leap-in transposase(R)

Efficient multi-site integration Genetic stability Unlimited payload Faster cell line development

Background

ATUM has developed a set of tools for genomic integration of DNA constructs based on novel transposons and their cognate transposases. This system features several properties that make it particularly well suited to engineering mammalian cell lines for bioproduction:

•• Integration sites are enriched in transcriptionally active chromatin. •• Multiple copies of a transposon are independently integrated into the legend

genome of a single cell. Transposon Recognition Site •• Transposition can be achieved in a + Transposase Inverted Terminal Repeat broad range of hosts. Expression Construct Transposase protein

•• Transposon excision perfectly Transfect restores the original genomic sequence. Multiple integrations by cut & paste •• The entire sequence between two transposon ends is integrated into Nucleus Nucleus a host genome, and there is no Cell Cell Chromosome Chromosome payload size limit. •• Transposon does not jump in the absence of cognate transposase. •• Multiple orthogonal transposases Alternative to AAV and Lentivirus gene delivery systems. are available.

Leap-in Transposase(R) Benefits

Application-optimized Shortened stable pool recovery Stable integration combined expression constructs times with structural integrity

Maximize expression levels Clonal productivity distribution Transposases integrate the using ATUM’s proprietary is characteristically higher and entire, intact transposon, thereby gene sequence and vector more uniform in stable pools. maintaining its structural optimization tools. integrity, and expression balance. Adjust and tune expression Fewer clones need to Clonal cell lines created using levels of your gene(s) by be screened to enable Leap-In transposases exhibit testing combinations of vector identification of highly genetic stability and stable elements. productive clonal cell lines. productivity over 60 population doublings. Express multichain proteins Initiate process development Control integration copy (such as bispecific antibodies) earlier with more representative number by manipulating dose or multiple genes within a pool derived reagents. of transposase and synthetic pathway. transposon. Footprint-free excision of transposon from host genome. Maintaining Structural Integrity

Leap-in Transposase(R)

The main limitation in creating cell lines from multi-ORF constructs is what happens to the DNA when it gets into the cell. Typically DNA is randomly fragmented and those fragments are integrated with additional rearrangements and concatemerization which may limit the benefits of providing the genes in a single construct.

Designed Construct

ITR HC LC GS ITR

+ Transposase - Transposase multiple independent integrations truncations of expression cassette of complete expression cassette plus random concatemerization and scrambling

ITR HC LC GS ITR HC LC GS

ITR HC LC GS ITR HC GS

ITR HC LC GS ITR HC

ITR HC LC GS ITR HC LC

ITR HC LC GS ITR LC GS

ITR HC LC GS ITR GS

ITR HC LC GS ITR HC LC GS LC GS

Structural integrity of the complete expression cassette is truncations plus random concatemerization and scrambling of maintained in the presence of transposase as shown in the left the expression cassette. HC: Heavy Chain, LC: Light Chain, ITR: panel. In the absence of transposase (right panel), structural Inverted Terminal Repeat, GS: Glutamine synthetase. integrity of the expression cassette is compromised as seen by

Leap-in transposases integrate the entire transposon into the expression host genome, thereby maintaining the structural integrity of the construct, consistent linkage of all genes, and the desired expression ratio between protein chains.

2 201903 www.atum.bio TRANSPOSASE

Antibody Expression

Although the Leap-in transposases share many desirable features of the piggyBac transposase from the looper moth Trichoplusia ni, they share only about 20% sequence identity and fall outside patent claims for the looper moth system. Five US patents protecting the Leap-in system have already issued (9,428,767, 9,534,234, 9,574,209, 9,580,697 and 10,041,077), with many more applications pending in the US and abroad.

The Leap-in transposase integrates all DNA between the two transposon ends. The selectable marker and all encoded open reading frames are therefore 100% linked. This is a very effective way to integrate genes that need to be expressed together, for example the heavy and light chains from monoclonal antibodies or the three or four chains of bispecific antibodies.

The relative expression levels of the different chains of an antibody often have a profound effect on the amount of antibody that can be produced. By using well characterized regulatory elements, ATUM can ensure optimal ratios of expression between different open reading frames within a construct. Multiple copies of a transposon within the genome arise from independent integrations at different sites. These are more stable than the concatamers that arise from random integration. Independently integrated transposons are also not susceptible to repeat- induced silencing.

