Manufacture of an Allogeneic CAR-T Stem Cell Memory ...
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Manufacture of an Allogeneic CAR-T Stem Cell Memory Product Candidate for Multiple Myeloma, P-BCMA-ALLO1, Is Robust, Reproducible and Highly Scalable Stacey A. Cranert , Maximilian Richter, Min Tong, Leslie Weiss, Yening Tan, Eric M. Ostertag, Julia Coronella, Devon J. Shedlock CONCLUSIONS Poseida Therapeutics, Inc., 9390 Towne Centre Drive, Suite 200, San Diego, CA, 92121 INTRODUCTION ABSTRACT METHODS & RESULTS Chimeric Antigen Receptor (CAR) T cell therapy has generated unprecedented efficacy in the treatment of multiple hematologic malignancies. For relapsed/refractory Multiple Myeloma (MM), autologous CAR-T products directed against the B cell maturation antigen (BCMA), such as Poseida’s P-BCMA-101, have demonstrated significant efficacy. P-BCMA-101 is comprised of a high-percentage of stem cell memory T cells (TSCM), resulting in a product that is much safer and potentially more durable than other anti-BCMA autologous product candidates. However, as individualized products, all autologous CAR-T products are expensive to manufacture and dependent upon patient T-cells of variable quality. We are developing P-BCMA-ALLO1, an off-the-shelf allogeneic (allo) BCMA-specific CAR-T product candidate derived from healthy donor material, which provides numerous advantages over autologous products, increasing patient access by being immediately available and greatly reducing manufacturing cost and variability. P-BCMA-ALLO1 is produced using two key platform technologies: the nonviral piggyBac® (PB) DNA Modification System and the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. The mRNA coding for hyperactive, or “Super PB” transposase (SPB), and CC enzymes are codelivered with the P-BCMA-ALLO1 PB-based DNA transgene via electroporation to healthy donor T cells to stably integrate the transgene, as well as to knockout (KO) several mediators of allo graft-versus-host and host-versus-graft responses to maximize patient safety and durability of response. The P-BCMA-ALLO1 transgene encodes three genes, a BCMA-specific single- domain variable heavy chain (VH)-CAR (VCAR) gene, a drug selection gene to generate a ~100% CAR+ product, as well as a caspase-based safety switch gene to reduce or eliminate the product in vivo, if desired. The CC System is used to KO the endogenous T Cell Receptor (TCR) and beta-2 microglobulin, thereby decreasing Major Histocompatibility Complex (MHC) class I expression. KO of these key targets is aimed to prevent graft-versus- host disease, as well as reduce host-versus-graft rejection of the product. The CC System can efficiently edit resting T cells, thereby maintaining a high-percentage of TSCM cells, and does not create unwanted off-target mutations, another important consideration when creating an allo product candidate. To maximize the number of doses produced from a single manufacturing run, we have developed a proprietary “booster molecule” that allows for significant expansion of TCR-KO CAR-TSCM cells to potentially produce hundreds of doses. To date, large-scale manufacturing of significant doses of potent allo CAR-T products has been challenging for the field. P-BCMA-ALLO1 manufacturing uses a potentially unlimited number of individual serial donors. We have currently produced P-BCMA-ALLO1 at both research and near-commercial scale from >35 donors with >97% manufacturing success. While a range of TCR-KO efficiencies was observed (~50-90%), the final product was always >99% homozygous TCR-KO after a purification step. Overall expansion of TCR-KO cells ranged from ~2-20 fold, and after removal of unedited TCR+ cells ~0.42-7.04x10e9 TCR-KO cells were recovered from 0.75x10e9 starting cells. However, working at clinical production scale (starting with ~3x10e9 cells), up to 250 doses of P- BCMA-ALLO1 could be manufactured per run, at a dose of 150x10e6 cells/patient. Importantly, with this level of donor and manufacturing robustness, no significant prior