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. 2021 Jul 2;29(12):3398–3409. doi: 10.1016/j.ymthe.2021.06.022

Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune rejection

Leila Peraro 1, Christopher M Bourne 1,2, Megan M Dacek 1,2, Enver Akalin 3, Jae H Park 1, Eric L Smith 1, David A Scheinberg 1,2,∗
PMCID: PMC8636170  PMID: 34217891

Abstract

Cellular therapies are engineered using foreign and synthetic protein sequences, such as chimeric antigen receptors (CARs). The frequently observed humoral responses to CAR T cells result in rapid clearance, especially after re-infusions. There is an unmet need to protect engineered cells from host-versus-graft rejection, particularly for the advancement of allogeneic cell therapies. Here, utilizing the immunoglobulin G (IgG) protease “IdeS,” we programmed CAR T cells to defeat humoral immune attacks. IdeS cleavage of host IgG averted Fc-dependent phagocytosis and lysis, and the residual F(ab′)2 fragments remained on the surface, providing cells with an inert shield from additional IgG deposition. “Shield” CAR T cells efficiently cleaved cytotoxic IgG, including anti-CAR antibodies, detected in patient samples and provided effective anti-tumor activity in the presence of anti-cell IgG in vivo. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of “off-the-shelf” allogeneic cellular therapies.

Keywords: CAR T cells, cell therapies, immunogenicity, humoral response, anti-CAR IgG, IdeS

Graphical abstract

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Cellular therapies are engineered using foreign and synthetic protein sequences. Utilizing the IgG protease “IdeS,” we programmed CAR T cells to defeat humoral immune attacks. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of “off-the-shelf” allogeneic cellular therapies.

Introduction

Chimeric antigen receptor (CAR) T cell therapies have been approved for the treatment of acute lymphoblastic leukemia (ALL) and diffuse large B cell lymphoma (DLBCL) with CD19-targeting CARs.1, 2, 3 More complex CAR T cell therapies have been engineered to secrete cytokines, single-chain variable fragments (scFv), and bispecific T cell engagers to potentiate their activity.4, 5, 6, 7 CRISPR-Cas9 technology also has been used for site-specific integration of the CAR.8, 9, 10, 11 “Off-the-shelf” allogeneic CAR T and natural killer (NK) cells are currently in clinical development.12, 13, 14 However, major challenges remain in the application of these technologies to other cancers. In addition, genetically modified cells rely on the use of foreign biological material or unnatural amino acid sequences, resulting in immunogenicity.15,16

Humoral responses and anaphylaxis to engineered cells have been reported in human trials.17, 18, 19, 20, 21, 22 Such antibodies may lead to limited persistence, poor engraftment, and reduced functionality of the CAR T cells.17,20,23 Whereas humanizing CARs renders them less immunogenic,24 not only are the antibody constructs potentially immunogenic but so are the artificial linkers used in the fusion sequences, as well as viral products used throughout the transduction process, which may be recognized as foreign.15,16

Although the immune response to engineered cells in the host can be both cellular and humoral, reported efforts have been directed at defining and possibly reducing the T cell response13 but not the immunoglobulin (Ig) response. To investigate the scope of this issue, in this work we first screened sera from 12 patients who received two or more rounds of 1928ζ CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC) and confirmed that host IgG responses were prevalent. Disease progression and outcomes for these patients were published recently.25 Our hypothesis was that anti-CAR IgG antibodies could prevent the expansion and function of the re-infusions of CAR T cells. We therefore designed an innovative approach based on the microbial immunoevasin IgG-degrading enzyme of Streptococcus pyogenes (IdeS)26 for arming engineered cells with an effective means to circumvent the host humoral immune response. IdeS is a cysteine protease with high specificity for human IgG that has already been safely used in highly sensitized patients to treat renal allograft rejection.26, 27, 28 The CAR T cells expressing IdeS, termed “Shield CAR T” cells, retained their original immune functions, cleaved IgG in vitro and in vivo, covered themselves in a protective coating of non-functional F(ab′)2, thereby preventing new attack by other IgG, and have improved persistence and efficacy over traditional CAR T cell therapies in the presence of antibodies directed to their cell surfaces.

Results

Murine CAR T cells induce a host IgG response in a syngeneic mouse model

To better understand the syngeneic anti-CAR responses observed in human trials,17, 18, 19, 20 we recapitulated a humoral response to a murine CAR in a syngeneic C57BL/6J mouse model in which weekly infusions of littermate-derived mouse m4H1128ζ CAR T cells led to the development of anti-CAR IgG responses (Figure 1A). m4H1128ζ CAR T cells have been previously established and tested in syngeneic systems targeting MUC16+ tumors.6 The 4H11 scFv is murine, as are the signaling domains. When we tested the mouse sera, we found that IgGs bound to a HEK293t-derived cell line that expressed the m4H1128ζ CAR construct (Figure 1B). Time-dependent increases in mouse IgG antibody titers were observed to the m4H1128ζ-expressing HEK cells, which was not observed in the control mice or the pre-bleed samples (Figure 1B) or HEK cells expressing an unrelated CAR, m1928ζ, which targets CD19+ cells (Figure 1C). These data showed that the polyclonal IgG contained in mouse sera specifically bound to sequences associated with the m4H1128ζ CAR. Binding of mouse IgG was also confirmed against m4H1128ζ CAR T cells (Figure 1D). These data demonstrated that CAR T cells were rapidly immunogenic even in syngeneic systems. These results were also confirmed in EL4 mCD19+ tumors and m1928ζ CAR T cells in naive mice and in cyclophosphamide-pre-treated mice (Figures S1A and S1B), the latter to mimic human clinical use. Thus, even mice lymphodepleted by cyclophosphamide could still mount an IgG response to CAR T cells.

