WHAT IS ALREADY KNOWN ON THIS TOPIC

The immune system of patients develops specific anti-chimeric antigen receptor (CAR) responses after autologous CAR-T cell administration, limiting CAR-T cell persistence and the administration of multiple doses. This is exacerbated for allogeneic CAR-T cell therapies.

WHAT THIS STUDY ADDS

This study proposes a simplified manufacturing strategy to decrease major histocompatibility complex I and II molecule expression, alleviating the immunogenicity of autologous and allogeneic CAR-T cells. This study also reveals an increased anti-CAR response in patients who received multiple doses of CAR-T cells.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

This study highlights an increased anti-CAR response in patients who received multiple CAR-T cell doses. This warrants the consideration of reducing the immunogenicity of autologous CAR-T cells in addition to the current efforts to develop allogeneic products that avoid elimination by the host immune system.

Background

Adoptive cell therapy, such as chimeric antigen receptor (CAR) CAR-T cell therapy, has improved patient outcomes for hematological malignancies.1 2 However, clinical studies demonstrate that autologous CAR-T cell products elicit humoral and cellular responses to the non-self components of the CAR in patients, thus limiting CAR-T cell persistence and the success of administering multiple doses.3 4 Allogeneic or “off-the-self” CAR-T cells originating from healthy donors are in clinical development but are even more prone to host rejection and, thus, may have drastically limited expansion and persistence—particularly in the absence of deep lymphodepleting regimens (such as alemtuzumab or equivalent).4 5

Currently, four of the six U.S. Food and Drug Administration (FDA)-approved CAR-T cell products (all autologous) use the FMC63-based αCD19 single-chain variable fragment (scFv), derived from a murine monoclonal antibody, as the extracellular binding domain.1 2 Clinical trials have indicated that treatment with FMC63-based autologous CAR-T cells elicits an immunological T cell response directed at specifically against the murine portions of scFv within the CAR and can limit CAR-T cell efficacy and persistence.6 Interestingly, human or humanized scFvs can also contain non-self sequences since the variable binding fragments are generated through multiple gene recombination events and somatic hypermutations. Additionally, the expression of proteins encoded by several human genes in a single peptide CAR chain creates fusion sequences at these junctions that do normally not exist and may be immunogenic.3 6–8 Patients with CD19-positive disease relapse could theoretically receive a subsequent infusion of αCD19 CAR-T cells, although clinical responses to second or subsequent infusions tend to be less effective, and cytotoxic T cells with specificity toward the CAR expand in patients after the initial infusion.3 9–12

For allogeneic CAR-T cell therapy, the issue of rejection is even greater due to HLA mismatch.4 13 This is thought to be detrimental to CAR-T cell therapy, as positive clinical outcomes of CAR-T cell therapy are strongly correlated with expansion and persistence of the infused cells.14–17 Solutions have included deep host immunosuppression,18 which unfortunately results in increased infectious complications, and/or complex gene-editing, which increases risks of off-target effects and translocations—especially with multiple genetic edits.19–22 Most of these allogeneic approaches have focused on the elimination of major histocompatibility complex (MHC) class I via gene knockout of beta-2-microglobulin (β2M) (CRISPR therapeutics),23 Precision Biosciences,24 as β2M is required for cell surface expression of MHC I, but this may also result in increased susceptibility of the therapeutic immune effector cells to NK cell-mediated rejection.25

In this study, we propose a simplified one-shot approach to easily generate autologous CAR-T cells with reduced cell surface expression of MHC class I and II by including the gene encoding Epstein-Barr virus (EBV) BNLF2a and an shRNA targeting vlass II transactivator (CIITA) in the CAR-transduction plasmid. This one-shot, additive stealth transgene approach generates functional CAR-T cells with reduced immunogenicity.

MethodsMice and cell lines

NSG mice were purchased from Jackson Laboratory and bred under pathogen-free conditions at the Center for Comparative Medicine at Massachusetts General Hospital (MGH). HEKT cells, NALM-6 (acute lymphoblastic leukemia), JeKo-1 (mantle cell lymphoma), and K562 (chronic myelogenous leukemia) were purchased from American Type Culture Collection, maintained as outlined by the supplier and, where indicated, transduced to express click beetle green-luciferase/enhanced green fluorescent protein (eGFP). Cell lines were authenticated by short tandem repeat profiling and routinely tested to exclude mycoplasma infection.