screening of donor material, other than to meet standard FDA requirements, would be needed. P-BCMA-ALLO1 made from multiple donors were comprised of an exceptionally high-percentage of the desirable TSCM cells (CD45RA+CD62L+CD45RO-) and had minimal to no expression of exhaustion markers, such as PD-1 or Lag3. Furthermore, P-BCMA-ALLO1 demonstrated potent efficacy in the RPMI-8226 xenograft model in NSG mice across multiple products generated from separate individual healthy donors. Altogether, these data demonstrate a robust, reproducible and highly scalable manufacturing process. Moreover, this manufacturing process can easily be expanded for use with additional CAR targets for treatment of other hematologic or solid tumor malignancies. Patient Patients Healthy Donor Anti-BCMA CAR TCR knock-out MHC I knock-out AUTOLOGOUS ALLOGENEIC CHALLENGES • Long vein-to-vein time • Variable potency & manufacturability of patient material • High cost of an individualized product ADVANTAGES • Healthy donor material for consistent potency & manufacturability • Universal, off-the shelf product for increased patient access & decreased cost • Large number of doses for numerous patients from single manufacturing run Apheresis Product Isolated T-Cells Nucleoporation, Gene Editing, and Gene Transposition Selection Expansion PB-CAR Transposon DNA Transposase mRNA + Cas-CLOVER TM mRNA + gRNAs + Cryostored CAR-T Cell Product TCR KO Purification Overview of P-BCMA-ALLO1 Manufacturing Process Adapted from Gattinoni et al. Nat. Med. 2017 CD62L CD45RA T SCM 89.9 % T CM 0.6% T EM 0.3% T EFF 9.4% Phenotype of P-BCMA-ALLO1 Product Fig. 1 Cas-CLOVER TM Gene Editing System combines the strengths of CRISPR/Cas9 and TALEN/ZFN editing approaches, and efficiently edits in resting T cells. A) The site-specific Cas-CLOVER TM Gene Editing System consists of a dimeric gRNA-guided nuclease. Editing activity is guided by a pair of gRNAs allowing for ease of design as well as multiplexing ability found in CRISPR/Cas9-based systems. At the same time editing activity is contingent on dimerization of two half domains, similar to TALENs or ZFNs, leading to exquisite editing specificity. B) Cas-CLOVER TM efficiently edits resting T cells. Upon editing, efficient disruption of surface expression of TCRα/β and CD3 was detected via flow cytometry and graphed. Cas-CLOVER™ Gene Editing System CRISPR/ Cas9 TALEN/ ZFN • Ease of use/design • Low cost • Multiplexing ability • Specificity A) B) 1 2 3 4 5 6 7 8 9 10 11 0 50 100 Before purification % TCR-KO Fig. 3 A Booster Molecule increases the yield of P-BCMA-ALLO1 cells while maintaining proliferative potential and the desirable early memory phenotype. P-BCMA-ALLO1 CAR-T cells were produced in presence or absence of the Booster Molecule. Addition of the Booster Molecule led to significant increases in cell expansion during production A) and increased the percentage of TCR KO cells at the end of production B) leading to increased product yield of P-BCMA-ALLO1. C) At the same time, P-BCMA-ALLO1 grown in the presence of the Booster Molecule maintained a high fraction of desirable T SCM cells and D) in an in vitro serial restimulation assay, P-BCMA-ALLO1 had a similar proliferative potential to TCR-replete (unedited) CAR-T. e e 0 5 10 15 20 25 Without With Fold Expansion Booster Molecule ~5x Expans ion Without Affectin g Profile A) B) C) Booster Molecule Increases Yield of P-BCMA-ALLO1 1 2 3 4 5 6 7 8 9 0 20 40 60 80 100 120 140 200 300 400 128 104 104 29 29 88 88 251 82 82 11 11 42 42 33 33 Donor Doses at 150x10 6 1 2 3 4 5 6 7 8 9 0 20 40 60 80 100 Donor %of Subset T SCM *T CM T EM T EFF *Includes both T CM and T SCM cells in transistion Unpurified β2M CD3 CD3- 84.2% Purified CD3- 99.9% β2M CD3 Anti-BCMA CAR 95.2% No CAR Control P-BCMA-ALLO1 A) B) Fig. 4 The final P-BCMA-ALLO1 product is >99% TCR KO and >95% CAR-positive. A) At the end of P-BCMA-ALLO1 production, a clinically-scalable purification step is added to deplete remaining TCR-positive CAR-T cells and yield a highly pure allogeneic CAR-T