Figure 1.

Figure 1

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CAR T cells induce IgG-mediated immune response in syngeneic mouse model

(A) Schematic of experimental timeline. Female C57BL/6 mice were inoculated with 1 × 106 EL4-MUC16 cells i.v. and then infused with 2 × 106 m4H1128ζ CAR T cells (n = 3) or untransduced mouse T cells (n = 3) once per week for 3 weeks. m4H1128ζ CAR T cells have been previously used to target MUC16-expressing tumors.6 To track the presence of CAR-binding IgG, cheek bleeds were performed throughout the experiment. (B) Mouse sera were analyzed for binding to m4H1128ζ-expressing HEK293t cells, detected with secondary anti-mouse AF647, and analyzed by flow cytometry. (C) Detection of non-specific binding was measured by looking at serum binding to m1928ζ-expressing HEK293t cells. m1928ζ is the construct targeting CD19+ targets. (D) Detection of mouse sera binding to m4H1128ζ CAR T cells. MFI, median fluorescence intensity. n = 3 mice per group; ∗p < 0.05; ∗∗p < 0.01;∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, Student’s t test.

Design and generation of cytotoxic Shield CAR T cells

Next, we invented a defensive system for the engineered cells by incorporating into them processive IdeS-mediated IgG cleavage, thus rendering local IgG immediately non-functional. Because IdeS works by cleaving off the Fc while retaining the F(ab′)2, the remaining Ig fragments bound to the cells serve as an inert barrier to further attacks on the same epitopes, hence forming a “shield.” We designed two “Shield CAR T” constructs: one in which IdeS is tethered to the membrane, IdeS-tm, and one where the enzyme is secreted, IdeS-sec (vector scheme and map in Figure 2A), both of which are translatable to human uses. At the outset we did not know which platform would provide the most potent or most localized protection from humoral attack or if there were pharmacological advantages to one form. The design of our Shield CAR cell vector constructs was based on previously reported methods, now widely used clinically.29 Efficient transduction of primary human T cells (Figure 2B) was achieved using Galv9 retroviral producer cell lines, as previously described.30,31 CAR expression on Shield CAR T was consistent with the parent 19BBζ CAR. Expression of membrane-bound IdeS-tm was confirmed by flow cytometry (Figure 2C; third panel) and was not seen in IdeS-sec cells (Figure 2C; fourth panel). Expression of IdeS was also demonstrated by immunoprecipitation of T cell lysates and medium supernatants with anti-hemagglutinin (HA) beads and subsequent western blot (WB) using an anti-IdeS antibody. IdeS-tm (only in cell lysate) and secreted form IdeS-sec (only in cell conditioned media) appeared at predicted molecular weights (MWs) of ∼45 and 37 kDa, respectively (Figure 2D).

Figure 2.

Figure 2

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Shield CAR T cells express IdeS and retain their cytotoxic activity in vitro

(A) Retroviral construct schema for traditional 19BBζ CAR T cells and Shield CAR T cells expressing membrane-bound IdeS (IdeS-tm) or secreted IdeS (IdeS-sec). (B) CAR expression detected by flow cytometry using anti-idiotype AF647 antibody. (C) Detection of membrane-bound IdeS on the same cells listed in (B) with anti-HA AF647. (D) Expression of IdeS detected in CAR T cell lysates and supernatant fluid by anti-HA immunoprecipitation and anti-IdeS immunoblot. Experiments were repeated at least two times. Representative data shown. (E) Cytotoxic activity of 19BBζ and Shield CAR T cells. Raji GFP/Luc were used as targets, and specific lysis was measured after 18 h in a luciferase-based lysis assay. Data shown are the mean of 3 independent donors. IP, immunoprecipitation; WB, western blot.

IdeS provides S. pyogenes evasion from antibody-mediated opsonization by cleaving IgG.26 We wanted to ensure that this bacterial enzyme expression within the T cells did not detract from their cytotoxic activity. No reductions in the cytolytic activity were observed in the two types of Shield CAR T cells (Figure 2E) at six different effector-to-target ratios (E:T) against Raji lymphoma targets. Cytotoxicity of the CAR T cells was also confirmed with mouse fibroblast NIH/3T3 target cells expressing hCD19 (Figure S2).

Shield CAR T cells express functional IdeS and form F(ab′)2 shield

Previous work has shown that IdeS selectively cleaves IgG below the hinge region, releasing Fc fragments, while the F(ab′)2 fragment remains intact.27 IdeS can cleave all isoforms of human IgG as well as rabbit IgG but, however, can only very minimally cleave some isoforms of mouse IgG.27,32,33 Therefore, all of our experiments with IdeS were performed with human or rabbit IgG.