(Stealth) CAR-T cell production

In brief, primary human T cells were purified from peripheral blood (Stem Cell Technologies) and activated on day 0 using CD3/CD28-Dynabeads (Life Technologies). Lentiviral transduction was performed on day 1, and on day 5 CD3/CD28-Dynabeads were removed. Where applicable, T cells were electroporated with Cas9 mRNA on day 5. In cases of flow-based sorting, T cells were sorted on day 8 using the eGFP marker and expanded until day 14 to be subsequently cryopreserved. When unsorted CAR-T cells were used, CAR-T cells were normalized for transduction efficiency using untransduced activated T cells from the same donor and expansion.

Cytotoxicity assay

CAR-T cells were incubated with luciferase-expressing tumor targets at indicated effector to target (E:T) ratios for 24 hours. The remaining luciferase activity was measured with a Synergy Neo2 luminescence microplate reader (Biotek). To assess the NK cell-mediated cytotoxicity, NK cells were purified from blood or frozen peripheral blood mononuclear cells (PBMCs) using an NK cell isolation kit (Stem Cell Technologies) and primed with 20 IU/mL hIL-2 and coincubated with CFSE-stained target cells (Life Technologies). After 3 hours, αCD107a antibodies were added. After a total of 4 hour, cells were centrifuged, resuspended with dead/alive marker SYTOXred (Life Technologies), and assessed by flow cytometer for viability and NK cell degranulation.

ELISpot assay

Plates with Immobilon-P membrane (Millipore) were activated and coated with anti-human IFNγ antibody (Clone NIB42, Biolegend). After blocking with 1% BSA, 5×105 PBMCs or 2×105 T cells were co-incubated with respective peptides, antigens, or stimulants. After 24 hours, the plate was washed and incubated with anti-human IFNγ antibody (Clone 4S.B3, Biolegend). After washing, the plate was incubated with avidin-HRP (Biolegend), developed using the BD Elispot AEC Substrate and analyzed with ImmunoSpot-Reader systems. All antibodies were used according to the manufacturers’ recommendation.

ELISA

Interferon ỿ from supernatants was measured following an overnight co-incubation of NLV responder T cells with target at a E:T ratio of 1:5 using Human DuoSet ELISA kits (R&D systems).

Flow cytometry

Cells were stained for 30 min at 4°C and washed twice with RPMI before analysis. SYTOXRed or SYTOXBlue (Life Technologies) was added as dead/alive marker, and singlet discrimination was performed on FSC and SSC detectors. Following antibodies were used according to the manufacturers’ recommendations in combination with their respective isotype control (online supplemental table 1). Antibody binding capacity was measured using Quantum Simply Cellar beads (Bangs laboratories). Analysis was performed by FlowJo software (BD Biosciences).

Mixed lymphocyte reaction assay

Stealth or CAR-T cells were stained with CFSE (Life Technologies) while autologous or allogeneic T cells were stained with CellTrace Violet (Life Technologies) before being co-incubated at a 4:1 ratio in the presence of 20 IU/mL hIL-2 and either isotype or MHC I (W6/32, Biolegend) or MHC II (Tü39, Biolegend) or both MHC I and II blocking antibodies. Fresh IL-2 was added every other day and the T cells were pulsed with new stealth T cells and blocking antibodies on days 7 and 14. On day 16, T responder cells were stained with SYTOXRed (viability) and assessed by flow cytometry for cell division. Allogeneicity of cells was assessed by PCR (American Red Cross) and a minimum of five out of six mismatched (HLA-A/B/C/DP/DQ/DR) were selected.

In vivo study

Luciferized NALM-6 or JeKo-1 cells were injected (1×106 cells per mouse) in NSG mice by tail vein. Tumor growth was confirmed by bioluminescence, at which time the mice were treated with an injection of 2×106 CAR-T cells in the tail vein. Tumor progression was evaluated by bioluminescence emission using an Ami HT optical imaging system (Spectral Instruments). At day 14 (or as indicated), the blood of the mice was collected and analyzed by flow cytometry for the presence of tumor and CAR-T cells per microliter of blood. For the allogeneic T cell mouse model, ‘activated’ allogeneic T cells were activated with CD3/CD28 beads and ‘Primed’ allogeneic T cells were pulsed twice with irradiated (100 Gy) PBMC originating from the CAR-T-cell donor and then expanded by a rapid expansion protocol.26 The allogeneic T cells were injected in NSG mice by tail vein 1 day prior to NALM-6 tumor cell injection.

