Development of Novel Non-Immunoglobulin Centyrin™-Based CARs

Burton E. Barnett, Jenessa B. Smith, Xinxin Wang, David Hermanson, Yening Tan, Eric M. Ostertag, Devon J. Shedlock

ABSTRACT

INTRODUCTION

CONCLUSIONS

Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapyand substantial progress has been made in developing adoptive T cell approaches for B cellmalignancies. B cell maturation antigen (BCMA) is an attractive target for patients withmultiple myeloma (MM) due to its high level of expression on tumor cells and restrictedexpression on normal tissues. In addition, BCMA is essential for cell growth and survival, andis therefore likely not susceptible to antigen escape. Traditionally, the antigen-binding domainof a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and lightchain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-basedCARs including the limited availability of scFv, their potential to elicit antibody responses, andtheir association with tonic signaling due, in part, to inherent instability and flexibility of thestructure and the potential for both HC/LC domain swapping and multimer formation throughframework region interactions. Thus, replacement with alternative binding technologies mayimprove CAR-T efficacy in the clinic.

Centyrins™ are alternative scaffold molecules that bind protein targets with high affinity andspecificity, similar to scFv molecules. However, unlike scFv, Centyrins™ are smaller, derivedfrom human consensus tenascin FN3 domains and are predicted to have decreasedimmunogenicity. Additionally, a monomeric Centyrin™ in CAR format (“CARTyrin” molecule) isless likely to engage in domain swapping or interact with other Centyrins™ at the cell surface,thereby limiting the potential for the tonic signaling that drives the functional exhaustion ofCAR T cells. Finally, as Centyrins™ are 1/3 the size of scFv molecules, they provide flexibilityfor the creation of multi-specificity CARs. Centyrins™ can be isolated against virtually anyantigen through ex vivo panning of an extensive Centyrin™ library, yielding many distinctbinders with a range of affinities and target epitopes.

We tested a panel of 12 anti-BCMA (11 monomeric and one multimeric “P9”) Centyrins™ bylinking them to a standard second-generation CAR scaffold. High quality mRNA of eachCARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expressionand functionality in human pan T cells against BCMA+ targets. We also constructed scFv-basedCARs against CD19 and BCMA for comparison. CD3/CD28-stimulated T cells wereelectroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, thefollowing day, analyzed for surface expression of CARTyrin and their ability to degranulateagainst BCMA+ tumor cells.

All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins impartedBCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionallycomparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in aparallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were furthercharacterized for functional avidity by determining their activity against a panel of target cellswith titrated levels of surface BCMA expression. To create this panel, various amounts of highquality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as afunction of CD107a expression. We observed a range of activities by each CARTyrin and showthat this assay can be utilized to determine the minimal effective dose of BCMA needed toinduce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specificCARTyrins are responsive to target cells with extremely low levels of surface BCMAexpression. The data shown in this poster are complemented by functional studies in vitroand in vivo that may be found in the posters listed below. These results confirm thatCentyrins™ are viable replacements for scFv in the construction of functional CARs andestablish their potential utility in generating novel BCMA-specific CAR molecules, as well asother novel targetable tumor antigens.

Poseida Therapeutics, Inc. 4242 Campus Point Court, Suite 700 San Diego, CA, 92121

(CARTyrins) Targeting Human BCMA

Signal PeptideTransmembrane

Hinge Costimulatory Signaling DomainCentyrin™

Figure 1: Surface CARTyrin Expression in RNA-electroporated human T cellsSurface expression of CARTyrin using a biotinylated BCMA-Fc protein and fluorescent streptavidin24 hours post electroporation in primary human T cells. T cells were previously stimulated withanti-CD3/CD28 mAb-coated beads, allowed to rest, and thawed O/N prior to electroporation with10ug CARTyrin mRNA. a) CARTyrin expression (y-axis). CARTyrin+ frequency (b) and meanfluorescence intensity (c). BCMA-specific CAR molecules (“scFv 1” and “scFv 2”) were included aspositive controls.