To evaluate whether the IdeS expressed within the CAR T cells was functionally active, we incubated 19BBζ, IdeS-tm 19BBζ, and IdeS-sec 19BBζ cells with human polyclonal IgG, which did not bind to the cells and would remain in solution (Figure 3A). Cleavage of the Fc fragments from the heavy chain was documented by the appearance of lower MW bands at 25 kDa, which were observed for both types of Shield CAR T cell constructs but not with the parental 19BBζ CAR T cells. Shield CAR T cells also could cleave high-titer rabbit-derived anti-thymocyte globulin (ATG) antibodies capable of binding to the cell surface of the CAR T cells themselves. ATG contains antibodies to CD3, CD4, CD8, as well as major histocompatibility complex (MHC) class I and II and β2m.34 Both types of Shield CAR T cells efficiently cleaved off the Fc portions of ATG from the cell surface (Figure 3B), and the Fc fragments were released into the supernatant fluid (Figure 3C). After ATG attack on the Shield CAR T cells, the cell surface showed loss of the Fc compared to 19BBζ (Figure 3D), with retention of ATG F(ab′)2 antibody fragments (Figure 3E), which was equivalent among all three CAR T cell groups (Figure 3E), not seen with the control parental CAR T cells or untransduced T cells (Figure 3D). This experiment demonstrated our hypothesis, that the Shield CAR T cells remained coated in a F(ab′)2 barrier, as shown in the schematic in Figure 3F. Interestingly, in these experiments the unsorted T cells in the population only had a transduction percentage of ∼30%, meaning that the majority of the cells in each Shield CAR T well were untransduced parental T cells. Yet, despite the heterogeneity, the untransduced majority bystander T cells were also protected by the minority of Shield CAR T cells in their midst. This occurred for both the membrane-bound and secreted forms of Shield cells. This result highlighted that Shield cells can also cleave the IgG in the microenvironmental milieu that might attack adjacent bystander cells and suggests that Shield CAR T cells might create a small local field of protection for other medical applications.

Figure 3.

Figure 3

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IdeS-expressing cells can efficiently cleave IgG antibodies, form a “shield,” and are protected from CDC and ADCC

(A) Human polyclonal IgG incubated with 19BBζ CAR T cells and Shield CAR T cells for 2 h and analyzed by WB using anti-Fc specific antibody. Hc, heavy chain. (B) 19BBζ and Shield CAR T cells incubated with ATG and analyzed at 3 and 18 h for cleavage off the cell surface by flow cytometry. (C) Supernatant fluid from experiment in (B) was analyzed by WB. Rb, rabbit. (D and E) MFI values corresponding to the presence of ATG on the surface of CAR T cells after overnight incubation analyzed with an anti-Fc specific antibody (D) and an anti-Fab specific antibody (E). (F) Schematic depiction of F(ab′)2 shielding mechanism (light blue is CAR T cell with blue CAR; red is IdeS; gray is residual F(ab′)2 fragments.) (G) 19BBζ and Shield CAR T cells were tested in a CDC assay where cells were incubated with 2 μg/mL ATG overnight and then incubated with rabbit complement for 1 h. Cell number was measured by Cell Titer-Glo. Data represent mean ± SD of triplicate samples. (H) ADCC of 19BBζ and Shield CAR T incubated with ATG was assessed by 51Cr 6 release assay. Data represent mean ± SD of triplicate samples. Experiments were repeated at least twice. Representative data are shown. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; Student’s t test.

We further examined whether the remaining F(ab′)2, which forms the shield, had any immunological or biological effects on the CAR with or without targets. To test this, we used a rabbit anti-mouse antibody that specifically binds mouse Fab and thus will bind the murine scFv of the 19BBζ CAR. This antibody was shown to bind equally well as an anti-idiotype CAR antibody (Figure S3A) and can be cleaved by IdeS efficiently (Figure S3B). We also ensured that the F(ab′)2 fragments remained bound to the cells as was shown above. We tested whether the activation and production of interferon (IFN)-γ by the cells would be affected by the presence of this CAR-binding antibody. CAR T cells and Shield CAR T cells were incubated with different concentrations of the antibody, with or without NIH/3T3 hCD19+ targets. Using an IFN-γ ELISA, we saw no impact on IFN secretion between antibody-treated groups and the controls, and, as expected, the levels of IFN increased in the presence of antigen-positive targets (Figure S4A). We also looked at the effects of the CAR-binding antibody on activation, by measuring expression of CD69. No significant increase was detected in % CD69+ cells, which suggested that the CAR-binding antibody and F(ab′)2 shield had no impact on CAR T cell activation (Figure S4B).

IdeS-expressing cells and bystanders are protected from Fc-mediated antibody killing

We also assessed whether Shield CAR T cells could protect themselves against antibody-mediated lysis in in vitro complement-dependent cytotoxicity (CDC) assays using ATG. The untransduced primary T cells and parental 19BBζ CAR T cells were lysed, whereas both forms of Shield CAR T cells retained full viability (Figure 3G). To test whether the retained F(ab′)2 fragments formed a “shield” on the cells, we repeated the CDC assay experiment, but this time added another aliquot of ATG while incubating with complement. Shield CAR T cells resisted lysis by the additional IgG-mediated complement fixation (Figure S5). Furthermore, although only 30% of the T cells were transduced with the CAR and IdeS, 100% of cells remained viable, meaning that both IdeS constructs (membrane tethered and secreted) could provide trans protection to nearby bystander cells.

Shield CAR T cells could also survive other modes of antibody-mediated killing, such as antibody-dependent cell-mediated cytotoxicity (ADCC) evaluated by use of a chromium release assay. Specific lysis of parental 19BBζ CAR T cells was observed at different concentrations of ATG, whereas the two forms of Shield CAR T cells were not lysed (Figure 3H).

