Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses

Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses Elvin Lauron, Kristen Zhang, Zhe Li, Mark O'Dair, David Qu, Adam Mealy, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6157466/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Allogeneic chimeric antigen receptor (CAR) T cells can achieve sustained clinical benefit in B cell malignancies and autoimmune diseases. Despite the many potential advantages over autologous products, allogeneic CAR T cells carry a higher risk of rejection, which may limit persistence and therapeutic efficacy. We report the design and evaluation of an optimized CD70 CAR that prevents rejection of allogeneic CAR T cells by targeting activated alloreactive lymphocytes. Co-expression of this CD70 CAR with a CD19 CAR resulted in sustained CAR T cell persistence in the presence of alloreactive lymphocytes and prolonged antitumor activity in a CD19 antigen escape model. In vivo, CD19/CD70 dual CAR T cells resisted rejection and eliminated B cells and CD70 + T cells from patients with systemic lupus erythematosus, lowering immunoglobulin production. An allogeneic CD19/CD70 dual CAR T cell therapy may therefore reduce the need for lymphodepleting conditioning regimens required prior to CAR T cell infusion. Health sciences/Molecular medicine Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma Biological sciences/Immunology/Applied immunology/Autoimmune diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Patient-derived T cells engineered to express a chimeric antigen receptor (CAR) have been shown to be well tolerated and highly effective in the treatment of hematologic malignancies and autoimmune disease (AID) 1 – 2 . Because autologous CAR T cells are derived from the patient’s T cells, there are several limitations preventing large-scale clinical application, including insufficient T cell yields, treatment delay due to length of production, cost of production, and limited manufacturing slots, among others 3 – 4 . These issues can potentially be circumvented by using T cells derived from healthy donors as the starting material for scalable manufacturing of allogeneic CAR T cells. However, T cell alloreactivity due to human leukocyte antigen (HLA) mismatching between allogeneic donor cells (herein referred to as the graft) and recipient (the host) can result in rapid rejection of the administered allogeneic CAR T cells by the patient’s immune system and/or induce graft-versus-host disease (GvHD), limiting therapeutic activity and increasing toxicity risks respectively 5 . Alloreactivity mediated by T cell receptor (TCR) recognition of non-self-HLA molecules can be avoided through several approaches. For example, ablating TCR expression on allogeneic CAR T cells using gene-editing technologies can successfully prevent GvHD 4 , 6 – 8 . Strategies to enhance the persistence and antitumor activity of allogeneic CAR T cells by either preventing or delaying host versus graft (HvG) alloreactivity are also being pursued 9 . A common approach to reduce host CD8 + T cell-mediated rejection of allogeneic CAR T cells is to generate HLA class I-deficient cells via genetic inactivation of the β2-microglobulin ( B2M) gene 10 . Although this approach is effective at reducing T cell-mediated allorejection, HLA-deficient cells become highly susceptible to ‘missing self’ killing by natural killer (NK) cells 11 – 12 . Overexpression of NK cell inhibitory ligands such as non-classical HLA molecules 13 and CD47 14 has been pursued as an attempt to endow allogeneic B2M knockout (KO) cells with resistance to both T and NK cell-mediated rejection. A limitation of this strategy, however, is that NK cells are heterogeneous and not all NK cells express the targeted inhibitory receptors. The balance between inhibition and activation of NK cells following allorecognition also needs to be considered since some NK cell inhibitory and activating receptors share the same ligand 15 . Successful inhibition of ‘missing self’ killing is likely to require extensive engineering of allogeneic B2M KO CAR T cells for the expression of multiple NK cell inhibitory ligands, which may be constrained by technical or manufacturing challenges and would likely increase the risk of genotoxicity. A promising alternative anti-rejection strategy that does not trigger ‘missing self’ killing is to selectively deplete alloreactive host lymphocytes. Since CD70 is transiently upregulated on activated lymphocytes, we reasoned that targeting CD70 on alloreactive host T and NK cells with an anti-CD70 CAR may be an effective approach for clinical application. CD70 antibody blockade has been demonstrated to prevent T cell-mediated rejection and promote allograft survival in mouse transplantation models 16 – 17 . CD70 expression in healthy tissues is largely restricted to activated lymphocytes and certain subsets of antigen presenting cells 18 , and targeting of CD70-expressing tumor cells with CAR T cells or monoclonal antibodies has so far shown to be safe and well tolerated in patients 19 – 21 . Furthermore, because CD70 is upregulated in numerous types of tumors and in T and B cells of patients with AID 22 – 23 , this approach could potentially enable improved targeting of heterogeneous tumors and elimination of autoreactive T and B cells. Here we demonstrate that a CD70 CAR, optimized for co-expression with a CD19 CAR, enhances allogeneic CAR T cell persistence through selective depletion of alloreactive CD70 + host immune cells, improves tumor control by preventing antigen escape in preclinical models of lymphoma, and enables enhanced reduction of autoantibodies in a mouse model of AID. Results CD70 CAR T cells show resistance to rejection in vitro CD70 expression is rapidly induced on T cells upon activation 24 and is a marker of alloreactive T cells in allogeneic hematopoietic stem cell transplantation recipients 25 . To test if CD70 is upregulated on alloreactive T cells in response to alloantigens in vitro , we co-cultured peripheral blood mononuclear cells (PBMCs) with allogeneic cells and assessed the expression of activation induced markers. CD70 was upregulated and maintained on alloreactive T cells at higher frequency compared to the activation induced markers 4-1BB, CD25, and CD69 (Fig. S1 ), confirming CD70 as an appealing target of activated alloreactive T cells. We therefore hypothesized that allogeneic CD70 CAR T cells could eliminate activated alloreactive T cells and thus avoid T cell-mediated rejection. To test this, allogeneic CD70 CAR T cells were generated with a previously-characterized anti-CD70 single-chain variable fragment (scFv) 26 and used in an in vitro model of CAR T cell rejection. PBMCs, representing patient ‘host’ cells, were cultured for 7 days with unedited HLA-mismatched ‘graft’ T cells derived from the same healthy donor used for allogeneic CAR T cell production. The resulting primed alloreactive host T cells were isolated to serve as a highly reactive cell population able to quickly recognize and reject allogeneic CAR T cells. Allogeneic CD70 CAR T cells were produced using lentiviral transduction as previously reported 26 , with knockout (KO) at the TCRα constant gene (TRAC) locus to prevent alloreactivity against the host T cells 27 . CD70 CAR-expressing or non-transduced (NTD) control T cells were then co-cultured with the primed host T cells (Fig. 1 A). In this model, expressing a CD70 CAR on graft T cells significantly reduced the percentage (Fig. 1 B- 1 C; p < 0.0001) and absolute numbers of CD70 + alloreactive host T cells (Fig. 1 D; p < 0.01) compared to non-transduced graft T cells. Expression of the CD70 CAR significantly enhanced the survival of graft T cells ( p < 0.0001), providing nearly complete resistance to rejection (Fig. 1 E). Conversely, the alloreactive host T cells efficiently rejected the non-transduced graft T cells. To further evaluate the anti-rejection activity of the CD70 CAR against a more diverse array of alloreactive host cells, PBMC MLR assays were performed. This assay allows the potential for both NK and T cell alloreactivity without ‘pre-priming’ the host cells (Fig. 1 F). In this model, CD70 + host T and NK cells were eliminated when co-cultured with CD70 CAR T cells (Fig. 1 G- 1 H). Only graft T cells expressing the CD70 CAR survived ( p < 0.0001) and resisted allorejection by host T and NK cells (Fig. 1 I). Notably, CD70 neg host T and NK cells were not negatively affected by the CD70 CAR (Fig. 1 J). These data suggest that the resistance of CD70 CAR T cells to allorejection is due to the ability to selectively eliminate alloreactive CD70 + host lymphocytes. Optimization of CD70 CAR expression and anti-rejection function We previously demonstrated the activity and specificity of this CD70 CAR in renal cell carcinoma and patient-derived xenograft models, tissue cross-reactivity studies, and a phase I clinical trial 21 , 28 . Importantly, allogeneic T cells expressing this CD70 CAR