Stealth CAR design

DNA constructs were synthesized and cloned into a second-generation lentiviral backbone under the regulation of a human EF-1α promoter and/or a human U6 promoter. EBV BNLF2a, herpes simplex virus (HSV) ICP47 and human cytomegalovirus (HCMV) transporter associated with antigen processing inhibitors (TAPi) sequences were synthesized in combination with eGFP by 2A self-cleaving peptide. Similarly, vectors with CRISPR/Cas9 guides for β2M and CIITA were constructed. The shRNA targeting CIITA was designed with software of Dharmacon and the Whitehead institute. The lentiviral vector expressing the combination of shRNA CIITA3, EBV BNLF2a and eGFP was also constructed. For CAR constructs, plasmids expressing the FMC63-based anti-CD19 CAR were synthesized.27

Statistical methods

All statistical analyses were performed with GraphPad Prism V.9 software. Data were presented as means±SEM with statistically significant differences determined by tests as indicated in figure legends.

ResultsExpression of viral TAP inhibitors in primary T cells decreases cell surface levels of MHC class I

Herpesviruses have convergently evolved to encode small proteins that inhibit TAP,28 a protein required for transporting cytoplasmic peptides across the endoplasmic reticulum and loading them for presentation on MHC class I molecules at the cell surface. Cells that lack expression of functional TAP complexes show a dramatic reduction in surface MHC I levels, substantially reducing their sensitivity to CD8+ T cells.29 We hypothesized that forced expression of viral TAP inhibitors (TAPi) would reduce MHC I expression in gene-modified cells, thereby preventing cell-mediated immune responses to foreign transgenes. To test if expression of herpesvirus TAPi reduced surface MHC I expression in primary T cells, bicistronic lentiviral constructs were generated to express HSV ICP47, HCMV US6, or EBV BNLF2a TAPi along with eGFP as transduction marker (figure 1A). Lentiviral constructs expressing sgRNA for β-2-microglobulin (β2M), without electroporated with Cas9 mRNA, were used as a positive control (β2M KO) or negative control (β2M−). At similar transduction efficiencies, TAPi-transduced cells had reduced levels of surface MHC I without affecting MHC class II upregulation on activation (figure 1B). Viral TAPi reduced total surface MHC I levels by at least one log-fold, which was maintained on additional stimulation by IFNγ or αCD3-antibody (figure 1C).