Transposase Speeds Recovery and Allows Increased Selective Pressure

A. B.

120 120

100 100 1

80 80

60 60 4 Viability

Viability 5 40 40 2 20 20 3 6 0 0 0 1 7 14 19 23 26 0 5 9 17 20 Time (Day) Time (Day)

Transposase speeds recovery. CHO K1 GS KO cell line from Horizon Discovery (catalog# HD-BIOP3) was co-transfected with transposon and transposase as shown. A. Under drug-free selection conditions (attenuated GS promoter), cell pools created using transposase (curve 1) recover viability in ~3 weeks, whereas cell pools created without transposase (curve 2) fail to recover. Curve 3 is the mock negative control. B. Attenuated GS cassettes recovered in the presence of 15mM MSX (curve 4), or 5mM MSX (curve 5). No recovery was seen when transposase was not used (curve 6).

3 Transposons with substantially attenuated selectable marker expression cassettes are integrated by transposases. When selectable marker expression from a single cassette is very low, recovery requires multiple copies to be integrated at sites in the genome favorable for gene expression. The combination of transposases and transposons with attenuated selectable marker cassettes thus produces highly productive pools of cells. Sample pool productivities are shown in the table below.

Examples of Stable Pool Productivity Cells are grown under selective conditions to produce stable pools. These are ranked by productivity and product quality. At this point material can be generated from the stable pools and single clone isolation is initiated.

GS volumetric specific HC promoter productivity productivity

IgG1-Hs ht 4.2 g/l 42 pcd IgG1-Hs ht 3.6 g/l 29 pcd HD-BIOP3 GS null CHOK1 cell line from Horizon Discovery was co-transfected with ATUM’s transposase IgG1-Hs ht 3.3 g/l 29 pcd and transposons with different vector and antibody IgG1-Hs ht 2.8 g/l 30 pcd combinations. Stable pools were established and productivity measured in non-optimized small scale IgG1-Hs hxt 4.2 g/l 33 pcd shake flask or deep-well cultures. IgG4-Hs hxt 5.0 g/l 43 pcd IgG4-Hs hxt 5.0 g/l 49 pcd

Examples of Stable Pool Productivity for Different Molecule Types

selection transposon specific protein stringency copy/cell productivity (pcd) 300 kDa glycoprotein-Fc fusion low 5 to 7 ~4 bispecific antibody medium ~16 7 to 12 antibody medium 20 to 25 35 to 50 antibody high 35 to 45 50 to 70

Stability of Stable Pools Leap-in transposase protein acts to integrate the transposon into the host genome. Because each integration contains a single copy of the transposon, there is no instability resulting from concatemer recombination or repeat-induced silencing. Productivity Maintained Copy Number Maintained

3500 50 45 3000 40 Graphs show the maintenance 2500 35 of productivity and transposon 2000 30 copy number in clonal cell lines 25 after 60 population doublings 1500 20 1000 15 (PD). A subset of data taken 10 from 47 clones created using 500

Copy number/reference 5 Productivity mg/L Productivity Leap-In technologies is shown. 0 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 All clones retained at least 70% Clones Clones of their initial productivity and T0 PD60-Gln PD60+Gln T0 PD60-Gln PD60+Gln transposon copy number. Productivity and copy number is maintained after 60 population doublings.

4 201903 www.atum.bio TRANSPOSONS AND TRANSPOSASES pD2500 & pD3600 Stable Vectors

ATUM offers two stable vector series: the pD2500s and pD3600s, which share a similar structure. The pD2500s are compatible with one of our Leap-in transposases (LPN-1), but they do not require a Marker promoter transposase for good levels of expression. The Mammalian marker Promoter 1 pD3600s are compatible with both of our ORF1 signal Leap-In transposases (LPN-1 and LPN-2), and Marker polyA we recommend that they only be used with ORF1 a transposase. This is because we have ORF1 poly A deliberately attenuated the selectable Insulator intervening sequence markers in the pD3600s, so multiple integration events are required for cell Leap-In Transposon viability under selection. The pD3600s P kanamycin are therefore capable of higher expression Promoter 2 yields. M_Kanamycin-r_* The pD2500 and pD3600 vectors are constructed in a modular way that allows ORF2 signal easy modification of component elements. Bacterial Origin ORF2

Versions are available that allow expression Transposon right border ORF2 polyA Insulator of up to four open reading frames in addition to the selectable marker.

Our stable vectors are built from several sets of mammalian functional elements:

⬤⬤ Metabolic selectable markers ⬤⬤ RNA processing elements Glutamine synthase (GS), dihydrofolate Introns, post-transcriptional response reductase (DHFR) elements

⬤⬤ Drug-resistance markers ⬤⬤ Insulators puromycin, blasticidin, hygromycin, neomycin, Flanking the construct, and isolating zeocin transcriptional units from each other

⬤⬤ Promoters EF1a, CMV (murine and human), hybrids

The total number of possible combinations of vector elements is very large, and ATUM does not have all of these built. However construction is modular, so it is easy for us to create custom combinations if we don’t have the combination you want.