are fratricide-resistant due to cis-masking of CD70 and can be manufactured in large scale 28 . Given that this unique CD70 CAR was initially assessed for manufacturability, safety, specificity, and anti-tumor activity, we aimed to optimize the newfound anti-rejection activity of the CAR. To do so we constructed a series of CD70 CAR variants (Fig. 2 A), which included CARs with a 4-1BB costimulatory domain (bbz) or without (z). In addition, we tested variants with different transmembrane domains and linker lengths. Linker lengths were adjusted by incorporating a CD34 epitope (Q) and an intra-CAR CD20-based off-switch (R), which were previously shown to influence CAR activity ​ 28 . T cells expressing the Qz, z, and Qbbz CD70 CAR variants had higher levels of transduction (Fig. 2 B) and CAR expression (Fig. 2 C) compared to the original CD70 CAR tested. However, the CD70Qz and CD70z CAR T cells exhibited reduced activation and differentiation compared to cells expressing variants with a costimulatory domain (Fig. 2 D- 2 E, and Fig. S2). To evaluate anti-rejection activity, alloreactive T and PBMC MLR assays were performed with CD70Qz and CD70z CAR T cells. The original CD70 CAR and the CD70Qbbz CAR were also included for comparison. CD70z CAR T cells showed significant survival in alloreactive T cell MLRs compared to non-transduced graft T cells (Fig. 2 F; p 0.05). In PBMC MLR assays, CD70Qz and CD70z CAR T cells showed enhanced survival ( p < 0.01) and suppressed host T and NK cell expansion (Fig. 2 G; p < 0.0001). Overall, the CD70z CAR T cells provided similar levels of resistance to allorejection compared to the original CD70 CAR yet showed higher CAR expression and an improved phenotype. We therefore chose the CD70z CAR variant for further characterization. Anti-rejection and anti-CD19 activity are maintained in allogeneic CD19 CAR T cells co-expressing a CD70 CAR To exploit the benefits of the CD70z CAR in mediating survival of allogeneic CAR T cells, the combination of an anti-rejection CD70z CAR with a CD19 CAR was examined. A bicistronic construct encoding a CD19/CD70 dual CAR was targeted to the TRAC locus using site-specific integration (SSI) to model an ‘off-the-shelf’ allogeneic CAR T cell product. Integration of the CD19/CD70 dual CAR transgene into the TRAC locus resulted in a high percentage of CD19 CAR/CD70 CAR double positive cells (Fig. S3). To rule out any negative effects of the CD70z CAR on CD19 CAR function, short-term killing assays were performed at different effector to target ratios (E:T) using CD19 + Raji lymphoma cells. Since Raji cells express high levels of CD70, CD70 KO Raji cells were also tested. The CD19/CD70 CAR T cells eliminated both CD70 + and CD70 KO Raji tumor cell targets and showed similar cytotoxicity compared to CD19 CAR T cells lacking the CD70z CAR (Fig. 3 A). Furthermore, there were no significant differences in effector cytokine production from CD19/CD70 CAR T cells and CD19 CAR T cells (Fig. S4A). CD19/CD70 CAR T cell cytotoxicity against CD19 + B cells in PBMCs from healthy and systemic lupus erythematosus (SLE) donors was also evaluated. Like CD19 CAR T cells, CD19/CD70 CAR T cells rapidly eliminated CD19 + B cells from both healthy and SLE donor PBMCs (Fig. 3 B and Fig. S4B). The possibility that the CD70z CAR may affect long-term CD19 CAR activity was also investigated using a serial restimulation assay. CAR T cells were co-cultured with Raji tumor cell targets and subsequently exposed to fresh target cells every 2–3 days until cytotoxic activity was no longer detectable. CD70 KO and CD19 KO Raji tumor cell targets were used to assess CD19 CAR and CD70 CAR function in isolation, respectively. Both CD19 CAR T cells and CD19/CD70 CAR T cells persisted and maintained CD19 CAR-mediated cytotoxicity against CD70 KO Raji tumor cells for up to 9 rounds of stimulation (Fig. S4C), indicating that co-expression of the CD70z CAR does not negatively impact short-term and long-term CD19 CAR function. CD19/CD70 CAR T cells also maintained CD70 CAR-mediated cytotoxicity against CD19 KO Raji tumor cells for up to 11 rounds of stimulation. Next, alloreactive T cell MLR assays were performed to assess the anti-rejection activity of the CD70z CAR in cells co-expressing a CD19 CAR. CD19/CD70 CAR T cells significantly reduced the number of CD70 + host T cells from healthy and SLE donors and were able to persist ( p < 0.0001), whereas CD19 CAR T cells lacking the anti-rejection CD70z CAR were eliminated by the host T cells and were unable to suppress expansion of CD70 + host T cells (Fig. 3 C- 3 D and Fig. S4D). These data show that the CD70z CAR and the CD19 CAR maintain independent functions when co-expressed on allogeneic T cells. Lastly, we evaluated the cytotoxic activity of CD19/CD70 CAR T cells in an in vivo orthotopic Raji lymphoma model. In mice bearing CD70 KO Raji tumors, CD19/CD70 CAR T cells exhibited a slight reduction in efficacy with only 3 of 10 mice showing tumor growth at approximately day 20 post CAR T cell treatment (Fig. 3 E and Fig. S5A). However, CD19/CD70 CAR T cells showed significantly higher peripheral expansion compared to CD19 CAR T cells (Fig. 3 F; p < 0.05). In mice bearing the parental Raji tumors, which express both CD19 and CD70, CD19/CD70 CAR T cells exhibited improved efficacy and significantly higher peripheral expansion compared to CD19 CAR T cells (Fig. 3 E-F and Fig. S5B; p < 0.0001), suggesting that dual targeting of CD19 and CD70 enhances CAR T cell efficacy. Dual targeting of antigenically heterogeneous tumors with CD19 CAR T cells co-expressing an anti-rejection CD70z CAR limits antigen escape CD70 is expressed in multiple hematologic malignancies including LBCL 29 and is emerging as a validated target for immunotherapies 29 – 32 . Therefore, we hypothesized that the CD70z CAR could enhance cytotoxicity against a heterogeneous population of Raji cells when co-expressed with a CD19 CAR. To test this, we used a mixed lymphoma model to simulate tumor antigen heterogeneity. CD19 KO and CD70 KO Raji cells were mixed at a 1:1 ratio prior to co-culturing with CAR T cells (Fig. 4 A). In this model, CD19 CAR T cells were unable to eliminate all the Raji tumor cells and CD19 neg Raji cells expanded (Fig. 4 B), whereas CD19/CD70 CAR T cells effectively eliminated all the Raji cells ( p < 0.001). These results were also observed in an antigenically heterogeneous Toledo lymphoma model (Fig. S6A), demonstrating enhanced tumor control of CD19/CD70 CAR T cells against multiple cancer cell lines in vitro . To evaluate the CD19/CD70 CAR T cell efficacy against antigenically heterogeneous tumors in vivo , NSG mice were implanted with a 1:1 mixture of CD19 KO and CD70 KO Raji cells, treated with either CD19/CD70 or CD19 CAR T cells, and monitored for tumor growth (Fig. 4 C). CD19/CD70 CAR T cells potently eradicated heterogeneous Raji tumors ( p < 0.01) and expanded in peripheral blood ( p < 0.0001), resulting in mice that remained in remission up to the end of the study (Fig. 4 D- 4 F, and Fig. S6B). CD19 CAR T cells also expanded in peripheral blood, although significantly less than CD19/CD70 CAR T cells. In contrast to CD19/CD70 CAR T cells, CD19 CAR T cells failed to control tumor growth. Taken together, these data demonstrate the enhanced efficacy of bispecific CAR T cells and the dual-purpose function of the CD70z CAR in CD19 + CD70 + malignancies. CD19/CD70 CAR T cells eliminate autoreactive CD19 + B cells, resulting in reduced immunoglobulin levels from donors with SLE Since CD19 is a promising target for allogeneic CAR T cell therapy against B cell mediated AID 33 , we evaluated the ability of CD19/CD70 CAR T cells to eliminate primary human B cells in a hematopoietic stem cell (HSC) humanized mouse model. CD34 + HSCs were engrafted into NSG mice (Hu-CD34 mice), resulting in multi-lineage reconstitution of human immune cell populations. Hu-CD34 mice received a single dose of 6 × 10 6 CAR + T cells or 6 × 10 6 non-transduced T cells per mouse (Fig. 5 A). Engrafted host B and T cells and CAR + T cells were monitored in peripheral blood by flow cytometry analysis. Host B cells were completely depleted by day 7 post CAR T cell injection but started to recover and reconstitute beginning at day 20 post CAR T cell injection (Fig. 5 B). No significant effects were observed on the host T cell population in this model (Fig. 5 B) and CD70 expression was not observed (Fig. S7A), which is likely attributed to the lack of thymic education and proper T cell development in Hu-CD34 mice 34 . CD19/CD70 CAR T cells were detectable in peripheral blood with peak expansion occurring on day 7 post CAR T cell injection, which was followed by contraction of the CAR T cells (Fig. 5 C). CD19/CD70 CAR T cells were nearly undetectable in peripheral blood by 30 days post-CAR T cell injection. These data suggest that the administration of CD19/CD70 CAR T cells into mice reconstituted with a human immune system result in deep B cell depletion without