Figure 1

Lentivirus transduction of viral ransporter associated with antigen (TAP) inhibitors decreases expression of major histocompatibility complex (MHC) class I on human primary T cells, reducing allogeneic T cell responses, moderately suppressing NK cell-mediated killing, and diminishing pre-existing antiviral T cell response. (A) Schematic overviewing the MHC class I antigen presentation pathway and design of the lentiviral constructs expressing the viral TAPi or a CRISPR-guide for β2M. (B) MHC I and MHC II cell surface expression on primary human T cells transduced with the lentiviral constructs shown in (A) as measured by flow cytometry. Histograms show a representative donor (n=3 donors in total), with the isotype control in light gray, untransduced (UTD) control in dark gray, and transduced cells in colored histograms. Bar graphs represent the mean+SEM of the median fluorescence intensity (MFI) fold change relative to the UTD control of all three donors. Dots represent the fold change in median fluorescence intensity (MFI) for individual donors. (C) MHC class I antibody binding capacity (ABC) of primary human T cells transduced with the lentiviral constructs shown in (A) with or without additional stimulation of αCD3 (OKT3) or IFNγ, quantified by flow cytometry. Bars represent the mean+SEM ABC of three donors. Dots represent the ABC of individual donors. (D) TAPi-expressing or B2M KO primary T cells were incubated with autologous IL2-stimulated NK cells. T cell viability was measured via flow cytometry for CD3+SytoxRed cells. Susceptibility to NK cell lysis is reported as the % of dead cells (CD3+SytoxRed+; labeled as %NK cell-mediated killing). NK cell degranulation was measured by staining for CD107a+cells. Dots represent the mean values of four donors from two technical replicates. Lines connect values from the same donor. (E) TAPi-expressing or β2M KO T cells generated from three donors were mixed with allogeneic T cells from one additional donor or autologous T cells (responder cells) labeled with Celltrace Violet and proliferation was measured by flow cytometry after 16 days. Bars represent the mean+SEM of three donors. Dots represent the mean values of individual donors from three technical replicates with the same allogeneic responder cells. (F) TAPi-transduced or UTD primary T cells from HLA-A2+ donors were transduced with a vector coding for HCMV pp65 (containing the NLV peptide) and incubated with autologous NLV-specific CD8+T cells generated by peptide pulsing isolated CD8+T cells with the NLV peptide. T cell activation was assessed by IFNγ production, measured by ELISA. Bars represent the mean+SEM of two donors. Dots represent the mean values of individual donors from three technical replicates (G) (upper) peripheral blood mononuclear cells (PBMCs) from different donors were assessed in an IFNγ ELISPOT for pre-existing antiviral cellular immunity against herpes simplex virus (HSV), Epstein-Barr virus (EBV), and/or human cytomegalovirus (HCMV) by coincubation with the respective immunogenic peptides. Heat map indicates the mean number of IFNγ-producing cells per million PBMCS measured in triplicate of each donor. Pictures show representative wells from one donor. Donors were considered to have pre-existing immunity if they had ≥100 IFNγ-producing cells per million PBMCs. (lower) CD8+ T cells from donors with a pre-existing antiviral cellular immunity were isolated and incubated with autologous TAPi-transduced T cells in an IFNγ ELISPOT. Heat map indicates the mean number of IFNγ-producing cells per million cells, from three technical replicates of each donor. Pictures show representative wells from one donor. For all panels: statistical significance was measured by Student’s t-test compared with UTD or B2M, as shown; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Since MHC I expression inhibits targeting by NK cells,30 we investigated the impact of MHC I downregulation on susceptibility to NK cell killing. Like previous reports,25 31 β2M KO T cells were susceptible to autologous NK cell lysis and induced NK cell degranulation, as measured by CD107a expression. Compared with β2M KO T cells, T cells expressing EBV viral TAPi triggered significantly reduced NK cell lysis or degranulation (figure 1D). Similarly, MHC I expression mediates allogeneic T cell responses due to a mismatch between the MHC and TCR. To measure the allogeneic response of TAPi-expressing T cells, a mixed lymphocyte reaction (MLR) was performed. Transduced T cells were incubated with autologous or allogeneic labeled responder T cells in the presence or absence of MHC I and II blocking antibodies. Responder T cell activation was measured by proliferation (figure 1E) and changes in CD69 and CD25 expression (online supplemental figure 1A,B). T cells transduced with viral TAPi, especially EBV BNLF2a, induced less allogeneic responder T cell activation, comparable to MHC I and/or MHC II blockade.

We next tested the ability of TAPi-expressing T cells to present cytoplasmic antigens by assessing the presentation of a peptide derived from the highly immunogenic HCMV pp65 protein. The immunogenic NLV peptide is presented on the HLA-A*02:01 allele and drives NLV-specific CD8+ T cells to secrete IFNγ.32 We first generated lines of NLV-specific ‘responder T cells’ by serial stimulation of PBMC derived from HLA-A*02:01 healthy donors who had evidence of CMV-specific memory responses. We then generated a panel of “stimulator T cells” derived from the same healthy donors, which were untransduced (UTD) or transduced with the constructs as shown (figure 1A), including the three different viral TAPi. Co-cultures of “stimulator cells” with “responder cells” demonstrated that viral TAPi expression, especially when derived from HSV or EBV, reduced antigen presentation, based on a reduction of IFNγ secretion in “responder T cells” (figure 1F). Despite reduced antigen presentation, using a viral protein to knock down the MHC I could lead to an immune response to its sequence. To measure the immunogenicity of viral TAPi transduction in T cells, we identified normal donors with pre-existing cellular immunity to the respective TAPi viruses. PBMCs from normal donors were screened with peptides known to be immunogenic and originating from HCMV, EBV, or HSV in an IFNγ ELISpot assay.33–36 T cells from normal donors with a detectable cellular response those viruses were then transduced with viral TAPi from the same virus and incubated with autologous CD8+ T cells. CD8 T cell activation was measured by IFNγ ELISpot (figure 1G). While T cells from an HCMV-responsive donor were activated in response to transduction with HCMV pp65, they did not respond to transduction with the CMV TAPi. Similarly, HSV-responsive and EBV-responsive donors did not produce IFNγ in response to HSV or EBV TAPi, indicating that these viral TAP inhibitors do not elicit T cell responses, despite the donors being responsive to other known immunogenic sequences from the same viruses.