cell product comprised of more than >99.9% TCR KO cells. B) The presence of a selectable marker in the P-BCMA-ALLO1 transposon allows for the generation of a P-BCMA-ALLO1 cell product that is >95% CAR-positive. P-BCMA-ALLO1 - a Highly Pure Allogeneic CAR-T Product Fig. 5 P-BCMA-ALLO1 exhibits potent anti-tumor effects in an in vivo mouse model of MM. P-BCMA-ALLO1 (ALLO) was tested in alongside with healthy donor non-edited cells (HDNE, CAR-positive/TCR-positive) in an in vivo model of MM tumor control. A) NGS mice were implanted sub- cutaneously with 1x10 7 RPMI-8226 BCMA+ tumor cells and tumors were established for 7 days before injection with 1x10 7 CAR-T cells. P-BCMA- ALLO1 exhibited potent and sustained anti-tumor efficacy that was comparable to non-edited control CAR-T cells. B-C) P-BCMA-ALLO1 also demonstrated robust in vivo proliferation that could be detected in the blood of treated animals by day 14 after T cell administration. C) The peak of expansion (Day 14) correlated with the timing of tumor control observed in A) and was similar to expansion levels observed for TCR-replete (unedited) CAR-T. P-BCMA-ALLO1 Exhibits Potent Anti-tumor Effects In Vivo 0 7 14 21 28 35 42 49 0 500 1,000 1,500 2,000 Days post CAR-T infusion Tumor Volume [mm 3 ] No CAR T Unedited anti-BCMA CAR-T Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 P-BCMA-ALLO1 Pre 7 14 21 28 35 42 49 1 10 100 1,000 10,000 100,000 Days post CAR-T infusion CAR+ cells/l blood Unedited anti-BCMA CAR-T P-BCMA-ALLO1 CellTrace TM Violet Unedited Control P-BCMA-ALLO1 40% 39% 2% 0% 0% 0% Donor #1 (PBMC) Donor #2 (PBMC) Autologous PBMC P-BCMA-ALLO1 Unedited Control 0.53% 0.01% 0.65% 0.29% 0.02% 0.39% 2.43% 83.0% 12.6% Unstimulated +Anti-CD3/CD28 +Anti-CD3/CD28 CD69 CD25 A) B) Fig. 6 P-BCMA-ALLO1 is not capable of mediating allogeneic responses. A) To show TCR KO in the P-BCMA-ALLO1 final product on a functional level, cells were exposed to an activation regiment comprised of anti-CD3 and anti-CD28 antibodies for 24 h. P-BCMA-ALLO1 did not show any upregulation of the activation markers CD25 and CD69 by flow cytometry. In contrast, unedited control CAR-T cells showed a strong induction of CD25 and CD69 in response to activation. B) P-BCMA-ALLO1 cells or donor-matched, unedited control CAR-T cells were exposed to PBMCs from mismatched donors as well as autologous PBMCs. While the unedited control CAR-T cells showed robust induction of CAR-T cell proliferation in response to the allogeneic PBMCs. In contrast, P-BCMA-ALLO1 cells did not proliferate, showing that P-BCMA-ALLO1 cannot mediate an allogeneic response. P-BCMA-ALLO1 Does not Mediate an Allogeneic Response • Highly desirable T SCM -rich product phenotype and potent proliferative potential • Allogeneic CAR-T equivalent or better than unedited healthy donor CAR-T in vitro & in vivo • Robust non-viral manufacturing process compatible with majority of healthy donors & ability to generate hundreds of doses per manufacturing run • Superior safety due to T SCM phenotype, no/low off-target gene editing, and safety switch • Results support rapid advancement of P-BCMA-ALLO1 into the clinic for treatment of MM Fig. 2 Cas-CLOVER TM editing in the TRAC and B2M loci shows no off-target editing activity by NGS. A) Cas-CLOVER TM was used to edit resting T cells using gRNA pairs targeting TRAC or B2M and potential off-target sites for different combinations of gRNAs were surveyed via NGS. B) NGS revealed efficient on-target editing for both TRAC and B2M loci while no above-background off-target editing events were detectable. Non-edited resting T cells from 3 donors were used to assess the level of non-editing related background mutations. Donor: A) B) Cas-CLOVER™ Shows no Off-Target Activity Cas-CLOVER™ Gene Editing System - The Best of Both Worlds No CAR T Auto Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 1 10 100 1,000 10,000 100,000 CAR+ cells/l blood Allo C) D) Unedited anti-BCMA CAR-T P-BCMA-ALLO1 TCR-α β2M
Transcript of Manufacture of an Allogeneic CAR-T Stem Cell Memory ...
