (HepaRG)
ceDNA ITR ceDNA 3000
% Day 1 Expression
% Day 1 Expression
% of GFP+ cells
100
structure is expression is
key to consistent 2000
accessing with non- 50
1000
the nucleus integrating
episomes 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Days Days
3
ceDNA-LNP shows durable expression in immunocompetent mice after a single IV
administration
Luciferase Factor IX
Control Control
10 8 Luc ceDNA/LNP 100 FIX ceDNA/LNP
Total Flux (photons/sec)
FIX (% WT activity)
10 7 10
10 6 1
10 5 0.1
0 50 100 150 200 0 7 14 21 28 35 42 49 56 63 70 77
Study Day Day
• Single IV administration at study day 0 • Single IV administration at study day 0
• Total flux measured by IVIS in vivo imaging • Factor IX activity calculated from protein ELISA
FIX used as surrogate for durability and redosing in wildtype mice because this human protein does not raise neutralizing antibodies in mice, unlike human FVIII
4ceDNA-LNP redosing achieves increased expression in immunocompetent mice
Luciferase Control Factor IX
Luc ceDNA/LNP Control
10 10 25 FIX ceDNA/LNP
Total Flux (photons/sec)
FIX (% WT activity)
10 9 20 Control
25 FIX ceDNA/LNP
10 8 15
FIX (% WT activity)
Control 20
10 7 10
Luc ceDNA/LNP
15
10 6 5
10 10
Total Flux (photons/sec)
10
0
1010
9 5
0 20 40 60 0 20 40 60
5
10 8 Study Day Day
0
10 7
• Single IV administration at study day 0 0 20• 40
Single IV administration60at study day 0
Day
• 6Re-dosed at day 35 with 10X higher dose
10 • Re-dosed at day 36 at same dose level
• Total flux measured by IVIS in vivo imaging • Factor IX activity calculated from protein ELISA
10 5
0 20 40 60
FIX used as surrogate for durability and redosing in wildtype mice because this human protein does not raise neutralizing antibodies in mice, unlike human FVIII
Study Day
5ceDNA Antibody Gene Transfer (AGT) enables persistent, hepatic
expression and secretion of monoclonal antibodies
Antibodies
Antibody Sequence
gaaggatatt aaagagcacc
tgcaggaatt ttttaagggg
atgccggggg aagggcttga
Hepatocyte
6Liver specific, dual ORF and dual vector designs encoding a SARS-CoV2 neutralizing
antibody yield highest expressing constructs
Vector Format Optimization
IgG Vector Format
Bicistronic Cassettes Equimol Hydrodynamic Injection (HDI)
Development/CMC risks? (C57Bl/6 mice; Day 7; Mean +/- SEM)
HC F/2A LC
500
Serum huIgG (μg/mL)
400
Anti-spike
Dual ORF cassettes 300
IRES vs 2A peptide(s)/linker? 200
HC LC
100
0
Dual vectors
1)
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2A
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Recombination/production risk of duplicate vs
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7ceDNA vector optimization across promoter, ORF codon usage, and signal peptides
identified high expressing dual vector and dual ORF designs
Codon Optimization Screen
Vector Cassette Optimization (1ug/an. HDI; C57Bl/6 mice; Mean +/- SEM)
50
risk of duplicate vs 40
Serum hIgG (μg/ml)
Vector Format Library 30
Anti-spike
(HC; LC; HC-2A-LC; HC/LC) 20
10
0
e
1
2
3
4
5
6
7
8
cl
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Codon Optimization library
C
C
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C
C
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Signal Peptide Screen
Promoter Screen (1ug/an. HDI; C57Bl/6 mice; Mean +/- SEM)
(1ug/an. HDI; C57Bl/6 mice; Mean +/- SEM)
40
80
30
60
Serum hIgG (μg/ml)
Serum hIgG (μg/ml)
Signal Pep library Promoter Library
Anti-spike
Anti-spike
40 20
20 10
Combinatorial Screening of
0 0
ORF/SigPep/Promoter Hits
1
2
3
4
5
6
7
8
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8Screening of Heavy and Light chain ratios suggests hepatic IgG expression with
ceDNA is enhanced with higher HC:LC molar ratios
In vitro Screen of HC:LC Ratios In vivo Fixed HC/LC Dose Screen of HC:LC Ratios
0.8 In vivo HC:LC Molar Ratio In vivo HC:LC Molar Ratio
LC Limiting HC Limiting Screen with Fixed LC Dose Screen with Fixed HC Dose
Secreted anti-spike hIgG (μg/ml)
(HDI; C57Bl/6 mice; Mean +/- SEM) (HDI; C57Bl/6 mice; Mean +/- SEM)
300 300
0.6
250 250
Serum hIgG (μg/ml)
Serum hIgG (μg/ml)
200 200
0.4
Anti-spike
Anti-spike
150 150
0.2 100 100
50 50
0.0 0 0
0.9:1 1.7:1 2.5:1 1:1.2 1:2.3 1:3.5
:1
10
1
1
1
1
2
3
5
:1
HC:LC Molar Ratio (Fixed LC Dose) HC:LC Molar Ratio (Fixed HC Dose)
5:
3:
2:
1:
1:
1:
1:
10
5
1:
1.