Expression of shRNA targeting CIITA decreases cell surface levels of MHC class II

Activated human T cells express high levels of MHC class II molecules. In gene-modified cells, high MCH II could trigger rejection via antigen cross-presentation of the genetic modifications.3 37 Similar to MHC class I, direct targeting of MHC class II expression with DNA-editing techniques is highly complex and potentially patient-specific, as these genes are highly polymorphic and harbor significant allelic variation.38 We sought to reduce MHC class II expression by targeting CIITA, the main regulatory factor that controls the transcription of MHC II genes.39 To avoid the use of gene-editing and double-strand breaks, we chose to encode an shRNA targeting CIITA driven by a U6 promoter, which is commonly used to transcribe small shRNA sequences,40 into our lentiviral vectors using a panel of shRNA sequences (figure 2A). We also compared our shRNA vectors to gene knockout of CIITA with CRISPR/Cas9. We noted that primary human T cells maintained an activated profile due to the initial activation by CD3/CD28 beads in the manufacturing process, as observed by a significant MHC II upregulation. Transduction with CIITA-targeting shRNA reduced the cell surface expression of MHC II, comparable to CIITA KO, without affecting MHC I expression (figure 2B). Both CIITA-targeting strategies, CRISPR/Cas9 and shRNA, rendered T cells with less than 20,000 MHC class II molecules on their surface, which was unaffected by additional stimulation with IFNγ or αCD3-antibody (figure 2C). However, only shRNA CIITA3 reduced MHC II expression without compromising T cell proliferation (figure 2D). In an MLR using allogeneic or autologous responder T cells, shRNA-mediated knockdown of CIITA reduced responder T cell proliferation (figure 2E, online supplemental figure 2A,B).

Figure 2

Lentiviral transduction of shRNA targeting class II MHC transactivator (CIITA) decreases expression of MHC class II on human primary T cells, reducing allogeneic responses. (A) Schematic overviewing the major histocompatibility complex (MHC) II antigen presentation pathway and design of the lentiviral constructs expressing shRNA targeting CIITA or CRISPR-guide for CIITA. (B) MHC I and MHC II cell surface expression on primary human T cells transduced with the lentiviral constructs shown in (A) as measured by flow cytometry. Histograms show a representative donor (n=3 donors in total), with the isotype control in light gray, untransduced (UTD) control in dark gray, and transduced cells in colored histograms. Bar graphs represent the mean+SEM of the median fluorescence intensity (MFI) fold change relative to the UTD control of all three donors. Dots represent the fold change in MFI for individual donors. (C) MHC class II antibody binding capacity (ABC) of primary human T cells transduced with the lentiviral constructs shown in (A) with or without additional stimulation of αCD3 (OKT3) or IFNγ, quantified by flow cytometry. Bars represent the mean+SEM of three donors. Dots represent the values of individual donors. (D) Primary T cells transduced with CIITA-targeting shRNA (on day 0) were sorted for GFP+ (vector-expressing) cells via FACS on day nine post-transduction. Proliferation following was assessed by cell count until the end of the manufacturing cycle (day 14). Dots represent the mean+SEM of three donors. Each donor was measured in technical triplicates. (E) CIITA shRNA3-expressing T cells or CIITA KO T cells generated from three donors were mixed with allogeneic T cells from one additional donor or autologous T cells (responder cells) labeled with Celltrace Violet and proliferation was measured by flow cytometry after 16 days. Bars represent the mean+SEM of three donors. Dots represent the mean values of individual donors from three technical replicates with the same allogeneic responder cells. For all panels: statistical significance was measured by Student’s t-test compared with UTD; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Expression of the viral TAP inhibitor EBV BNLF2a and an shRNA targeting CIITA can be combined in primary T cells to decrease cell surface levels of both MHC class I and class II.