Shield CAR T cells are resistant to anti-HLA allograft response in vitro

Anti-histocompatibility antigen (HLA) antibody attack is a major hurdle for allotransplants, and recombinant soluble IdeS infusions have been used in a humans to prevent acute graft rejection prior to HLA-mismatched allotransplants.28 Anti-HLA IgG is believed to be a key component in poor donor cell engraftment as well as antibody-mediated rejection of solid-organ allografts.35, 36, 37 We asked whether IdeS-expressing Shield cells could cleave human and rabbit anti-HLA antibodies. HEK293t cells were engineered to express IdeS-tm and IdeS-sec, as well as the inactive C94S mutant of IdeS. All four cell lines had equal levels of surface HLA by flow cytometry (Figure 4A). The added rabbit anti-HLA was cleaved from the cell surface by both IdeS-tm and IdeS-sec HEK cells. The control IdeS-tm(mut) HEK cells displayed levels of Fc similar to the wild-type (WT) HEK cells (Figures 4A and 4B). Therefore, HLA-targeting antibodies can be successfully cleaved by IdeS, whereas the catalytically inactive enzyme showed no cleavage.

Figure 4.

Figure 4

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Shield cells can cleave anti-HLA antibody and are protected from transplant patient hyper-immune HLA-targeting antibodies

(A) HEK293t cells stably expressing membrane-bound (tm) and secreted (sec) IdeS and the inactive point mutant C94S, IdeS-tm(mut), were incubated with mouse anti-HLA AF647 and rabbit anti-HLA detected with anti-Fc secondary. (B) Ratios of rabbit to mouse anti-HLA antibody MFI. (C) Serum sample from human renal transplant patient with high levels of anti-A02 IgG antibodies showed binding to A02+ cell lines (A-375 and OVCAR) and A02+ and non-A02 primary T cells by flow cytometry using anti-hu Fc specific AF647. (D and E) A02+ Shield CAR T cells (both tm and sec) can cleave IgG in transplant patient serum at several dilutions, whereas IgG in 19BBζ remains intact after overnight incubation at 37°C. (F) Shield CAR T cells are protected from CDC mediated by hyperimmune IgG in human renal transplant patient serum after overnight incubation at 37°C. Cell number was measured by Cell Titer-Glo. Data represent mean ± SD of triplicate samples. All experiments were repeated at least twice. Representative data are shown. ∗p < 0.05, ∗∗p < 0.01; Student’s t test.

To model a human anti-HLA immune response to allogeneic CAR T cells, we obtained serum samples from kidney transplant patients exhibiting high levels of donor-specific anti-A02 antibodies. We found one patient sample that showed high levels of binding on all cell types (Figure 4C). We further tested the transplant patient sample in an IgG-cleavage assay using A02+ T cells transduced with 19BBζ, IdeS-tm 19BBζ and IdeS-sec 19BBζ cells at various dilutions. Cleavage of the antibodies in the serum was observed in the Shield CAR T cell samples by flow cytometry (Figure 4D), while IgG remained intact on parental 19BBζ CAR T cells (Figures 4D and 4E). With the same method described above, the 19BBζ and Shield CAR T cells were tested in a CDC assay using the transplant patient serum. The data obtained recapitulated the data obtained with ATG (Figures 3G and 3H), where reduced viability was observed for parental 19BBζ and the Shield CAR T cells resisted CDC (Figure 4F).

Shield CAR T cells cleave IgG and provide antibody-mediated depletion in vivo

Next, we tested the efficacy of Shield CAR T cells in a xenograft mouse model. IdeS does not cleave mouse IgG, thus not allowing us to directly test Shield CAR T cells in a syngeneic mouse model. Immunodeficient mice (NSG) were engrafted intraperitoneally (i.p.) with antigen-positive 3T3 cells expressing hCD19, to provide a target in vivo for CAR T cell proliferation and expansion (Figure 5A). Next, 2 × 106 19BBζ, IdeS-tm 19BBζ, or IdeS-sec 19BBζ cells were injected i.p. Mice were then injected with rabbit or human IgG, and after 16 h the ascites harvested from the mice could be analyzed by immunoblot, as previously shown.6 In the mice injected with Shield CAR T cells, but not parental controls, cleavage of the IgG was observed, as seen by the appearance of lower MW Fc fragments (Figures 5B and 5C).

Figure 5.

Figure 5

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Shield CAR T cells cleave IgG in vivo, survive antibody-mediated cytotoxicity, and kill their target

(A) Diagram showing experimental setup to detect IgG cleavage in vivo. NSG mice were engrafted i.p. with 0.5 × 106 3T3 hCD19+ cells; the next day 2 × 106 19BBζ and Shield CAR T cells were injected i.p. CAR T cells were allowed to expand for 1 day, and then 20 μg of polyclonal human or rabbit IgG was added i.p. To measure cleavage of IgG, 0.5 mL of PBS was used for an i.p. lavage; 50–100 μL of i.p. fluid was analyzed by WB. (B) WB of rabbit polyclonal IgG cleavage recovered from i.p. cavity (arrows show Hc and Fc). (C) Western blot of human polyclonal IgG cleavage recovered from i.p. cavity. (D) Diagram showing experimental setup to test resistance of Shield CAR T cells to antibody-mediated killing in vivo. NSG mice were engrafted i.p. with 1 × 105 NIH/3T3 hCD19+ FLuc target cells. The next day, mice were imaged and randomized by BLI. Mice were then injected with 2 × 106 19BBζ and Shield CAR T cells in one group and 19BBζ and Shield CAR T cells coated with ATG in another group (incubated with ATG ex vivo). Tumor progression was followed weekly by imaging. (E) BLI (total flux) at days 0, 3, and 13 showed that ATG suppressed the therapeutic effect of the WT CAR T cells but not the Shield CAR T cells. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; Student’s t test.