Dual Vectors HC:LC Molar Ratio
3:2 HC to LC molar ratio is optimal for dual At a fixed dose of LC or HC ceDNA, increasing dose of
vector ceDNA construct pairs HC improves expression while increase LC dose has
limited effect
HepG2 cells; supernatant collected at 72hr
9ceDNA format of optimized designs show dose dependent expression, with best dual
vector designs yielding 5-10x higher activity relative to dual ORF designs
ceDNA Dose Response
(Hydrodynamic Injection; C57Bl/6 mice; Mean +/- SEM)
1000
100
Serum hIgG (μg/ml)
Anti-spike
10
1
0.1
0.01
0.001 0.01 0.1 1 10
Dose (g/an.)
ceDNA-1 (Dual Vectors; 3:2 HC/LC)
ceDNA-2 (Dual ORF)
10LNP delivery of dual vectors achieved persistent, therapeutically relevant
concentrations of anti-SARS-CoV2 hIgG in mice
Vectorized IgG Expression after LNP delivery
(i.v. infusion; Rag2 mice; Mean +/- SEM)
100000
10000
Serum hIgG (ng/ml)
Anti-spike
1000
100
LLOQ
1
0 10 20 30 40
Study Day
ceDNA-1; 2mg/kg (Dual Vectors)
ceDNA-2; 2mg/kg (Dual ORF)
11Serum from ceDNA treated animals shows potent neutralization of SARS-CoV-2
pseudovirus, equivalent to the recombinantly produced antibody
SARS-CoV-2
Pseudovirus Neutralization
IC50 (ng/ml)
59.58
28.39
39.59
37.42
36.62
Serum neutralizing activity of individual animals
treated with ceDNA encoding anti-SARS-CoV2
mAb (HDI; C57Bl/6 mice) or recombinant mAb
spiked into mouse serum at a comparable dilution
All neutralization assays performed by collaborators at VIR Biotechnology
(VeroE6 cells; CoV2 Spike (D19) pseudotyped VSV-Luc)
12Evolution of gene therapy manufacturing – novel enzymatic process with potential for
improved scaling and cost effectiveness
Benefit for antibody gene transfer, with potential to impact millions of patients
UPSTREAM DOWNSTREAM PURIFICATION FORMULATION PAYLOAD/PURITY PAYLOAD SCALE
Sf9 cell Budded virus harvested: Multi-column purification:
STEP STEP STEP
1 production and 2 Scale limited due to 3 full versus empty only
then encapsidation capsid instability
Tens of
AAV INCONSISTENT thousands
Mixture of full/empty Payload & Quality
10s of Thousands
capsids, complete and
incomplete genomes
Suspension ceDNA ceDNA drug STEP High-purity
STEP STEP STEP
Sf9 cell harvested substance 4 ceDNA
1 2 3 packaged Millions
production from cells purified
CONSISTENT
ceDNA Payload & Quality
STEP Enzymatic ceDNA manufacture (cell free) Hundreds
1 One day process from pDNA
of Millions
13Data demonstrate early capabilities of non-viral AGT platform and provide a path for
non-viral delivery of antibodies for disease prevention and treatment
• Encoding HC and LC on separate ceDNA vectors and delivering both in a 3:2 molar ratio is optimal format for hepatic
expression
• Combinatorial optimization of regulatory sequences and heavy and light chain codons of anti-SARS-CoV2 mAb resulted
in highly expressing constructs
• LNP co-formulation and delivery of paired vectors achieved therapeutically relevant levels of mAb in mice (~8ug/ml)
• Antibodies produced in situ retained binding and functional activity, and demonstrated comparable ex vivo
neutralization activity relative to recombinant control
• Non-viral delivery of ceDNA encoding neutralizing, monoclonal antibody has potential for persistent expression and
long-term protection against SARS-CoV2 with a single dose
• Enzymatic synthesis may further unlock scaled manufacturing, potentially enabling AGT to reach global populations for
SARS-CoV2 and future pandemics
Acknowledgements: The authors wish to acknowledge our generous collaborators at Vir Biotechnology, including
Fabio Benigni, Dora Pinto, Michael Housley, and Laura Rosen.
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