Base-edited CAR T cells for combinational therapy against T cell malignancies

Abstract

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

Fig. 1. Generation of ‘T v T’ fratricide resistant CAR T cells.

A Schema of base editing for T cells employing 3rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C->U->T conversion (G->A antisense strand) resulting in STOP codon. B Lentiviral transduction of edited cells from step 1 using 3rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5′ region; DU3: delta untranslated 3′ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.

Fig. 2. 3CAR and 7CAR primary T cells evade fratricide during production.

A Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited (n = 4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products (n = 2) resulted in enriched TCR⁻CD7⁻ 3CAR/7CAR cells (red box). B Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR (n = 4), 7CAR (n = 4) production or untransduced (UTD) (n = 4) cells. Error bars represent SEM across (n = 4) donors. C Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5′ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G > A (antisense) and C > T changes within editing window highlighted by red vs blue colour. E Percentage of G > A conversions throughout TRBC-targeting protospacer sequence (left), and C > T conversions throughout CD7-targeting protospacer sequence (right).

Fig. 3. Multiplexed cytidine deamination reduces frequency of dsDNA break-mediated chromosomal translocations.

A Schematic of TRBC locus within chromosome 7 q-arm and CD7 locus within chromosome 17 q-arm highlighted by red line. Four predicted chromosomal translocations generated following simultaneous dsDNA-mediated cleavage at TRBC and CD7 loci. B Gel electrophoresis of DNA products from either untransduced (UTD) or mixed 3CAR/7CAR co-cultures edited with SpCas9 or coBE3 mRNA following PCR amplification with TRBC Fwd - CD7 Fwd, TRBC Rev – CD7 Fwd, TRBC Rev – CD7 Rev and TRBC Fwd – CD7 Rev primer combinations. Positive bands detected at ~250 bp. Control bands are PCR amplicons from of synthesised fusions. C Histogram showing percentage of digital droplet PCR (ddPCR)-based quantification of four possible predicted translocations (T1-T4) in DNA from mixed 3CAR/7CAR co-cultures edited with SpCas9 or coBE3 mRNA.

Fig. 4. 3CAR/7CAR base edited T cells mediate potent killing of T-ALL cells in vitro.

In vitro cytotoxicity of base edited 3CAR and 7CAR cells against T-ALL cell lines and primary T-ALL targets. A ⁵¹Cr labelled Jurkat T cells modified to express CD3⁺CD7⁺, CD3⁺CD7⁻, CD3⁻CD7⁺ or CD3⁻CD7⁻ were co-cultured with either 3CAR (white squares), 7CAR (grey squares), mixed 3CAR/7CAR (black squares) or untransduced (white circles) cells at an increasing ratio of effectors:targets (E:T). Error bars represent SEM of (n = 3) technical replicates. B Cytotoxic activity of 3CAR, 7CAR, mixed 3CAR/7CAR or untransduced primary T cell controls against primary patient T-ALL (T-ALL #7) cells. Representative flow cytometry plots gated on CFSE⁺ live T-ALL tumour cells (top panel). Frequency of surface antigens CD3, CD7 (middle panel) and CD19, CD56, (lower panel) gated on CFSE + live Jurkat cells.

Fig. 5. Base edited 3CAR and 7CAR cells demonstrate comparable anti-tumour clearance in vivo to CRISPR-Cas9 edited cells.

A Experimental timeline of GFP⁺LUC⁺ Jurkat T cell injection (Day 0) and effector T cell injection (Day 4) in n = 28 NOD/SCID/γc^–/– (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3–31). Organ harvest post mortem for flow-based characterisation (Day 31). B n = 28 NOD/SCID/γc^–/– (NSG) mice were intravenously injected with 10 × 10⁶ GFP⁺LUC⁺ Jurkat T cells flow sorted for CD3⁺CD7⁺ expression (Day 0) prior to receiving an IV infusion of effector T cells (Day 4). Mice received either 10 × 10⁶ SpCas9 edited TCR⁻CD7⁻ 3CAR (n = 3), 10 × 10⁶ SpCas9 edited TCR⁻CD7⁻ 7CAR (n = 3), 10 × 10⁶ SpCas9 edited TCR⁻CD7⁻ 3CAR/7CAR (n = 3), 10 × 10⁶ coBE3 edited TCR−CD7⁻ 3CAR (n = 5), 10 × 10⁶ coBE3 edited TCR⁻CD7⁻ 7CAR (n = 5), or 10 × 10⁶ coBE3 edited TCR⁻CD7⁻ 3CAR/7CAR (n = 5) effectors. Control mice received untransduced (UTD) cells (n = 3) or PBS (n = 1). Leukaemic progression was monitored by serial bioluminescent imaging (BLI) until day 31 when all groups were sacrificed. C Bioluminescence signal of each animal plotted as Average radiance [photons/s/cm²/sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent median with interquartile range. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc. D Number of CD2⁺GFP⁻ effector events or CD2⁺GFP⁺ tumour events per 1 × 10⁴ acquired bone marrow events. Error bars represent SEM. ***P < 0.001 ****P < 0.0001.

Fig. 6. 3CAR and 7CAR cells effectively clear T cell malignancy in vivo.

A NSG mice were infused with 1 × 10⁷ GFP + LUC + Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of (n = 5) CD3⁻CD7⁻, (n = 4) CD3⁺CD7⁻, (n = 5) CD3⁻CD7⁺ or (n = 5) CD3⁻CD7⁻ and imaged on day 3 prior to infusion of 1 × 10⁷ TCR⁻CD7⁻ 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR⁻7CAR⁻) and in animals engrafted with antigen-negative (CD3⁻CD7⁻) leukaemia. B Bioluminescence signal of each animal plotted as Average radiance [photons/s/cm²/sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc****P < 0.0001. C Example of day 24 flow cytometry-based detection in bone marrow of mCD11b⁻/hCD45⁺ effector T cells (CD2⁺GFP⁻) in 3CAR/7CAR treated animals and residual leukaemia (CD2⁺GFP⁺) in antigen-negative and untransduced groups. D Frequency of hCD45⁺CD2⁺GFP⁻ effector events or hCD45⁺CD2⁺GFP⁺ Jurkat events per 10⁴ acquired bone marrow events.

Fig. 7. Cytidine deamination does not compromise integrity of antigen specificity of CAR sequences.

Serial examination of 3CAR or 7CAR scFv RNA sequences 48 h and 96 h after electroporation with SpCas9 (SP3/SP7) or coBE3 (BE3/BE7) mRNA and again at end of production on d14. A Amplicons of 3CAR (left) and 7CAR (right) vH and vL sequences with antigen-binding regions (ABR) displayed mapped as a Heatmap in R using the gplots library for C>N conversion rates at the marked sites. B 3CAR (left) and 7CAR (right) scFv ABR mapped as a Heatmap for C > T conversion rates. C Stacked histogram showing <2% C > T changes in each region (ABR1H, ABR2H, ABR3H etc and outside the binding regions – (‘rest’) in SpCas-CAR3, SpCas-CAR7, BE-CAR3, BE-CAR7 or untransduced (UTD) samples.