CAR T cells have been shown to expand in vivo and kill their target within the first week of engraftment.38 To test the resilience of Shield CAR T cells to antibody-mediated killing in vivo, we used potent ATG as the cytotoxic antibody (as shown in Figure 3). NSG mice were engrafted with 1 × 105 NIH/3T3 hCD19+/GFP+/ luciferase+ cells, and the next day mice were imaged for tumor engraftment and randomized based on the tumor bioluminescence imaging (BLI) (Figure 5D). In this experiment the membrane-bound version of Shield CAR T cells was used, as design of an assay in this model with the secreted form was not feasible. To ensure that sufficient ATG was added to deplete the T cells, CAR T cells in the ATG group were incubated with ATG ex vivo for 30 min to allow for binding and subsequently injected into the mice. Although the tumor burden in all groups was initially equal, by day 13 the 19BBζ and Shield CAR T cell groups that had not been exposed to ATG had lower BLIs, meaning that they had killed tumor cells in vivo (Figure 5E; BLI raw images in Figure S6). However, in the ATG-treated groups only Shield CAR T cells continued to control the tumor burden. In this way, Shield CAR T cells resisted ATG attack and functioned normally, whereas parental CAR T cells bound by ATG were depleted.

Shield cells resisted killing mediated by anti-CAR IgG detected in CAR T cell patient sera

Although many current research efforts are working on improving CAR T cell potency,39 a notable limitation of CAR T cell efficacy is limited persistence inside patients, raising the concern for anti-CAR immune responses.20,40 We obtained serum samples from 12 patients (Table S1) who had been treated with two infusions of 1928ζ CAR T cells. For two of the patients (1 and 4), we tested two different time points after the second infusion. Further detail can be found in Table S1. For all samples, we tested the presence of anti-CAR IgG in an ELISA where cell lysates from WT and 1928ζ-expressing HEK293t-derived producer cells were used as the antigen. Cell lysates were adsorbed directly onto the plate, and, notably, the only difference between the cell lysates was the expression of the 1928ζ CAR. Serial dilutions of sera were applied, and IgG was detected with an anti-human horseradish peroxidase (HRP)-labeled secondary (Figure 6A). Four out of 12 patients produced anti-CAR IgGs after the second cell infusion. A WB confirmed that serum samples 4a and 4b, corresponding to the samples with the highest titers detected by ELISA, also showed bands corresponding to the MW of the CAR (Figure 6B), found only in the 1928ζ CAR HEK lysates. To evaluate the cytotoxicity of the CAR T cell patient serum samples, we used a CDC assay with rabbit complement. More than 75% killing of 1928ζ HEK cells was observed in 5 out of the 14 samples tested (33% of patients) (Figure 6C), whereas WT HEK cells remained viable. Patients who had antibodies to CARs showed no expansion of CAR T cells in their blood (Table 1), whereas 4 out of 6 patients with no detected antibodies had substantial expansion.25

Figure 6.

Figure 6

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Shield cells survive antibody-mediated cytotoxicity from CAR T cell patient sera, shown to have CAR-binding IgG

(A) Serum samples from 12 ALL patients treated with 2 infusions of 1928ζ CAR T cells were analyzed for binding to the 1928ζ CAR by ELISA. For patients 1 and 4, two samples at different time points (“a” and “b”) were tested after the second CAR T cell infusion. Details regarding the time points can be found in Table S1. Lysates from WT HEK293t and 1928ζ-expressing HEK cells were used as the target cell antigen. A binding ratio of 1928ζ to WT HEK cells was used to exclude non-specific binding. Samples are numbered for each unique patient and labeled a and b to designate samples obtained from the same patient at different intervals after CAR T cell infusion. (B) Patient sera were used as primary antibodies in a WB assay. WT and 1928ζ HEK lysates were run on SDS-PAGE gel and then incubated with diluted patient sera overnight. Anti-human HRP-labeled secondary was used for detection. (C) CDC assay to test the cytotoxicity of the patient serum samples. WT and 1928ζ HEK producer cells were used as targets, incubated with CAR T cell patient sera and rabbit complement, and cell number was measured by Cell Titer-Glo. (D) Patient sera 1a and 4a inhibited the binding of CAR anti-idiotype AF647-antibody as measured by flow cytometry. (E) Supernatant fluid from experiment in (D) was analyzed by WB, revealing cleavage of IgG in CAR T cell patient sera by the Shield cells (arrow shows Fc fragment). (F) CDC assay to test cytotoxicity against Shield HEK cells. Cells were incubated with diluted patient sera 1a and 4a for 2 h and then incubated with rabbit complement. Cell number was measured by Cell Titer-Glo. Data represent mean ± SD of triplicate samples. All experiments were repeated at least twice. Representative data are shown. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; Student’s t test.

Table 1.

Summary of antibody detection and CAR T cell expansion data from 9 patients

Antibodies detected Expansion > 1,000 vcn/mL No expansion
Yes 0 3
No 4 2
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Expansion and clinical outcome data analyzed in Wudhikarn et al.25 Patients 1, 8, and 10 omitted from analysis. Chi-square test, p = 0.06.