prolonged B cell aplasia. Additionally, CD19/CD70 CAR T cells engrafted, expanded, and transiently depleted B cells in Hu-CD34 NSG mice without the need for prior lymphodepletion or pre-conditioning of the mice. Next, we sought to develop a humanized PBMC mouse model in which PBMCs with functional T cells can be engrafted rapidly from a variety of human donors to assess the efficacy of CD19/CD70 CAR T cells. NSG mice, aged 7 to 8 weeks, were pre-conditioned with 1 Gy irradiation and 2 x 10 7 PBMCs isolated from healthy human donors were adoptively transferred by intravenous (i.v.) injection (Fig. 6 A). In this model, adoptively transferred host B cells successfully engraft (Fig. 6 B-C) and rapidly transition from a predominantly naïve B cell phenotype to a more differentiated phenotype in the absence of CAR T cells (Fig. S7B-C). Furthermore, host B cells produced detectable levels of human immunoglobulin G (IgG) and M (IgM) in the serum of mice (Fig. 6 D), allowing us to determine the effects of CAR T cell-mediated B cell depletion on immunoglobulin production. Three days after adoptive transfer of PBMCs, mice were randomized and received a single dose of 1 × 10 6 CAR + T cells, 4 × 10 6 CAR + T cells, or were left untreated. CD19/CD70 CAR T cells effectively depleted host B cells at both dose levels, resulting in a significant reduction in human IgG and IgM production (Fig. 6 B-D; p < 0.0001). CD19/CD70 CAR T cells expanded in a dose-dependent manner in both the spleen and peripheral blood following target exposure (Fig. 6 E- 6 F). A minor population of CD70 low T cells was observed in the spleen of untreated control mice on day 10 but was absent in mice receiving CD19/CD70 CAR T cells (Fig. 6 G and 6 H). Lastly, we used PBMCs from SLE donors in the model described above to test the ability for CD19 and CD19/CD70 CAR T cells to eliminate autoreactive B cells and abrogate autoantibody production. NSG mice were engrafted with PBMCs isolated from SLE donors and received a single dose of 5× 10 6 CD19/CD70 CAR T cells, CD19 CAR T cells, or non-transduced control T cells (Fig. 7 A). As expected, CD19/CD70 and CD19 CAR T cells depleted host B cells and proliferated (Fig. 7 B-E). CD19/CD70 CAR T cells remained detectable in the spleen on day 14 post CAR T cell injection, whereas CD19 CAR T cells did not (Fig. 7 C and 7 D). Although a reduction in immunoglobulin production was observed in mice receiving CD19 CAR T cells, CD19 CAR T cells did not significantly reduce the levels of α-dsDNA autoantibodies, IgG, and IgM detected in the serum when compared to untreated mice on day 14 post-CAR T cell injection (Fig. 7 F). These data suggest that CD19 CAR T cells were rejected by host T cells following activation and expansion, resulting in reduced therapeutic efficacy compared to CD19/CD70 CAR T cells. For one of the two SLE host donors tested (SLE D2), two mice receiving CD19 CAR T cells developed GvHD and were euthanized before day 14 post CAR T cell injection. Nonetheless, similar results were observed on day 7 post CAR T cell injection for both SLE host donors tested. In contrast, CD19/CD70 CAR T cells significantly reduced the production of human immunoglobulins and persisted throughout the duration of the study ( p < 0.05- p < 0.0001) (Fig. 7 C-F), likely due to the reduction of host T cells in mice dosed with CD19/CD70 CAR T cells (Fig. 7 D). Taken together, these data highlight the ability of the CD70z CAR to enhance therapeutic efficacy through anti-rejection activity and the potential of allogeneic CD19/CD70 CAR T cells to treat AID driven by autoreactive B cells and T cells. Discussion Delivering the promise of allogeneic CAR T cell therapies may require strategies to improve intrinsic CAR T cell function, including cell persistence, to achieve the desired efficacy outcomes without the risk of increased toxicity that may result from increased lymphodepletion intensity. This is particularly true in AID, where combination chemotherapy is rarely used. Our results demonstrate a simple and effective approach for boosting allogeneic CAR T cell activity and persistence by targeting both B cells and activated alloreactive T cell subsets marked by expression of CD70. We show that allogeneic CD19 CAR T cells expressing an anti-rejection CD70z CAR maintain anti-CD19 function and selectively eliminate alloreactive cells to evade rejection, while sparing CD70 neg host lymphocytes. Furthermore, combination of this CD70z CAR with a CD19 CAR improves therapeutic efficacy in tumor models of antigen loss and in an AID model in which antibody titers are suppressed to a greater degree by the dual CAR T cells as compared to CD19-only CAR T cells. An additional critical factor that may limit clinical responses to allogeneic CAR T cells is target antigen loss or downregulation, which is emerging as a major mechanism of resistance in malignancies treated with CD19 CAR T cells 35 . Given the modular nature of this platform, the enhanced tumor control and potential to prevent antigen escape conferred by the CD70z CAR may well extend to other tumor types characterized by stable or induced expression of CD70. CD70 is expressed in various hematologic malignancies including 71% percent of diffuse large B-cell lymphomas 29 , 36 , and is an attractive immunotherapeutic target due to its limited expression in normal tissues. CD70 is also expressed on stimulated plasmacytoid dendritic cells (pDCs), which play an important role in the pathogenesis of AID, including SLE. pDCs can be stimulated by DNA-containing immune complexes, a hallmark of SLE, to produce IFN-α and IL-6. pDC-derived IFN-α and IL-6 subsequently drive autoreactive B cell proliferation and the accumulation of autoantibody producing cells 37 . Stimulated pDCs can also induce antibody production by direct contact with B cells through a CD70-CD27 interaction 38 – 39 . The CD70z CAR could therefore provide additional benefit to AID patients through elimination of CD70 + pDCs. CD70 may also be transiently expressed on virus-specific T cells, and it is therefore possible that endogenous T cell responses may be dampened in patients receiving CD70 CAR T cells. However, data to date remains equivocal on this point. For example, human cytomegalovirus-specific T cells have been reported to have low levels of CD70 expression following cognate antigen stimulation and expansion 40 . Importantly, allogeneic T cells expressing a chimeric receptor specific for the activation marker CD70 (Fig. S8), 4-1BB or OX40 did not completely reduce the cytotoxic activity of virus-specific T cells 41 – 42 . These findings suggest that targeting activation-induced markers on T cells may not necessarily abrogate productive antiviral T cell responses. Some clinical studies evaluating CD70 CAR T cells and anti-CD70 antibodies have reported overall adequate safety profiles with no increased risk of infection 19 – 21 , 43 . Importantly, treatment with CAR T cells conveys a risk of infection 44 , including from viral reactivation, and adequate viral prophylaxis and patient monitoring will be required. While the main purpose of the anti-rejection CD70z CAR approach is to improve persistence of allogeneic CAR T cells by eliminating alloreactive host lymphocytes, engagement of the CD70z CAR with its target on alloreactive or autoreactive host immune cells may also stimulate CAR T cell activation and expansion. Studies have shown that providing CAR T cells with immediate target stimulation by locoregional delivery can enhance CAR T cell activity, avoiding anergy prior to encountering target 45 – 46 . Therefore, the presence of a CD70z CAR would not only preserve allogeneic CAR T cells but may contribute to expanding them through target-mediated activation, creating a primed population of cells capable of seeking out and destroying cancer cells or autoreactive cells. Preliminary data from clinical studies in renal cell carcinoma patients indicate that allogeneic CD70 CAR T cells undergo peripheral expansion at levels not previously seen with other allogeneic CAR T cell therapies, presumably due to target mediated expansion and the avoidance of allorejection 21 , providing an early clinical validation of the findings described in this study. Materials and methods Construction of lentiviral and adeno-associated virus vectors encoding anti-CD70 and anti-CD19 CARs The pLVX lentiviral vector (LVV) was employed for cloning and expression of the CD70 CAR or CD70 CAR variants. An adeno-associated virus (AAV) plasmid containing two inverted terminal repeats was modified to incorporate two homology arms, each 300–400 bp in length corresponding to the genomic integration site at the TRAC locus, which flank the nuclease target site. The AAV cDNA sequences corresponding to the CD19 CAR or CD19/CD70 dual CAR were cloned downstream of the human Pgk-1 promoter and are followed by the bovine growth hormone polyA signal. All CD70 CAR constructs contained an scFv specific for CD70 28 followed by an extracellular spacer or “hinge” (H) and transmembrane (TM) domain derived from either human CD8α or human CD28, and the intracellular domains of 4-1BB (bb) and CD3ζ (z), or the CD3ζ domain only for constructs without a costimulatory domain. Some variants contained short mimotopes derived from either CD20 (R mimotope) and/or CD34 (Q mimotope) 28 , 47 that were inserted in the extracellular domain of the CD70 CAR to increase the length of the spacer. The CD19 CAR contained the FMC63 scFv, CD8α hinge and transmembrane domain, and cytoplasmic domains derived from 4-1BB and CD3ζ. Cell and viruses PBMCs were sourced from Stemcell Technologies and T cells were isolated using the human pan T Cell isolation kit (Miltenyi Biotec). To generate Raji and Toledo knockout cell lines, Cas9 ribonucleoproteins (RNPs) were prepared by incubating 60 ng of Cas9 (IDT) with 35 pmol sgRNA (Synthego) in Neon electroporation buffer (Invitrogen). Luciferase-GFP-expressing Raji cells were mixed with Cas9 RNPs and electroporated with a Neon transfection kit and device (Invitrogen). Transfected cells were expanded in RPMI 1640 medium (Invitrogen) containing 10% FBS (GE Healthcare) and KO cells were isolated by FACS. Cell sorting was performed on a BD FACS Aria Fusion sorter. To produce LVV, HEK293T cells were transfected with lentiviral transfer vectors, psPAX2, and pMD.2G (École Polytechnique Fédérale de Lausanne, Switzerland) using Lipofectamine 2000 (Thermo Fisher Scientific). 24 hours after transfection, the medium was replaced with X-Vivo 15 medium (Lonza), supplemented with 10% FBS. Supernatant was then harvested and passed through a 0.45 µM filter 24 hours later. All AAV vectors used in this study were produced using a triple transfection with AAV-MAX system according to the manufacturer's protocol (Thermo Fisher). Briefly, the pRC6, pHelper, and transgene plasmids were mixed at the molar ratio of 1:1:1 for gene transfection. After 72 hours, cells were lysed with the AAV-MAX lysis buffer for 2 hours and cell debris were removed by centrifugation and filtration with 0.2 µM filter (Nalgene). The clarified lysate was loaded on a HiTrap AVB column (Cytiva) and column-bounded AAV particles were eluted with 0.1 M NaOAc, 0.5 M NaCl, pH 3.0. The elution solution was immediately neutralized with 1M Tris-HCl, pH 8.8, further concentrated, and exchanged to PBS. The titers of the concentrated AAVs were determined by qPCR (Takara). CAR T cell production The CD70 CAR T cells used in Figs. 1 and 2 were produced with LVV as previously described 28 . To generate CAR T cells via site-specific integration, human T cells were first activated with T cell TransAct reagent (Miltenyi) as recommended by the manufacturer protocol and cultured in X-Vivo15 medium supplemented with 5% human AB serum (Gemini Bio-Products) and 100 IU/mL IL-2 (Miltenyi Biotech). Two days post-activation, cells were washed in PBS and electroporated using the AMAXA 4D nucleofector electroporation apparatus with 15 µM of RNP, TRAC crRNA combined with ABR-001 nuclease, for every 3–12 x 10 6 cells. ABR-001 gene-editing technology was developed by Arbor Biotechnologies, Inc 48 . Following electroporation, T cells were transduced with AAV at an MOI of 15,000 and incubated at 30°C overnight. Cells were then returned to 37°C and were supplemented with IL-2 every 2–3 days. At day 14 post-activation, TCRα/β depletion was performed using EasySep™ human TCRα/β depletion kit (STEMCELL Technologies) as instructed by the manufacturer’s protocol. T cells were cryopreserved in 90% FBS/10% DMSO or CS5 freezing media using rate-controlled freezing chambers and stored in liquid nitrogen vapor phase. Cytotoxicity and cytokine-release assay For short-term cytotoxicity assays, CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells or with primary target cells at defined E:T ratios for 24 hours in RPMI 1640 medium supplemented with 10% FBS. Target cell killing was determined using Bright-glo reagent (Promega) or flow cytometric analysis. For cytokine-release assays, CAR T cells were co-cultured with Raji target cells at an E:T ratio of 1:4. At 24 hours, cell culture supernatant was assayed for cytokines using the MSD U-PLEX platform (Meso Scale Diagnostics). For long-term cytotoxicity assays, CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells at an initial E:T ratio of 8:1 for 48 hours in RPMI 1640 medium supplemented with 10% FBS. Half of each well was then removed to measure target cell killing as determined using Bright-glo reagent (Promega). The remaining cells of each well were passaged on to fresh Raji cells and this process was repeated every 2–3 days until cytotoxic activity was no longer detected. The percentage killing was determined relative to target-only control wells. Flow cytometry, staining, and antibodies Mouse peripheral blood, bone marrow, and spleen samples were lysed using ACK lysing buffer (Lonza). Samples for flow cytometric analyses were stained on ice with Fixable Viability Ghost Dye (Tanbo) before cell surface staining. An anti-idiotype antibody against the CD70 scFv was raised in Balb/c mice and was purified from hybridoma cell culture supernatants. Antibodies were labeled with R-PE (Prozyme) using sulfhydryl-maleimide reaction chemistry. The following antibodies were purchased from BD Biosciences, eBioscience, BioLegend, ACROBiosystems, or ThermoFisher: CD3 (UCHT1), CD56 (5.1H11), CD19 (SJ25C1), CD20 (2H7), CD70 (113 − 16), PD-1 (EH12.2H7), CD137 (4B4-1), CD25 (M-A251), CD69 (FN50), TCRαβ (IP26), CD8 (SK1), CD4 (SK3), CD62L (BDB565219), CD45RO (UCHL1), HLA-A2 (BB7.2), HLA-ABC (W6/32), CD45 (HI30), FMC63 (Y45), CD27 (O323), CD38 (HB-7), CD138 (MI15), IgD (IA6-2) and CD45.1 (A20). Cells were fixed in BD Cytofix fixation buffer (BD Biosciences) prior to analysis in a CytoFLEX LX flow cytometer. CAR T cells were enumerated using 123count eBeads counting beads™ (Invitrogen). Mixed lymphocyte reaction (MLR) assays For PBMC MLR assays, host PBMCs were co-cultured with TRAC KO graft T cells at a ratio of 10:1 in 200 µL RPMI medium supplemented with 10% FBS and 20 U/mL of recombinant human IL-2 in round-bottomed 96-well plates. For alloreactive T cell MLR assays, unedited graft donor T cells were irradiated at 30 Gy and co-cultured with host PBMCs at a ratio of 1:1 in RPMI supplemented with 10% FBS and 20 U/mL of recombinant human IL-2, IL-7, and IL-15. On day 4 of the co-culture, 50% of the cell culture medium was replaced with fresh RPMI supplemented with 10% FBS. On day 7, primed T cells were isolated using EasySep™ human T cell isolation kit (STEMCELL Technologies) as instructed by the manufacturer’s protocol. Primed alloreactive T cells were then co-cultured with TRAC KO graft T cells at a 1:1 ratio in 200 µL RPMI medium supplemented with 10% FBS and 20 U/mL of recombinant human IL-2 in round-bottomed 96-well plates. If MLR co-cultures exceeded 4 days, 50% of the medium was replaced on day 4. Medium was then replaced every 2–3 days after. Cells were analyzed by flow cytometry at the indicated time points. Graft CAR T and host cells were differentiated based on expression of HLA-A2 (Fig. S9). Antigen escape assays To model antigenic tumor heterogeneity, CD19 KO and CD70 KO target cells were first mixed at 1:1 ratio and then co-cultured with CAR T cells at an E:T ratio of 1:1 or 1:4. Cells were cultured in RPMI medium supplemented with 10% FBS in 96-well or 24-well G-Rex plates (Wilson Wolf). On days 2, 50% of the cell culture medium was replaced with fresh RPMI supplemented with 10% FBS. At the indicated time points, 100 µL samples of the heterogeneous cell mixtures were removed and analyzed by flow cytometry. Target cells were enumerated using CountBright™ absolute counting beads (Invitrogen). Virus-specific T cell tri-culture Virus-specific T cells (VSTs) and autologous B-lymphoblastoid cell lines (BLCLs) were purchased from Charles River Laboratories. CD19/CD70 double knockout (dKO) BLCLs, herein referred to as antigen presenting cells (APCs), were generated as described above. APCs were incubated with 1 µg/mL of pp65 peptides (Charles River Laboratories) for 1 h at 37°C. Peptide-pulsed APCs were washed extensively prior to tri-culture with VSTs and CAR T cells. Surviving cells in tri-cultures were quantified by flow cytometry at 48 h. Orthotopic mouse model All procedures performed on animals were reviewed and approved by an Institutional Animal Care and Use Committee and were conducted in accordance with established guidelines. Female NSG mice 8 to 12 weeks of age were obtained from The Jackson Laboratory. Parental, CD70 KO , or a 1:1 mixture of CD19 KO and CD70 KO luciferase-GFP-expressing Raji cells were prepared, and 1 x 10 5 cells were administered to mice by i.v. injection. Tumor burden was measured using an IVIS Spectrum instrument (PerkinElmer) twice weekly. Mice were randomized based on total body bioluminescence 3 days after tumor cell injection. CAR T cells were injected immediately after thawing, 4 days after tumor cell injection. Total T cells administered