Manufacture of an Allogeneic CAR-T Stem Cell Memory Product Candidate for Multiple Myeloma, P-BCMA-ALLO1, Is Robust, Reproducible and Highly ScalableStacey A. Cranert, Maximilian Richter, Min Tong, Leslie Weiss, Yening Tan, Eric M. Ostertag, Julia Coronella, Devon J. Shedlock
CONCLUSIONS
Poseida Therapeutics, Inc., 9390 Towne Centre Drive, Suite 200, San Diego, CA, 92121
INTRODUCTION
ABSTRACT
METHODS & RESULTS
Chimeric Antigen Receptor (CAR) T cell therapy has generated unprecedented efficacy in the treatment ofmultiple hematologic malignancies. For relapsed/refractory Multiple Myeloma (MM), autologous CAR-T productsdirected against the B cell maturation antigen (BCMA), such as Poseida’s P-BCMA-101, have demonstratedsignificant efficacy. P-BCMA-101 is comprised of a high-percentage of stem cell memory T cells (TSCM), resultingin a product that is much safer and potentially more durable than other anti-BCMA autologous productcandidates. However, as individualized products, all autologous CAR-T products are expensive to manufactureand dependent upon patient T-cells of variable quality. We are developing P-BCMA-ALLO1, an off-the-shelfallogeneic (allo) BCMA-specific CAR-T product candidate derived from healthy donor material, which providesnumerous advantages over autologous products, increasing patient access by being immediately available andgreatly reducing manufacturing cost and variability.
P-BCMA-ALLO1 is produced using two key platform technologies: the nonviral piggyBac® (PB) DNA ModificationSystem and the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. The mRNA coding forhyperactive, or “Super PB” transposase (SPB), and CC enzymes are codelivered with the P-BCMA-ALLO1 PB-basedDNA transgene via electroporation to healthy donor T cells to stably integrate the transgene, as well as toknockout (KO) several mediators of allo graft-versus-host and host-versus-graft responses to maximize patientsafety and durability of response. The P-BCMA-ALLO1 transgene encodes three genes, a BCMA-specific single-domain variable heavy chain (VH)-CAR (VCAR) gene, a drug selection gene to generate a ~100% CAR+ product, aswell as a caspase-based safety switch gene to reduce or eliminate the product in vivo, if desired. The CC Systemis used to KO the endogenous T Cell Receptor (TCR) and beta-2 microglobulin, thereby decreasing MajorHistocompatibility Complex (MHC) class I expression. KO of these key targets is aimed to prevent graft-versus-host disease, as well as reduce host-versus-graft rejection of the product. The CC System can efficiently editresting T cells, thereby maintaining a high-percentage of TSCM cells, and does not create unwanted off-targetmutations, another important consideration when creating an allo product candidate. To maximize the numberof doses produced from a single manufacturing run, we have developed a proprietary “booster molecule” thatallows for significant expansion of TCR-KO CAR-TSCM cells to potentially produce hundreds of doses.