5 One Vector, Two Open Reading Frames Co-transfection of separate vectors containing genes for heavy and light chains is a common method for producing antibody from transiently transfected cells. This approach is less desirable for stable cell line generation. The production of properly assembled antibody requires sufficient light chain to help the heavy chain fold. However a great excess of light chain diverts cellular resources away from producing enough heavy chain to obtain high titers. If genes encoding the two chains are incorporated into the production host genome independently, the number of gene copies integrated, and integration position effects, will influence the relative expression of the two chains. This results in a higher downstream screening burden during the cell line development process.

A preferable method is to express both genes from the same construct. In this case regulatory elements can be chosen to produce a desired expression ratio between the two chains. The table below shows 9 different construct configurations. The total productivity differs by less than 30% for any of the constructs, but ratios of between 1:1 and 3:1 can be achieved. We find that this ratio is influenced by the 3´ sequences of the first gene, as well as the promoter sequences driving the second.

first second gene ID first promoter polyA promoter chain ratio productivity heavy chain

269998 EF1a-Hs a A 2.96 24,164 6,126 269986 EF1a-Hs a B 2.86 24,442 6,339 269996 EF1a-Hs a C 2.53 30,799 8,608 270003 EF1a-Hs b D 1.97 24,182 7,846 270005 EF1a-Hs c C 1.81 33,991 12,129 270000 EF1a-Hs d E 1.78 23,058 8,037 269984 EF1a-Hs b C 1.17 25,303 11,699 269976 EF1a-Hs d F 1.10 32,845 15,887 270006 EF1a-Hs e B 1.01 30,603 15,548

Genes encoding DasherGFP and CayenneRFP were cloned into proteins (in arbitrary units). The total fluorescent protein expression pD3641h in 9 different dual-promoter configurations as shown is shown in the “productivity” column. Expression of the lower above. Constructs were transfected with LPN-1 transposase mRNA expressing proteins is shown in the “heavy chain” column. The into CHO-K1 GS KO cells, and selected in media without glutamine. ratio of expression of the two proteins is shown in the “chain ratio” After recovery, DasherGFP and CayenneRFP fluorescence were column. measured and used to calculate the relative expression of the two

ORF1 ORF2 ORF2:ORF1 Ratio 0.9 3

0.8 2.5 0.7 Two identical ORFs coding for a secreted protein were 0.6 2 tagged with V5 (ORF1) or FLAG (ORF2). A combination 0.5 of vector elements were utilized to control the ratio of 1.5 0.4 expression of expression of ORF2 to ORF1. Expression ratios

Relative expression expression Relative 0.3 1 of ORF2 : ORF1 between 2.5 : 1 and 1 : 1.5 were demonstrated. 0.2 0.5 0.1

0 0 expression ORF2:ORF1 Ratio of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Constructs

Similar combinations of control elements can be used to create constructs carrying 3 or 4 open reading frames, enabling expression of all of the chains for a bispecific at controlled ratios.

6 201903 www.atum.bio TRANSPOSASE AND CELL LINE DEVELOPMENT

One Vector, Three or Four Open Reading Frames

Combinations of control elements can be used to create constructs carrying 3 or 4 open reading frames, enabling expression of all of the chains for a bispecific at controlled ratios.

kan promoter Left transposon end Kan promoter Left transposon end Kanamycin R Marker polyA Kanamycin R pUC ori pUC ori Insulator M_Glutamine synthetase_* Marker polyA Right transposon end

Right transposon end

Mammalian marker Promoter for Marker Insulator

ORF3 polyA ORF4 polyA Promoter for Marker ORF1 polyA 3 Slot Co-linear 4 Slot Divergent ORF3 ORF4 ORF1 12373bp 18357bp

ORF1 Promoter ORF4 Promoter ORF1 Promoter

ORF3 Promoter

ORF3 polyA

ORF1 ORF3 ORF2 polyA ORF2 polyA

ORF2 ORF1 polyA ORF2 ORF3 promoter Spacer ORF2 Promoter ORF 2 Promoter

Constructs with 3 open reading frames (ORFs) in a co-linear Constructs with 4 open reading frames (ORFs) in a divergent configuration with combinations of control elements enables configuration with combinations of control elements enables expression of all 3 ORFs at controlled ratios. expression of all 4 ORFs at controlled ratios.