were kept constant in all groups by adding non-transduced T cells from the same donor. Peripheral blood was collected into EDTA-coated tubes using submandibular or retro-orbital bleeding and 20 µL was used for flow cytometric analyses. Mice were euthanized when disease model-specific endpoints were exhibited, including hind-leg paralysis, ruffled fur, or 20% reduction in body weight. Humanized mouse models Hu-CD34 mice, aged 16 to 17 weeks, were obtained from The Jackson Laboratory. Hu-CD34 mice received a single dose of CAR T cells or non-transduced control T cells via i.v. injection. Human B and T cells and CAR + cells were monitored in peripheral blood by flow cytometry analysis weekly. For the PBMC mouse model, NSG mice aged 7 to 8 weeks were pre-conditioned with 1 Gy irradiation and 10–20 x 10 6 human PBMCs isolated from healthy donors or SLE donors were adoptively transferred via i.v. injection. Three or seven days after PBMC transfer, mice were randomized and received a single dose of CAR T cells via i.v. injection or were left untreated. Cells from spleen and blood were analyzed by flow cytometry at indicated time points post treatment. Engrafted B and T cells and total CAR + cells were assessed and quantified using 123count eBeads. Human α-dsDNA antibody, IgG, and IgM levels in the plasma were measured by enzyme-linked immunosorbent assay. Statistical analysis Data are shown as mean ± SEM. The data were analyzed with an unpaired Student t test or one-way and two-way ANOVA followed by Dunnett’s or Tukey’s multiple comparisons test using Prism GraphPad software. Asterisks indicate statistical significance and p values are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Declarations Acknowledgments We thank Janette Sutton and Jerick Sanchez for support with animal studies, and Yanqi Chang for the helpful suggestions and for providing support with AAV reagents. Author contributions EJL and CS designed the studies and interpreted the data. KZ, ZL, MKO, AM, DN, HC, DH, DQ, and SG performed experiments. EJL, ZJR, and CS wrote the manuscript and are responsible for the integrity of the work as a whole. Competing Interests All authors are current employees of Allogene Therapeutics, Inc. Materials and Correspondence The data that support the findings of this study are available from the corresponding author upon reasonable request. References Sadelain, M. CD19 CAR T Cells. Cell 171 , 1471 (2017). Georg, S. CAR T-cell therapy in autoimmune diseases. Lancet (2023) doi:10.1016/S0140-6736(23)01126-1. Zhao, J., Lin, Q., Song, Y. & Liu, D. Universal CARs, universal T cells, and universal CAR T cells. J Hematol Oncol 11 , (2018). Hoffmann, J.-M. et al. Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients. Front Immunol 8 , (2018). Depil, S., Duchateau, P., Grupp, S. A., Mufti, G. & Poirot, L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 19 , 185–199 (2020). Voravit, R. et al. Phase III Study Comparing Methotrexate and Tacrolimus (Prograf, FK506) With Methotrexate and Cyclosporine for Graft-Versus-Host Disease Prophylaxis After HLA-Identical Sibling Bone Marrow Transplantation. Blood 92 , 2303–2314 (1998). Torikai, H. et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood 119 , 5697–5705 (2012). A., K. N. et al. Clonable T Lymphocytes in T Cell-Depleted Bone Marrow Transplants Correlate With Development of Graft-v-Host Disease. Blood 68 , 770–773 (1986). Li, W. et al. Simultaneous editing of TCR, HLA-I/II and HLA-E resulted in enhanced universal CAR-T resistance to allo-rejection. Front Immunol 13 , (2022). Wang, D., Quan, Y., Yan, Q., Morales, J. E. & Wetsel, R. A. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells. Stem Cells Transl Med 4 , 1234–1245 (2015). Kärre, K., Ljunggren, H. G., Piontek, G. & Kiessling, R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319 , 675–8 (1986). Hans-Gustaf, L. & Klas, K. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today 11 , 237–244 (1990). Jo, S. et al. Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Nat Commun 13 , (2022). Deuse, T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 37 , 252–258 (2019). Siemaszko, J., Marzec-Przyszlak, A. & Bogunia-Kubik, K. NKG2D Natural Killer Cell Receptor-A Short Description and Potential Clinical Applications. Cells 10 , 1420. Yamada, A. et al. CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo. J Immunol 174 , 1357–1364 (2005). Zhao, J. et al. Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells. Front Immunol 11 , (2021). Borst, J., Hendriks, J. & Xiao, Y. CD27 and CD70 in T cell and B cell activation. Curr Opin Immunol 17 , 275–281 (2005). Tu, S. et al. CD19 and CD70 Dual-Target Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Relapsed and Refractory Primary Central Nervous System Diffuse Large B-Cell Lymphoma. Front Oncol 9 , (2019). Riether, C. et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat Med 26 , 1459–1467 (2020). Srour, S. et al. Abstract CT011: A phase 1 multicenter study (TRAVERSE) evaluating the safety and efficacy of ALLO-316 following conditioning regimen in pts with advanced or metastatic clear cell renal cell carcinoma (ccRCC). Cancer Res 83 , CT011–CT011 (2023). Han, B. K., Olsen, N. J. & Bottaro, A. The CD27–CD70 pathway and pathogenesis of autoimmune disease. Semin Arthritis Rheum 45 , 496–501 (2016). Flieswasser, T. et al. The CD70-CD27 axis in oncology: the new kids on the block. J Exp Clin Cancer Res 41 , (2022). García, P. et al. Signalling via CD70, a member of the TNF family, regulates T cell functions. J Leukoc Biol Suppl 76 , 263–270 (2004). Verma, K. et al. CD70 identifies alloreactive T cells and represents a potential target for prevention and treatment of acute GVHD. Blood Adv 8 , 4900–4912 (2024). Panowski, S. et al. Investigation of Allocar TTM Targeting CD70 As a Potential Therapy for an Array of Hematological Malignancies. Blood 134 , 5157–5157 (2019). Poirot, L. Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies. Cancer Res 75 , 3853–3864 (2015). Panowski, S. H. Preclinical Development and Evaluation of Allogeneic CAR T Cells Targeting CD70 for the Treatment of Renal Cell Carcinoma. Cancer Res 82 , 2610–2624 (2022). Deng, W. et al. CD70‐targeting CAR‐T cells have potential activity against CD19‐negative B‐cell Lymphoma. Cancer Commun (Lond) 41 , 925–929 (2021). J., J. et al. CD70: An emerging target in cancer immunotherapy. Pharmacol Ther 155 , 1–10 (2015). Jacobs, J. et al. Unlocking the potential of CD70 as a novel immunotherapeutic target for non-small cell lung cancer. Oncotarget 6 , 13462–13475 (2015). Sauer, T. et al. CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity. Blood 138 , 318–330 (2021). Wang, X. et al. Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis. Cell (2024) doi:10.1016/j.cell.2024.06.027. Watanabe, Y. et al. The analysis of the functions of human B and T cells in humanized NOD/shi-scid/γcnull (NOG) mice (hu-HSC NOG mice). Int Immunol 21 , 843–858 (2009). Spiegel, J. Y. et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nat Med 27 , 1419–1431 (2021). Flieswasser, T. et al. Screening a Broad Range of Solid and Haematological Tumour Types for CD70 Expression Using a Uniform IHC Methodology as Potential Patient Stratification Method. Cancers 11 , 1611 (2019). Davison, L. M. & Jorgensen, T. N. New Treatments for Systemic Lupus Erythematosus on the Horizon: Targeting Plasmacytoid Dendritic Cells to Inhibit Cytokine Production. J Clin Cell Immunol 08 , (2017). Zhang, H. et al. A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes. Proc Natl Acad Sci U S A 114 , 1988–1993 (2017). Shaw, J., Wang, Y.-H., Ito, T., Arima, K. & Liu, Y.-J. Plasmacytoid dendritic cells regulate B-cell growth and differentiation via CD70. Blood 115 , 3051–3057 (2010). Lu, J. et al. Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults. Immun Ageing 19 , (2022). Mo, F. et al. Engineered off-the-shelf therapeutic T cells resist host immune rejection. Nat Biotechnol 39 , 56–63 (2021). Mo, F. et al. Engineering T cells to suppress acute GVHD and leukemia relapse after allogeneic hematopoietic stem cell transplantation. Blood 141 , 1194–1208 (2023). Iyer, S. P. et al. S262: THE COBALT-LYM STUDY OF CTX130: A PHASE 1 DOSE ESCALATION STUDY OF CD70-TARGETED ALLOGENEIC CRISPR-CAS9–ENGINEERED CAR T CELLS IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) T-CELL MALIGNANCIES. in HemaSphere vol. 6 163–164 (2022). Kampouri, E. et al. Infections after chimeric antigen receptor (CAR)‐T‐cell therapy for hematologic malignancies. Transpl Infect Dis 25 , (2023). Adusumilli, P. S. A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease, in Combination with the Anti–PD-1 Agent Pembrolizumab. Cancer Discov 11 , 2748–2763 (2021). Donovan, L. K. et al. Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma. Nat Med 26 , 720–731 (2020). Valton, J. et al. A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies. Sci Rep 8 , (2018). McGaw, C. et al. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. Nat Commun 13 , (2022). Additional Declarations There is NO Competing Interest. Supplementary Files ZhangetalSupplementalMaterials2025NC.docx Supplementary Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6157466","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430219574,"identity":"cab509bc-5555-412c-b1b3-bcd695193481","order_by":0,"name":"Elvin 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rejection by alloreactive T and NK cells. \u003c/strong\u003e\u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of alloreactive T cell MLR assay. (\u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry plots of CD70 expression on host CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells at 48 hours. (\u003cstrong\u003eC\u003c/strong\u003e) Percentage and (\u003cstrong\u003eD\u003c/strong\u003e) absolute numbers of CD70\u003csup\u003e+\u003c/sup\u003e host T cells at 48 hours. (\u003cstrong\u003eE\u003c/strong\u003e) Percent survival and absolute numbers of graft CAR T cells compared to non-transduced control T cells. Symbols represent unique graft-host donor pairs (n = 8). Representative of two independent experiments. (\u003cstrong\u003eF\u003c/strong\u003e) Schematic of PBMC MLR assay. (\u003cstrong\u003eG\u003c/strong\u003e) Representative flow cytometry plots of CD70 expression on host T and NK cells at day 9. (\u003cstrong\u003eH\u003c/strong\u003e) Absolute numbers of CD70\u003csup\u003e+\u003c/sup\u003e host T and NK cells are shown. (\u003cstrong\u003eI\u003c/strong\u003e) Absolute numbers of graft CAR T cells are shown. (\u003cstrong\u003eJ\u003c/strong\u003e) Percentage of CD70\u003csup\u003eneg\u003c/sup\u003e host CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and NK cells is shown. Symbols represent unique graft-host donor pairs (n = 6).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/55e03b06ef80c370dab18a99.png"},{"id":78888346,"identity":"4418267d-78e4-4a23-9e30-89b16fedbdcd","added_by":"auto","created_at":"2025-03-20 10:00:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":937647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and functional screening of T cells expressing anti-rejection CD70 CAR variants. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of CD70 CAR variant architecture. (\u003cstrong\u003eB\u003c/strong\u003e) Frequencies of CD70 CAR-expressing cells (day 14), (\u003cstrong\u003eC\u003c/strong\u003e) CD70 CAR expression level (day 14), (\u003cstrong\u003eD\u003c/strong\u003e) expression of activation markers CD25 and 4-1BB (day 9), and (\u003cstrong\u003eE\u003c/strong\u003e) differentiation status (day 14) as determined by flow cytometry. Circles and triangles represent first generation (without a 4-1BB signaling domain) and second generation (with a 4-1BB signaling domain) variants, respectively. All results are shown as mean ± SEM. Symbols represent unique donors (n = 2). (\u003cstrong\u003eF\u003c/strong\u003e) Percent survival and absolute numbers of graft CAR T cells in alloreactive T cell MLRs. (\u003cstrong\u003eG\u003c/strong\u003e) Graft CAR T cell, host T cell, and host NK cell absolute numbers on day 9 of PBMC MLR assays are shown. Data are the combined results from 5-6 unique graft-host donor pairs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/03b7d70be85273e0f441e0f0.png"},{"id":78887048,"identity":"021ef8ae-605b-41db-9344-e697f1b4a212","added_by":"auto","created_at":"2025-03-20 09:52:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":625257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD70z CAR and CD19 CAR maintain independent functions when co-expressed on allogeneic T cells.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Percent killing of Raji cells in a 24-hour killing assay with allogeneic TRAC\u003csup\u003eKO\u003c/sup\u003e CAR T cells. Data are shown as mean ± SEM of two technical replicates. (\u003cstrong\u003eB\u003c/strong\u003e) Flow cytometry plots showing T and B cells from healthy or SLE donors after a 24-hour co-culture with CAR T cells. \u003cstrong\u003e(C)\u003c/strong\u003e Number of host T cells from healthy donors and CAR T cells over time in alloreactive T cell MLR assays. (\u003cstrong\u003eD\u003c/strong\u003e) Number of host T cells from SLE donors and CAR T cells over time in alloreactive T cell MLR assays. MLR data are representative of 4 unique graft-host donor pairs tested. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of tumor growth bioluminescence (n = 10) in an \u003cem\u003ein vivo\u003c/em\u003e orthotopic Raji lymphoma model. (\u003cstrong\u003eF\u003c/strong\u003e) CAR T cell expansion in peripheral blood of mice (n = 10).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/69cfc6f67036cdcbb4120177.png"},{"id":78887049,"identity":"72c6b047-c899-41fb-a102-d85f819d5b01","added_by":"auto","created_at":"2025-03-20 09:52:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":667901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-expression of CD19 CAR and CD70z CAR limits CD19 antigen escape in Raji lymphoma cells. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of antigen escape assay. (\u003cstrong\u003eB\u003c/strong\u003e) Absolute numbers of mixed Raji cells over time from antigen escape assays. (\u003cstrong\u003eC\u003c/strong\u003e) Schematic of \u003cem\u003ein vivo\u003c/em\u003e antigen escape model. (\u003cstrong\u003eD\u003c/strong\u003e) Representative bioluminescence images of tumor burden in control and treated mice (n=10). (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of bioluminescence shown in panel D (n = 10). (\u003cstrong\u003eF\u003c/strong\u003e) CAR T cell expansion in peripheral blood (n = 10).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/48f3cdca6de460f3ceec794d.png"},{"id":78887071,"identity":"429c4fcc-2dee-4703-b076-57a63fe2d2d3","added_by":"auto","created_at":"2025-03-20 09:52:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":433829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAllogeneic CD19/CD70 CAR T cells transiently deplete B cells in a humanized CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e NSG mouse model.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic of Hu-CD34 mouse model. (\u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry plots of host T and B cells from peripheral blood over time. Quantification of host B cells (top) and host T cells (bottom) in peripheral blood over time (n = 3). (\u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry plots of graft CAR T cells on day 7 and quantification of graft CAR T cells in peripheral blood over time (n = 3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/084534680a98321534365dbe.png"},{"id":78887061,"identity":"43a5294f-55f6-4e87-857b-7e012a22ef16","added_by":"auto","created_at":"2025-03-20 09:52:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":510876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAllogeneic CD19/CD70 CAR T cells deplete human B cells and CD70\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, resulting in reduced immunoglobulin production.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic of PBMC humanized mouse model. (\u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry plots of host B and T cells in the spleen on day 7. (\u003cstrong\u003eC\u003c/strong\u003e) Absolute numbers of CD19\u003csup\u003e+\u003c/sup\u003e host B cells in the spleen. (\u003cstrong\u003eD\u003c/strong\u003e) Measurement of human IgG and IgM in the serum. (\u003cstrong\u003eE\u003c/strong\u003e) Representative flow cytometry plots of CAR T cells in the spleen on day 7. (\u003cstrong\u003eF\u003c/strong\u003e) Absolute numbers of CAR T cells in the spleen and peripheral blood. (\u003cstrong\u003eG\u003c/strong\u003e) Representative flow cytometry plots of CD70 expression on host T cells in the spleen on day 7. (\u003cstrong\u003eH\u003c/strong\u003e) Absolute numbers of CD70\u003csup\u003e+\u003c/sup\u003e host T cells in the spleen. Symbols represent individual mice (n = 4).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/925af85bbd4edd3f1740421d.png"},{"id":78887063,"identity":"4c9bbb53-eabe-483f-8bac-3b31f93c717a","added_by":"auto","created_at":"2025-03-20 09:52:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":708152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAllogeneic CD19/CD70 CAR T cells deplete autoreactive B cells, reduce autoantibody production, and resist rejection \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic of PBMC humanized mouse model. (\u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry plots of host B and T cells in the spleen on day 14. (\u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry plots of CAR T cells in the spleen on day 14. (\u003cstrong\u003eD\u003c/strong\u003e) Absolute numbers of CD19\u003csup\u003e+\u003c/sup\u003e host B cells, CAR\u003csup\u003e+\u003c/sup\u003e T cells, and host T cells in the spleen. (\u003cstrong\u003eE\u003c/strong\u003e) CAR T cell expansion in peripheral blood. (\u003cstrong\u003eF\u003c/strong\u003e) Measurement of human α-dsDNA antibodies, IgG, and IgM in the serum. Symbols represent individual mice (n = 3).