HEK293t cells highly expressing the 19BBζ CAR and IdeS were used to show binding of antibodies contained in patient sera to the CAR in an inhibition-based assay. Shifted binding curves after blocking with patient sera 1a and 4a indicated inhibition of binding of the anti-idiotype antibody, which was observed to different extents compared with the control (Figure 6D). This implied that anti-idiotype IgGs were present in patient sera 1a and 4a. The IgGs in the sera were also analyzed for cleavage by WB (Figure 6E). The WB confirmed that samples incubated with IdeS-expressing HEK cells resulted in cleavage of IgG, as observed by the appearance of low MW Fc fragments, and inhibition of anti-idiotype binding was observed (Figure 6D). This suggested that anti-CAR F(ab′)2 fragments remained bound to the CAR on the surface of the cells.

We finally wanted to determine whether IdeS-expressing cells could survive CDC killing mediated by anti-CAR IgGs present in CAR T cell patient samples. HEK 19BBζ, IdeS-tm 19BBζ, and IdeS-sec 19BBζ cells were incubated for 2 h with diluted patient sera 1a or 4a or a healthy donor. HEK 19BBζ cells were only 50% viable, whereas IdeS-expressing HEK cells fully retained their viability (Figure 6F), suggesting that IdeS could cleave the human patient anti-CAR specific IgG and protect the cells from CDC.

At this point we have shown that Shield CAR T cells can cleave antibodies directed to the CAR or the cell itself, but an outstanding question is whether Shield CAR T cells can still lyse targets in the presence of CAR-binding antibodies. We tested this in two different ways. First, we used the rabbit anti-mouse Fab-specific antibody, which we showed binds the CAR and can be cleaved by IdeS (Figure S3). At different concentrations of the antibody, the 19BBζ CAR can still kill NIH/3T3 hCD19+ cells (Figure S7A). Next, we tested whether CAR T cells could lyse their targets in the presence of patient sera 1a and 4a, which were previously shown to bind and kill CAR-expressing cells by CDC (Figure 6). Cytotoxicity assays were performed with patient sera 1a and 4a at different dilutions, and CAR T and Shield CAR T cells were still able to kill antigen-positive cells (Figure S7B).

Discussion

The potential for neutralizing humoral immunogenicity of engineered cell therapies and particularly the risk of host humoral responses has not been studied, nor have effective solutions been devised. Poor expansion and low persistence of CAR T cells, especially with repeat infusions,40 and the presence of CAR-reactive IgG in patient sera all suggest that this is a difficult problem that may worsen with the increase in use of allogeneic cells in patients. Interestingly, most successes in CAR T cell therapies thus far have been in patients with B cell neoplasms, where the host often has an underlying immune deficiency and the CAR T cells themselves effectively kill the normal CD19+ B cells, the source of anti-CAR antibodies. In addition, these patients also often have been extensively treated with lymphocytotoxic drugs, further suppressing a potential immune response.41 Despite these features of the currently treated patient populations, we still observed a high rate of humoral response to the CAR T cells, as have others.17, 18, 19, 20 The immunogenicity of infused cells may be an even more important hurdle to overcome for the treatment of non-B/plasma cell blood cancers and solid tumors.

We sought to defeat the host humoral response by mimicking a highly potent, evolutionarily optimized microbial system using the IdeS enzyme, to avoid opsonization by human IgG and destroy host antibodies. This study highlights two major findings: (1) the existence of anti-CAR T cell humoral responses in patients receiving CAR T cells, and in murine models that recapitulated the effect, and (2) the ability to reprogram cell therapies to be resistant to this IgG-mediated recognition and killing. No patient who mounted anti-CAR IgG responses, detected at various time points after their second CAR T cell infusion, demonstrated expansion of the CAR T cells upon re-infusion (Table 1). Although the patient cohort is small, this finding highlights both the prevalence of anti-CAR T antibodies that has been previously anecdotally reported and its detrimental effects.

IdeS has been used effectively as a systemically infused enzyme to block allograft rejection, but because the infusion is systemic, this also results in rapid and persistent loss of all IgGs from the patients’ sera.28 Our Shield cells could successfully cleave IgG in vitro and in vivo, rendering them protected from IgG-mediated CDC and ADCC, and were protected from antibody-mediated depletion in a xenograft model in mice. The cells were effective at depleting polyclonal IgG, ATGs reactive with multiple cell surface targets, human anti-HLA antibodies found in allograft patients, and human anti-CAR antibodies found in patients treated with CAR T cells.

Although both Shield cell types worked, the membrane-tethered IdeS may provide one ideal design for this type of cell protection, as it would limit systemic circulation of the enzyme. On the other hand, local secretion of the IdeS might provide a small field effect that could be useful in protecting larger regions, such as in an allograft, or autoimmune disease. The choice of Shield CAR T cell form may depend on the application of the cells and type of tumor system. In vitro and in mice, it is hard to accurately compare the activity between the membrane-bound and secreted forms because the kinetics of each form is so different. That is, the membrane form appears immediately and is stable, whereas the secreted form slowly accumulates in the media. Further experiments are required to test the benefits of each form of Shield CAR T cells in other model systems. Because IdeS is not active against rodent IgGs, some of these models are difficult to construct and are beyond the scope of the current work.