To date, large-scale manufacturing of significant doses of potent allo CAR-T products has been challenging for thefield. P-BCMA-ALLO1 manufacturing uses a potentially unlimited number of individual serial donors. We havecurrently produced P-BCMA-ALLO1 at both research and near-commercial scale from >35 donors with >97%manufacturing success. While a range of TCR-KO efficiencies was observed (~50-90%), the final product wasalways >99% homozygous TCR-KO after a purification step. Overall expansion of TCR-KO cells ranged from ~2-20fold, and after removal of unedited TCR+ cells ~0.42-7.04x10e9 TCR-KO cells were recovered from 0.75x10e9starting cells. However, working at clinical production scale (starting with ~3x10e9 cells), up to 250 doses of P-BCMA-ALLO1 could be manufactured per run, at a dose of 150x10e6 cells/patient. Importantly, with this level ofdonor and manufacturing robustness, no significant prior screening of donor material, other than to meetstandard FDA requirements, would be needed.P-BCMA-ALLO1 made from multiple donors were comprised of an exceptionally high-percentage of the desirableTSCM cells (CD45RA+CD62L+CD45RO-) and had minimal to no expression of exhaustion markers, such as PD-1 orLag3. Furthermore, P-BCMA-ALLO1 demonstrated potent efficacy in the RPMI-8226 xenograft model in NSG miceacross multiple products generated from separate individual healthy donors. Altogether, these data demonstratea robust, reproducible and highly scalable manufacturing process. Moreover, this manufacturing process caneasily be expanded for use with additional CAR targets for treatment of other hematologic or solid tumormalignancies.
Patient
Patients
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Anti-BCMACAR
TCR knock-outMHC I knock-out
AUTOLOGOUS ALLOGENEIC
CHALLENGES• Long vein-to-vein time• Variable potency & manufacturability of patient
material• High cost of an individualized product
ADVANTAGES• Healthy donor material for consistent potency &
manufacturability• Universal, off-the shelf product for increased patient
access & decreased cost• Large number of doses for numerous patients from
single manufacturing run
ApheresisProduct
IsolatedT-Cells
Nucleoporation, Gene Editing, and
Gene Transposition
Selection Expansion
PB-CAR Transposon DNA
Transposase mRNA+
Cas-CLOVERTM mRNA + gRNAs
+
Cryostored CAR-T Cell Product
TCR KOPurification
Overview of P-BCMA-ALLO1 Manufacturing Process
Adapted from Gattinoni et al. Nat. Med. 2017 CD62L
CD
45R
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Phenotype of P-BCMA-ALLO1 Product
Fig. 1 Cas-CLOVERTM Gene Editing System combines the strengths of CRISPR/Cas9 and TALEN/ZFN editing approaches, and efficiently edits in restingT cells. A) The site-specific Cas-CLOVERTM Gene Editing System consists of a dimeric gRNA-guided nuclease. Editing activity is guided by a pair of gRNAsallowing for ease of design as well as multiplexing ability found in CRISPR/Cas9-based systems. At the same time editing activity is contingent ondimerization of two half domains, similar to TALENs or ZFNs, leading to exquisite editing specificity. B) Cas-CLOVERTM efficiently edits resting T cells.Upon editing, efficient disruption of surface expression of TCRα/β and CD3 was detected via flow cytometry and graphed.
Cas-CLOVER™ Gene Editing System
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Fig. 3 A Booster Molecule increases the yield of P-BCMA-ALLO1 cells while maintaining proliferative potential and the desirable early memoryphenotype. P-BCMA-ALLO1 CAR-T cells were produced in presence or absence of the Booster Molecule. Addition of the Booster Molecule led tosignificant increases in cell expansion during production A) and increased the percentage of TCR KO cells at the end of production B) leading toincreased product yield of P-BCMA-ALLO1. C) At the same time, P-BCMA-ALLO1 grown in the presence of the Booster Molecule maintained a highfraction of desirable TSCM cells and D) in an in vitro serial restimulation assay, P-BCMA-ALLO1 had a similar proliferative potential to TCR-replete(unedited) CAR-T.