Examples of expression ratios in 3 and 4 open reading frame (ORF) constructs

0.35 ORF1 ORF2 ORF3 ORF4 0.8 0.30 ORF 1 ORF 2 ORF 3 0.7 0.25 0.6 0.20 0.5 0.4 0.15 0.3 0.10

0.2 expression Relative 0.05 0.1 Relative expression 0 0.00 1 2 3 4 5 6 Constructs Construct

Genes encoding fluorescent proteins were cloned into pD3641h in 3 or 4 slot co-linear ratio of expression of the 3 or 4 proteins is shown. For the 3-ORF vector, various ratios or divergent multi-promoter configurations as shown above and page 8. Constructs of all three ORFs were achieved. For the 4-ORF vector, a 4-fold range of expression were transfected with LPN-1 transposase mRNA into CHO-K1 GS KO cells, and selected was observed between the different ORFs. Transposase allows insertion of the entire in media without glutamine. After recovery, fluorescence were measured and used expression cassette enabling expression of each ORF at controlled ratios. to calculate the relative expression of the fluorescent proteins (in arbitrary units). The

Regulatory elements can be chosen to produce a desired expression ratio between the proteins. The graphs above show examples of expression ratios in constructs with 3 and 4 co-linear or divergent multi-promoter configurations. Similarly, different expression ratios can be achieved by combinations of control elements, enabling expression of all of the chains for a bispecific at controlled ratios.

7 Monitoring Leap-in Transposase(R) Activity

Genetic stability is established by copy number, productivity and consistent growth rate. Hypothetic transposase activity acting on the stably integrated transposons could potentially induce instability and integration site change. We have demonstrated using TLA (Targeted Locus Amplification) based integration site mapping that there was no change in numbers and genomic positions of integrated trangenes over >100 population doublings.

Leap-in transposase mediates footprint-free excision

To charactertize (qualify) a host cell line, extended passage cultures can be monitored using a functional assay using a reporter construct utilizing the footprint-free excision feature of Leap-in transposases as shown.

A synthetic transposon (RFP gene Excision Efficiency of Transposase flanked by Leap-in 1 ITR’s inside a GFP gene flanked by Leap-in 2 3000 ITRs) was integrated into CHO cells 2 ug 6 ug 18 ug 2500 using Leap-in 2 transposase. Cells exhibited red fluorescence as RFP is 2000 expressed. 1500

1000

500 Cells transfected with Leap-in GFP Fluorescence 0 mRNA. Leap-in 1 transposase excised 1 2 3 4 5 6 7 RFP at cognate ITRs. Transposase variants

Cells exhibited green fluorescence as intact GFP is expressed Readouts: • Green fluorescence upon RFP cassette excision. • No green fluorescence detected after 3 months of cell culture.

Leap-in transposase mRNA in cells is transient

1800

1600

1400 NG GS KO CHO cells were transfected with LPN-1 mRNA, samples were taken from different time points post- 1200 ) transfection. Total RNA was extracted from NG GS KO CHO fg 1000 cells for RT ddPCR. FAM labeled primers targeted the LPN- 800

RNA ( y = 1133.8e-0.057x 1 RNA and HEX labeled primers for mouse actin used as 600 positive control. 400

200

0 0 20 40 60 80 100 120 140 160

Hours post transfection

8 201903 www.atum.bio TRANSPOSASE AND CELL LINE DEVELOPMENT

Transfected Leap-in transposase mRNA in cells is transient and is below the detection limit (~0.1 molecules of transposase mRNA per cell) in ~150 hours.

Transfected Leap-in transposase DNA in cells is undetectable (detection limit is ~0.002 molecules of transposase DNA per cell) in established clones.

Flexible Licensing Options for Leap-in Transposase(R)

Bring the advantages of Leap-in technology to your in-house cell line development. We offer three tiers of licensing to meet your research and commercialization needs. Under all licenses, Leap-in mRNA and synthetic transposon DNA are purchased from ATUM. Genes and vectors designed and synthesized by ATUM.

Evaluation Test and optimize the Leap-in technology in your labs. Convert to a full research license after 6 months. Research Annual license fee. Access to all new transposon and transposase technology developed at ATUM. Commercialization Payable on cell lines that enable commercialization of your therapeutic, diagnostic or product. Pay annually, as a single lump sum, or attached to clinical milestones.

Contact us for information on licensing options & cell line development services

+1 650 853 8347 ext. 200 +1 877 DNA TOGO ext. 200 [email protected]

9 +1 877 DNA TOGO +1 650 853 8347 [email protected]

research. create. break through.