Both strategies to downregulate the cell surface expression of MHC I or II were effective separately, but the question remained as to whether these strategies could be combined. The TAPi EBV BNLF2a was selected to be combined with the shRNA CIITA3. This TAPi reduced sufficient MHC I at the cell surface to suppress antigen presentation while the remaining MHC I at the cell surface can potentially suppress NK cell activation. These MHC I and II downregulation strategies were combined by including both EBV TAPi and shRNA CIITA3 into one lentiviral vector (figure 3A). When transduced into primary human T cells, the combined EBV-TAPi/shRNA-CIITA3 vector reduced MHC I and II expression (figure 3B,C) and reduced proliferative responses in MLRs (figure 3D, online supplemental figure 3A,B). This demonstrates that gene-modified primary T cells can successfully evade cellular immune responses by our proposed MHC class I and II downregulation strategies, creating “stealth” T cells.

Figure 3

Combining Epstein-Barr virus (EBV) transporter associated with antigen processing (TAPi) with class II MHC transactivator (CIITA)-targeting shRNA decreases major histocompatibility complex (MHC) I and II expression on primary human T cells, reducing allogeneic T cell responses. (A) Schematic overviewing the lentiviral construct combining EBV TAPi with shRNA CIITA3 and the control, single-expression constructs. (B) MHC I and MHC II expression on primary human T cells transduced with the lentiviral constructs shown in (A) as measured by flow cytometry. Histograms show a representative donor (n=3 donors in total), with the isotype control in light gray, untransduced (UTD) control in dark gray, and transduced cells in colored histograms. Bar graphs represent the mean+SEM of the median fluorescence intensity (MFI) fold change relative to the UTD control of all three donors. Dots represent the fold change in MFI for individual donors. (C) MHC class I (upper graph) and II (lower graph) antibody binding capacity (ABC) of human primary T cells transduced with the lentiviral constructs shown in (A) with or without additional stimulation of αCD3 (OKT3) or IFNγ, quantified by flow cytometry. Bars represent the mean+SEM of three donors. Dots represent the values of individual donors. (D) T cells expressing EBV TAPi and/or shRNA targeting CIITA cells generated from three donors were mixed with allogeneic T cells from one additional donor or autologous T cells (responder cells) labeled with Celltrace Violet and proliferation was measured by flow cytometry after 16 days. Bars represent the mean+SEM of three donors. Dots represent the mean values of individual donors from three technical replicates with the same allogeneic responder cells. For all panels: statistical significance was measured by Student’s t-test compared with UTD; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Stealth-enabled αCD19 CAR-T cells are functional in vitro and in vivo