IdeS is derived from S. pyogenes, and it would likely be immunogenic itself. However, the enzyme would be constitutively produced, locally secreted or tethered to the membrane, and has the ability to cleave IgG to also protect itself from the host immune response, which is its natural evolved goal. Future enhancements of this system might involve gating IdeS expression to further localize its actions.42, 43, 44 The limited peripheral persistence of CAR T cells in humans has also been attributed to cellular responses, where CAR T cells are targeted by endogenous T cells,19,45 not addressed by our approach. Other approaches to suppress T cell responses are under investigation.13,19,46

We also would expect that a true anti-idiotype antibody to the CAR could block recognition by the CAR of its target. Because the IdeS would not necessarily change that type of binding by cleaving the IgG, IdeS may not resolve this problem. We do not know if such blocking antibodies commonly exist in patients. However, if the primary failure of the immunogenic CAR T cells in the patients is a result of the clearance, opsonization, or killing of the cells by host IgG reacting with a number of cell surface epitopes, the IdeS enzyme should prevent this clearance. Another potential pitfall with our approach is that the IdeS itself may be immunogenic to T cells, along with all of the other foreign material, and other solutions described immediately above may be needed to address this potential issue.

Shield cells may have other important applications beyond traditional and allogeneic CAR T cell therapies, such as local elimination of anti-HLA antibodies, which are responsible for acute renal allograft rejection. In addition, engineered cell therapies are now also being tested against autoimmune diseases, such as for the treatment of pemphigus vulgaris, systemic lupus erythematosus, idiopathic thrombocytopenia, Goodpasture’s disease, and arthritis.27,33,47, 48, 49 Therefore, localized use of IgG-depleting Shield cells might have advantages in these IgG-mediated autoimmune diseases as well, by providing long-term highly targeted cell protection and depletion of pathogenic IgG.

Materials and methods

T cell isolation and CAR T cell generation

Vector constructs and retroviral producers were generated as previously shown.29,50 Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient separation from blood from healthy donors according to the institutional review board (IRB) protocol. PBMCs were activated with 50 ng/mL OKT-3 (Miltenyi Biotec) and 100 IU/mL recombinant human interleukin (IL)-2 for 48 h. Primary T cells were transduced with Gavl9-produced virus in retronectin-coated 6-well plates at 2,000 × g for 1 h. Two spinfections were performed, and cells were checked for CAR expression after 72 h. Mouse T cells were isolated mechanically from mouse spleens and activated with CD3/CD28 magnetic beads (Gibco, Thermo Fisher) and 100 IU/mL IL-2 in mouse media. After 2 days, beads were removed and cells were transduced by spinfection in retronectin-coated plates with Phoenix-ECO virus.

Mouse systemic syngeneic model

Seven- to 12-week old C57BL/6 mice (Jackson Laboratory) were inoculated intravenously (i.v.) with 1 × 106 EL4-MUC16 cells. The next day, one group of mice was treated i.v. with 2 × 106 m4H1128ζ mouse CAR T cells and the other group with 2 × 106 untransduced mouse T cells. The mice were bled once a week by venipuncture of the facial vein (cheek bleed). Serum samples were analyzed by flow cytometry. Animal experiments were conducted under MSCKCC IACUC approved protocols.

Flow cytometry

Flow cytometry was used to measure transduction efficiency using anti-idiotype conjugated with Alexa Fluor 647. For the 19BBζ CAR clone 19E3 was used. The following antibodies were used: anti-mouse AF647 (Jackson ImmunoResearch), anti-HA AF647 (2-2.2.14) (Invitrogen), and anti-rabbit Fc-specific AF647, anti-rabbit Fab-specific AF488, and anti-human Fc-specific AF647 (Jackson ImmunoResearch).

Complement-dependent and antibody-dependent cell-mediated cytotoxicity assays

CDC was performed as previously shown.51 Cells were seeded at 2.5 × 104/well in a 96-well plate. ATG was added at different concentrations in serum-free RPMI and incubated overnight. Rabbit complement (Bio-Rad) was added, and cells were further incubated for 1 h at 37°C. Cell viability was measured by Cell Titer-Glo assay measuring luminescence. For the additional ATG CDC experiment, an equal amount of ATG was added to the wells prior to incubation with the rabbit complement. ADCC was measured with chromium-release assays as previously shown.52 1 × 106 cells per group were incubated with different concentrations of ATG overnight or medium in 24-well plates. Cells were washed and labeled with 50 μCi of 51Cr and incubated for 2 h at 37°C. Cells were washed 2× with 5% fetal bovine serum (FBS)-RPMI and then plated, and PBMCs isolated from a healthy donor were added at 50:1 E:T. Wells without effectors were used as negative control. 1% SDS was used as 0% viability, and cells with only media were used for 100% viability.

Anti-HLA antibody experiments

HEK293t WT and expressing IdeS-tm, IdeS-tm(mut), and IdeS-sec were incubated with rabbit anti-HLA, washed, and then incubated with anti-rabbit Fc-specific AF647 and analyzed by flow cytometry. HEK cells were also tested with mouse anti-HLA AF647. Serum samples from kidney transplant patients were obtained from the Montefiore Medical Center. Samples were tested for binding by incubating at serial dilutions of the sera with A-375, OVCAR cell lines, and primary T cells obtained from A02+ and A02− healthy donors. Binding was analyzed with an anti-human Fc-specific AF647 and flow cytometry. Cleavage of serum IgG was evaluated by incubating 19BBζ and Shield CAR T cells with sera (diluted in Opti-MEM) overnight at 37°C and then analyzed with anti-human Fc-specific AF647 and flow cytometry.