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/bd46d93d4d42f3e962356a18.png"},{"id":78889123,"identity":"78b90511-83c4-4bc3-b8ef-8fea6baf215a","added_by":"auto","created_at":"2025-03-20 10:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5838019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/5136e9a9-96d3-4b7c-8d6a-e6b5a58482fc.pdf"},{"id":78887054,"identity":"c03675aa-d7f6-44cf-ad06-eb8339ec5ecb","added_by":"auto","created_at":"2025-03-20 09:52:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2414567,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"ZhangetalSupplementalMaterials2025NC.docx","url":"https://assets-eu.researchsquare.com/files/rs-6157466/v1/433b5a30a8b99ffe4b59fe91.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePatient-derived T cells engineered to express a chimeric antigen receptor (CAR) have been shown to be well tolerated and highly effective in the treatment of hematologic malignancies and autoimmune disease (AID)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Because autologous CAR T cells are derived from the patient\u0026rsquo;s T cells, there are several limitations preventing large-scale clinical application, including insufficient T cell yields, treatment delay due to length of production, cost of production, and limited manufacturing slots, among others\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These issues can potentially be circumvented by using T cells derived from healthy donors as the starting material for scalable manufacturing of allogeneic CAR T cells. However, T cell alloreactivity due to human leukocyte antigen (HLA) mismatching between allogeneic donor cells (herein referred to as the graft) and recipient (the host) can result in rapid rejection of the administered allogeneic CAR T cells by the patient\u0026rsquo;s immune system and/or induce graft-versus-host disease (GvHD), limiting therapeutic activity and increasing toxicity risks respectively\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlloreactivity mediated by T cell receptor (TCR) recognition of non-self-HLA molecules can be avoided through several approaches. For example, ablating TCR expression on allogeneic CAR T cells using gene-editing technologies can successfully prevent GvHD\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Strategies to enhance the persistence and antitumor activity of allogeneic CAR T cells by either preventing or delaying host versus graft (HvG) alloreactivity are also being pursued\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. A common approach to reduce host CD8\u003csup\u003e+\u003c/sup\u003e T cell-mediated rejection of allogeneic CAR T cells is to generate HLA class I-deficient cells via genetic inactivation of the β2-microglobulin (\u003cem\u003eB2M)\u003c/em\u003e gene\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although this approach is effective at reducing T cell-mediated allorejection, HLA-deficient cells become highly susceptible to \u0026lsquo;missing self\u0026rsquo; killing by natural killer (NK) cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Overexpression of NK cell inhibitory ligands such as non-classical HLA molecules\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and CD47\u003csup\u003e14\u003c/sup\u003e has been pursued as an attempt to endow allogeneic \u003cem\u003eB2M\u003c/em\u003e knockout (KO) cells with resistance to both T and NK cell-mediated rejection. A limitation of this strategy, however, is that NK cells are heterogeneous and not all NK cells express the targeted inhibitory receptors. The balance between inhibition and activation of NK cells following allorecognition also needs to be considered since some NK cell inhibitory and activating receptors share the same ligand\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Successful inhibition of \u0026lsquo;missing self\u0026rsquo; killing is likely to require extensive engineering of allogeneic \u003cem\u003eB2M\u003c/em\u003e KO CAR T cells for the expression of multiple NK cell inhibitory ligands, which may be constrained by technical or manufacturing challenges and would likely increase the risk of genotoxicity.\u003c/p\u003e \u003cp\u003eA promising alternative anti-rejection strategy that does not trigger \u0026lsquo;missing self\u0026rsquo; killing is to selectively deplete alloreactive host lymphocytes. Since CD70 is transiently upregulated on activated lymphocytes, we reasoned that targeting CD70 on alloreactive host T and NK cells with an anti-CD70 CAR may be an effective approach for clinical application. CD70 antibody blockade has been demonstrated to prevent T cell-mediated rejection and promote allograft survival in mouse transplantation models\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. CD70 expression in healthy tissues is largely restricted to activated lymphocytes and certain subsets of antigen presenting cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and targeting of CD70-expressing tumor cells with CAR T cells or monoclonal antibodies has so far shown to be safe and well tolerated in patients\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, because CD70 is upregulated in numerous types of tumors and in T and B cells of patients with AID\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, this approach could potentially enable improved targeting of heterogeneous tumors and elimination of autoreactive T and B cells. Here we demonstrate that a CD70 CAR, optimized for co-expression with a CD19 CAR, enhances allogeneic CAR T cell persistence through selective depletion of alloreactive CD70\u003csup\u003e+\u003c/sup\u003e host immune cells, improves tumor control by preventing antigen escape in preclinical models of lymphoma, and enables enhanced reduction of autoantibodies in a mouse model of AID.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCD70 CAR T cells show resistance to rejection\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCD70 expression is rapidly induced on T cells upon activation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and is a marker of alloreactive T cells in allogeneic hematopoietic stem cell transplantation recipients\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To test if CD70 is upregulated on alloreactive T cells in response to alloantigens \u003cem\u003ein vitro\u003c/em\u003e, we co-cultured peripheral blood mononuclear cells (PBMCs) with allogeneic cells and assessed the expression of activation induced markers. CD70 was upregulated and maintained on alloreactive T cells at higher frequency compared to the activation induced markers 4-1BB, CD25, and CD69 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), confirming CD70 as an appealing target of activated alloreactive T cells. We therefore hypothesized that allogeneic CD70 CAR T cells could eliminate activated alloreactive T cells and thus avoid T cell-mediated rejection. To test this, allogeneic CD70 CAR T cells were generated with a previously-characterized anti-CD70 single-chain variable fragment (scFv)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and used in an \u003cem\u003ein vitro\u003c/em\u003e model of CAR T cell rejection. PBMCs, representing patient \u0026lsquo;host\u0026rsquo; cells, were cultured for 7 days with unedited HLA-mismatched \u0026lsquo;graft\u0026rsquo; T cells derived from the same healthy donor used for allogeneic CAR T cell production. The resulting primed alloreactive host T cells were isolated to serve as a highly reactive cell population able to quickly recognize and reject allogeneic CAR T cells. Allogeneic CD70 CAR T cells were produced using lentiviral transduction as previously reported\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, with knockout (KO) at the TCRα constant gene (TRAC) locus to prevent alloreactivity against the host T cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CD70 CAR-expressing or non-transduced (NTD) control T cells were then co-cultured with the primed host T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In this model, expressing a CD70 CAR on graft T cells significantly reduced the percentage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and absolute numbers of CD70\u003csup\u003e+\u003c/sup\u003e alloreactive host T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to non-transduced graft T cells. Expression of the CD70 CAR significantly enhanced the survival of graft T cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), providing nearly complete resistance to rejection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Conversely, the alloreactive host T cells efficiently rejected the non-transduced graft T cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the anti-rejection activity of the CD70 CAR against a more diverse array of alloreactive host cells, PBMC MLR assays were performed. This assay allows the potential for both NK and T cell alloreactivity without \u0026lsquo;pre-priming\u0026rsquo; the host cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In this model, CD70\u003csup\u003e+\u003c/sup\u003e host T and NK cells were eliminated when co-cultured with CD70 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Only graft T cells expressing the CD70 CAR survived (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and resisted allorejection by host T and NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Notably, CD70\u003csup\u003eneg\u003c/sup\u003e host T and NK cells were not negatively affected by the CD70 CAR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). These data suggest that the resistance of CD70 CAR T cells to allorejection is due to the ability to selectively eliminate alloreactive CD70\u003csup\u003e+\u003c/sup\u003e host lymphocytes.