The murine scFv FMC63, recognizing CD19 and used in four of the six FDA-approved CAR-T cell products, has been reported to elicit autologous T cell responses in patients.3 6 Thus, we tested our stealth strategy in the context of FMC63 CARs to verify they retain function and avoid eliciting cellular immunity. The stealth FMC63-based αCD19 CAR was generated by incorporating both the EBV TAPi and shRNA CIITA3 (figure 4A). The transduction efficiency of the stealth CAR-T cells was reduced compared with the αCD19 CAR-T cells (as measured by the %GFP+cells), likely due to the increased construct size. However, there was no significant difference in GFP MFI between the stealth and αCD19 CAR-T cells, suggesting similar CAR transgene expression between the vectors (online supplemental figure 4A-C). The stealth αCD19 CAR-T cells had reduced MHC I and II molecules on their cell surface compared with the T cells transduced with the αCD19 CAR alone and had robust expression of EBV TAPi and reduced CIITA mRNA expression compared with the αCD19 CAR alone by qPCR (figure 4B). Interestingly, this reduction of MHC I molecules at the cell surface did not increase NK cell cytotoxicity, and proliferation of the CAR-T cells was unchanged compared with the untransduced T cells (figure 4C, D). Additionally, phenotypic analysis by CD4, CD8, CCR7, and CD45RA further showed no differences in CD4/CD8 ratios and memory phenotypes comparing the αCD19 CAR-T cells without the stealth technology (figure 4E). The stealth αCD19 CAR-T cells also maintained their ability to target tumor cells in vitro. When co-incubated with luciferase-expressing acute lymphoblastic leukemia (ALL) NALM6 cells or mantle cell lymphoma JeKo-1 cells, stealth αCD19 CAR-T cells reduced tumor cell viability to the same extent as αCD19 CAR-T cells (figure 4F). The in vivo functionality was also investigated. After tumor engraftment with NALM6 cells or JeKo-1, mice were left untreated or injected with αCD19 CAR-T cells with or without stealth technology. CAR-T cell expansion in the blood was assessed by flow cytometry, and tumor clearance was measured by bioluminescence imaging (BLI) (figure 5A,E). Mice treated with αCD19 CAR-T cells with or without stealth technology showed comparable tumor clearance while tumors vastly expanded in untreated mice by BLI (figure 5B,F). Both αCD19 CAR-T cells and stealth αCD19 CAR-T cells expanded similarly in the blood, as observed at day 14 by the presence of GFP+CD3+ cells (figure 5C,G). Tumor cells (GFP+CD3 cells) were absent or minimally present in the blood of CAR-T cell-treated groups, while a large expansion was found in the untreated group, similar to the BLI imaging. Kaplan-Meier survival curves demonstrated no difference in the survival of mice treated with αCD19 CAR-T cells with or without the additional stealth technology (figure 5D,H). In summary, stealth αCD19 CAR-T cells retained their ability to recognize and clear CD19-expressing cells both in vitro and in vivo.

Figure 4

Stealth αCD19 chimeric antigen receptor (CAR) T cells have a similar phenotype and function compared with control αCD19 CAR T cells in vitro. (A) Schematic overviewing the control (αCD19 CAR alone) and stealth (coexpression of Epstein-Barr virus (EBV) transporter associated with antigen processing (TAPi) and CIITA3 shRNA) αCD19 CAR lentiviral constructs. (B) (Left) Major histocompatibility complex (MHC) I and MHC II expression on primary human T cells transduced with the lentiviral constructs shown in (A) as measured by flow cytometry. Bar graphs represent the mean+SEM of the median fluorescence intensity (MFI) fold change relative to the UTD control of all three donors. Dots represent the fold change in MFI for individual donors. (right) EBV TAPi and CIITA expression in transduced T cells measured by qRT-PCR. Bars represent the mean+SEM of three donors. Dots represent the mean values of individual donors from three technical replicates. (C) UTD, control, or stealth CAR T cells were incubated with autologous IL2-stimulated NK cells. T cell viability was measured via flow cytometry for CD3+SytoxRed cells. Susceptibility to NK cell lysis is reported as the % of dead cells (CD3+SytoxRed+; labeled as % NK cell-mediated killing). K562 cells (lacking MHC I expression) were used as a positive control. Bars represent the mean+SEM of three donors. Dots represent the mean values of individual donors from three technical replicates. (D) Primary T cells transduced with the control or stealth constructs (on day 0) were sorted for GFP+ (vector-expressing) cells via FACS on day nine post-transduction. Proliferation following was assessed by cell count until the end of the manufacturing cycle (day 14). Dots represent the mean+SEM of three donors. Each donor was measured in technical triplicates. (E) (upper) CD4:CD8 ratios of UTD and αCD19 CAR-T cells measured by flow cytometry. Dots represent the mean values of four donors. Lines connect values from the same donor. (lower) Pie charts of T cell memory phenotypes measured by flow cytometry according to CD45RA and CCR7 expression. Pie sections represent the mean percentage of each population from three donors. Naïve, CD45RA+CCR7+; EMRA, terminally differentiated effector memory cells re-expressing CD45RA, CD45RA+CCR7−; TCM, central memory, CD45RA-CCR7+; TEM, effector memory, CD45RA-CCR7−. (F) The ALL cell line NALM-6 or the mantle cell lymphoma cell line JeKo-1 were mixed with αCD19 CAR T cells with or without stealth technology and cytotoxicity was measured by luciferase expression retaining in live cells (reported as % viability). Dots represent the mean±SEM of two donors from three technical replicates. For all panels: statistical significance was measured by Student’s t-test compared with UTD or control αCD19 CAR, as shown; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.