In vivo IgG cleavage assay and persistence experiments

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory) were engrafted with 5 × 105 NIH/3T3 hCD19+ i.p. The next day mice were injected with 2 × 106 CAR T and Shield CAR T cells. Cells were allowed to expand for 1 day, and then the mice were injected with 20 μg of rabbit or human polyclonal IgG. Eight to 10 h later 0.5 mL pf PBS was used for an i.p. lavage and analyzed by WB. To test efficacy and persistence, NSG mice were engrafted with 1 × 105 NIH/3T3 hCD19+ GFP+/FLuc+ cells i.p. Mice were imaged the next day and randomized based on BLI. Mice were injected with 2 × 106 CAR T cells or 2 × 106 CAR T cells pre-incubated with 50 μg/mL ATG. 3T3 progression was monitored by BLI imaging.

CAR T cell patient sera experiments

Patient and healthy donor samples were obtained, and patient sera were de-identified, on an MSKCC IRB-approved protocol after written informed consent prior to the study. WT and 1928ζ Galv9 producer cells were lysed with RIPA buffer. Plates were coated with 10 μg/mL lysate in coating buffer overnight at 4°C, then blocked with 1% BSA-TBST solution; samples were diluted 1:100 in TBST and incubated for 1 h at room temperature (RT). An anti-human HRP-labeled antibody (Jackson ImmunoResearch) and 3,3′,5,5″- tetramethylbenzidine (TMB) solution (Thermo Fisher) were used to measure signal. The ratio of Abs at 450 nm of binding to 1928ζ over WT lysate was used to avoid non-specific binding. For detection of binding to CAR lysates of CAR T cell patient sera, WT and 1928ζ Galv9 lysates were separated by SDS-PAGE, and patient sera were used as primary antibodies at 1:500 dilution. A secondary anti-human Fc-specific conjugated with HRP was used. CDC assay was performed as described above with patient sera diluted 1:100 in serum-free medium. The binding inhibition assay was performed with HEK cells expressing the 19BBζ CAR. Sera were incubated with HEK cells at 1:100 dilution, then washed and incubated with anti-19CAR idiotype AF647 labeled antibody and analyzed by flow cytometry. Cleavage of the sera IgG was evaluated by WB as described above. Cytotoxicity of the CAR T cells in the presence of CAR-binding antibodies was tested in two ways. CAR T cells and Shield CAR T cells were incubated with rabbit anti-mouse Fab-specific antibody (Jackson ImmunoResearch) and 1.5 × 104 NIH/3T3 hCD19+ GFP+/FLuc+ cells at 1:1 E:T. Cells were incubated overnight at 37°C, and target viability was measured via luciferase-based lysis assay. Cytotoxicity of CAR T cells was also measured in the presence of patient sera 1a and 4a at different dilutions at 1:1 E:T.

IFN-γ ELISA and CD69 assays

The Invitrogen IFN-γ ELISA kit was used to measure IFN-γ secretion. Briefly, 2–3 × 105 CAR T cells and Shield CAR T cells were seeded in a 96 U-bottom plate with different concentrations of rabbit anti-mouse Fab-specific antibody (Jackson ImmunoResearch) in complete RPMI medium. Cells were incubated overnight at 37°C, and then the supernatant was analyzed by ELISA. Cells were washed and then stained for CAR and CD69 expression and analyzed by flow cytometry.

Acknowledgments

This work was supported by NIH R01 CA55349, R35 CA241894, P01 CA23766, F31 CA239511, F31 CA254331, NIH P30 CA008748, and the Tudor Fund.

Author contributions

L.P. and D.A.S. designed the study and cowrote the manuscript. L.P., C.M.B., and M.M.D. performed experiments. E.A. provided samples and expertise for anti-HLA-based experiments. E.L.S. and J.H.P. provided samples and expertise for experiments with CAR T cell patient sera. C.M.B., M.M.D., E.A., and E.L.S. edited the manuscript.

Declaration of interests

MSKCC has filed for patent protection for D.A.S., M.M.D., and L.P. for work related to this paper. D.A.S. has an equity interest in, consults for, or is on the Board of Sellas Life Sciences, Pfizer, Oncopep, Actinium, Co-Immune, Eureka, Repertoire, Sapience, Iovance, and Arvinas. J.H.P. receives funding from the Conquer Cancer Foundation of ASCO, a Leukemia and Lymphoma Society Career Development Grant, the Geoffrey Beene Cancer Foundation, a National Comprehensive Cancer Center Young Investigator Award, and an American Society of Hematology Scholar Junior Faculty Award. J.H.P. has consulted and provided an advisory role for Amgen, Novartis, Kite Pharma, Incyte, Allogene, Autolus, Intellia, Artiva, AstraZeneca, Pfizer, Takeda, and Servier. E.L.S. received funding from NCI K08 CA241400-02. E.L.S. has licensed patents and royalties with BMS, as well as consulting and receiving research funding. E.L.S. has consulted for Fate Therapeutics and Chimeric Therapeutics.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2021.06.022.

Supplemental information

Document 1. Figures S1–S7 and Table S1
mmc1.pdf (537.9KB, pdf)
Document 2. Article plus supplemental information
mmc2.pdf (1.7MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document 1. Figures S1–S7 and Table S1
mmc1.pdf (537.9KB, pdf)
Document 2. Article plus supplemental information
mmc2.pdf (1.7MB, pdf)

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