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Fig. 4 The final P-BCMA-ALLO1 product is >99% TCR KO and >95% CAR-positive. A) At the end of P-BCMA-ALLO1 production, a clinically-scalablepurification step is added to deplete remaining TCR-positive CAR-T cells and yield a highly pure allogeneic CAR-T cell product comprised of more than>99.9% TCR KO cells. B) The presence of a selectable marker in the P-BCMA-ALLO1 transposon allows for the generation of a P-BCMA-ALLO1 cellproduct that is >95% CAR-positive.
P-BCMA-ALLO1 - a Highly Pure Allogeneic CAR-T Product
Fig. 5 P-BCMA-ALLO1 exhibits potent anti-tumor effects in an in vivo mouse model of MM. P-BCMA-ALLO1 (ALLO) was tested in alongside withhealthy donor non-edited cells (HDNE, CAR-positive/TCR-positive) in an in vivo model of MM tumor control. A) NGS mice were implanted sub-cutaneously with 1x107 RPMI-8226 BCMA+ tumor cells and tumors were established for 7 days before injection with 1x107 CAR-T cells. P-BCMA-ALLO1 exhibited potent and sustained anti-tumor efficacy that was comparable to non-edited control CAR-T cells. B-C) P-BCMA-ALLO1 alsodemonstrated robust in vivo proliferation that could be detected in the blood of treated animals by day 14 after T cell administration. C) The peak ofexpansion (Day 14) correlated with the timing of tumor control observed in A) and was similar to expansion levels observed for TCR-replete(unedited) CAR-T.
P-BCMA-ALLO1 Exhibits Potent Anti-tumor Effects In Vivo
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Fig. 6 P-BCMA-ALLO1 is not capable of mediating allogeneic responses. A) To show TCR KO in the P-BCMA-ALLO1 final product on a functional level,cells were exposed to an activation regiment comprised of anti-CD3 and anti-CD28 antibodies for 24 h. P-BCMA-ALLO1 did not show anyupregulation of the activation markers CD25 and CD69 by flow cytometry. In contrast, unedited control CAR-T cells showed a strong induction ofCD25 and CD69 in response to activation. B) P-BCMA-ALLO1 cells or donor-matched, unedited control CAR-T cells were exposed to PBMCs frommismatched donors as well as autologous PBMCs. While the unedited control CAR-T cells showed robust induction of CAR-T cell proliferation inresponse to the allogeneic PBMCs. In contrast, P-BCMA-ALLO1 cells did not proliferate, showing that P-BCMA-ALLO1 cannot mediate an allogeneicresponse.
P-BCMA-ALLO1 Does not Mediate an Allogeneic Response
• Highly desirable TSCM-rich product phenotype and potent proliferative potential• Allogeneic CAR-T equivalent or better than unedited healthy donor CAR-T in vitro & in vivo• Robust non-viral manufacturing process compatible with majority of healthy donors &
ability to generate hundreds of doses per manufacturing run• Superior safety due to TSCM phenotype, no/low off-target gene editing, and safety switch• Results support rapid advancement of P-BCMA-ALLO1 into the clinic for treatment of MM
Fig. 2 Cas-CLOVERTM editing in the TRAC and B2M loci shows no off-target editing activity by NGS. A) Cas-CLOVERTM was used to edit resting T cellsusing gRNA pairs targeting TRAC or B2M and potential off-target sites for different combinations of gRNAs were surveyed via NGS. B) NGS revealedefficient on-target editing for both TRAC and B2M loci while no above-background off-target editing events were detectable. Non-edited resting T cellsfrom 3 donors were used to assess the level of non-editing related background mutations.
Donor:
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Cas-CLOVER™ Shows no Off-Target Activity
Cas-CLOVER™ Gene Editing System - The Best of Both Worlds
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