• All CARTyrins tested were expressed on the surface of primary human T cells• All BCMA-specific CARTyrins (except F9 comprised of multimeric Centyrin™) exhibited specific

effector function against BCMA+ tumor cells, demonstrating comparable functionality toBCMA scFv-based CARs

• BCMA-specific CARTyrins exhibit high functional avidity and a titratable dose response againstBCMA antigen, demonstrating a range of potency down to picogram amounts of BCMA

• Testing RNA CARs is an effective screening platform for identification of lead CAR candidates• The potency exhibited by Centyrin™-based CARTyrins combined with their advantages in size,

immunogenicity, flexibility, and lack of tonic signaling suggest they may provide an excellentalternative to scFv based CARs

Figure 3: Electroporation with lower levels of CARTyrin mRNA permit greater functional distinctionbetween CARTyrin candidates(a) Surface expression of in T cells 24 hours post electroporation with either 10 ug or 5ug of CARTyrin mRNA. (b) Expression of CD107a(y-axis) and CD8 (x-axis) in live (7AAD-), CD3+ T cells 4 hours post co-culture with tumor cells at an effector to target ratio of 1:4. Each barrepresents one CARTyrin molecules, in order of P5, P12, P8, P2, P11. Assays were performed 24hrs post electroporation.

Figure 4: Comparative analyses of BCMA-specific CARTyrins against BCMA+ tumor cells(a) Surface expression of CARTyrin in T cells electroporated with 5ug of CARTyrin mRNA. (b) Frequency of CD107a+ inlive, CD3+, CD8+ T cells 4 hours post co-culture with tumor cells at an effector to target ratio of 1:4. Assays wereperformed 24hrs post electroporation. Error bars represent 3 independent experiments.

Centyrin™ is a registered trademark of Janssen Pharmaceuticals, Inc. Poseida has licensed certain rights to the

Centyrin™ technology platform from Janssen Pharmaceuticals, Inc. for use in autologous T cell therapeutics

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Figure 2: T cells electroporated with BCMA CARTyrins demonstratespecific activity against BCMA+ tumor cells(a) Expression of CD107a (x-axis) in live (7AAD-), CD3+ T cells 4 hours post co-culturewith tumor cells at an effector to target ratio of 1:4. Frequencies of CD107a+ cells isshown on the plots. (b) Bar graphs represent CD107a+ frequency of CD8+ T cells.Assays were performed 24 hours post electroporation with BCMA-specific CARTyrins.(c) BCMA expression via flow cytometry in the indicated tumor cells. A CD19-specificscFv CAR (“CD19”) was included as a positive control for its activity against Nalm6 cells.

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Figure 5: BCMA-specific CARTyrins functionagainst picogram amounts of BCMA(a) Expression of CD107a (y-axis) in CD8+ T cells (pre-gated onlive (7AAD-), CD3+) 4 hours post co-culture with K562 tumorcells electroporated with the indicated amount of BCMA mRNA(x-axis). Effector to target ratio of 1:4. (b) Expression of BCMAon BCMA-electroporated K562 cells. Mean fluorescenceintensity (MFI) of BCMA expression is quantified on the y-axis.Assays were performed 24 hours post electroporation withBCMA-specific CARTyrins. L.O.D., limit of detection.

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I. PiggyBac™-Produced CAR-T Cells Exhibit Stem-Cell Memory Phenotype Session 703 Poster III. A Novel Bcma-Specific, Centyrin™-Based CAR-T Product for the Treatment of MultipleMyeloma Session 653 Poster I

CD107a

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P9 P10 P11 P12 scFv 1 scFv 2 CD19 Mock

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METHODS & RESULTS

scFv Centyrin™

Size: ~250 aa ~90 aa

Components: Heavy & Light ChainVariable Regions,

Flexible Linker

Fibronectin type III domain

Derived: Mouse/RodentAbs Human

Stability: Not as stable as original IgG format

Stable