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of CD70 CAR expression and anti-rejection function\u003c/h2\u003e \u003cp\u003eWe previously demonstrated the activity and specificity of this CD70 CAR in renal cell carcinoma and patient-derived xenograft models, tissue cross-reactivity studies, and a phase I clinical trial\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Importantly, allogeneic T cells expressing this CD70 CAR are fratricide-resistant due to cis-masking of CD70 and can be manufactured in large scale\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Given that this unique CD70 CAR was initially assessed for manufacturability, safety, specificity, and anti-tumor activity, we aimed to optimize the newfound anti-rejection activity of the CAR. To do so we constructed a series of CD70 CAR variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which included CARs with a 4-1BB costimulatory domain (bbz) or without (z). In addition, we tested variants with different transmembrane domains and linker lengths. Linker lengths were adjusted by incorporating a CD34 epitope (Q) and an intra-CAR CD20-based off-switch (R), which were previously shown to influence CAR activity\u003cb\u003e​\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. T cells expressing the Qz, z, and Qbbz CD70 CAR variants had higher levels of transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and CAR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) compared to the original CD70 CAR tested. However, the CD70Qz and CD70z CAR T cells exhibited reduced activation and differentiation compared to cells expressing variants with a costimulatory domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, and Fig. S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate anti-rejection activity, alloreactive T and PBMC MLR assays were performed with CD70Qz and CD70z CAR T cells. The original CD70 CAR and the CD70Qbbz CAR were also included for comparison. CD70z CAR T cells showed significant survival in alloreactive T cell MLRs compared to non-transduced graft T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). CD70Qz also showed improved survival compared to non-transduced graft T cells, yet the differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In PBMC MLR assays, CD70Qz and CD70z CAR T cells showed enhanced survival (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and suppressed host T and NK cell expansion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Overall, the CD70z CAR T cells provided similar levels of resistance to allorejection compared to the original CD70 CAR yet showed higher CAR expression and an improved phenotype. We therefore chose the CD70z CAR variant for further characterization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-rejection and anti-CD19 activity are maintained in allogeneic CD19 CAR T cells co-expressing a CD70 CAR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo exploit the benefits of the CD70z CAR in mediating survival of allogeneic CAR T cells, the combination of an anti-rejection CD70z CAR with a CD19 CAR was examined. A bicistronic construct encoding a CD19/CD70 dual CAR was targeted to the \u003cem\u003eTRAC\u003c/em\u003e locus using site-specific integration (SSI) to model an \u0026lsquo;off-the-shelf\u0026rsquo; allogeneic CAR T cell product. Integration of the CD19/CD70 dual CAR transgene into the \u003cem\u003eTRAC\u003c/em\u003e locus resulted in a high percentage of CD19 CAR/CD70 CAR double positive cells (Fig. S3).\u003c/p\u003e \u003cp\u003eTo rule out any negative effects of the CD70z CAR on CD19 CAR function, short-term killing assays were performed at different effector to target ratios (E:T) using CD19\u003csup\u003e+\u003c/sup\u003e Raji lymphoma cells. Since Raji cells express high levels of CD70, CD70\u003csup\u003eKO\u003c/sup\u003e Raji cells were also tested. The CD19/CD70 CAR T cells eliminated both CD70\u003csup\u003e+\u003c/sup\u003e and CD70\u003csup\u003eKO\u003c/sup\u003e Raji tumor cell targets and showed similar cytotoxicity compared to CD19 CAR T cells lacking the CD70z CAR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, there were no significant differences in effector cytokine production from CD19/CD70 CAR T cells and CD19 CAR T cells (Fig. S4A). CD19/CD70 CAR T cell cytotoxicity against CD19\u003csup\u003e+\u003c/sup\u003e B cells in PBMCs from healthy and systemic lupus erythematosus (SLE) donors was also evaluated. Like CD19 CAR T cells, CD19/CD70 CAR T cells rapidly eliminated CD19\u003csup\u003e+\u003c/sup\u003e B cells from both healthy and SLE donor PBMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig. S4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe possibility that the CD70z CAR may affect long-term CD19 CAR activity was also investigated using a serial restimulation assay. CAR T cells were co-cultured with Raji tumor cell targets and subsequently exposed to fresh target cells every 2\u0026ndash;3 days until cytotoxic activity was no longer detectable. CD70\u003csup\u003eKO\u003c/sup\u003e and CD19\u003csup\u003eKO\u003c/sup\u003e Raji tumor cell targets were used to assess CD19 CAR and CD70 CAR function in isolation, respectively. Both CD19 CAR T cells and CD19/CD70 CAR T cells persisted and maintained CD19 CAR-mediated cytotoxicity against CD70\u003csup\u003eKO\u003c/sup\u003e Raji tumor cells for up to 9 rounds of stimulation (Fig. S4C), indicating that co-expression of the CD70z CAR does not negatively impact short-term and long-term CD19 CAR function. CD19/CD70 CAR T cells also maintained CD70 CAR-mediated cytotoxicity against CD19\u003csup\u003eKO\u003c/sup\u003e Raji tumor cells for up to 11 rounds of stimulation.\u003c/p\u003e \u003cp\u003eNext, alloreactive T cell MLR assays were performed to assess the anti-rejection activity of the CD70z CAR in cells co-expressing a CD19 CAR. CD19/CD70 CAR T cells significantly reduced the number of CD70\u003csup\u003e+\u003c/sup\u003e host T cells from healthy and SLE donors and were able to persist (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas CD19 CAR T cells lacking the anti-rejection CD70z CAR were eliminated by the host T cells and were unable to suppress expansion of CD70\u003csup\u003e+\u003c/sup\u003e host T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Fig. S4D). These data show that the CD70z CAR and the CD19 CAR maintain independent functions when co-expressed on allogeneic T cells.\u003c/p\u003e \u003cp\u003eLastly, we evaluated the cytotoxic activity of CD19/CD70 CAR T cells in an \u003cem\u003ein vivo\u003c/em\u003e orthotopic Raji lymphoma model. In mice bearing CD70\u003csup\u003eKO\u003c/sup\u003e Raji tumors, CD19/CD70 CAR T cells exhibited a slight reduction in efficacy with only 3 of 10 mice showing tumor growth at approximately day 20 post CAR T cell treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and Fig. S5A). However, CD19/CD70 CAR T cells showed significantly higher peripheral expansion compared to CD19 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In mice bearing the parental Raji tumors, which express both CD19 and CD70, CD19/CD70 CAR T cells exhibited improved efficacy and significantly higher peripheral expansion compared to CD19 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F and Fig. S5B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), suggesting that dual targeting of CD19 and CD70 enhances CAR T cell efficacy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual targeting of antigenically heterogeneous tumors with CD19 CAR T cells co-expressing an anti-rejection CD70z CAR limits antigen escape\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCD70 is expressed in multiple hematologic malignancies including LBCL\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and is emerging as a validated target for immunotherapies\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that the CD70z CAR could enhance cytotoxicity against a heterogeneous population of Raji cells when co-expressed with a CD19 CAR. To test this, we used a mixed lymphoma model to simulate tumor antigen heterogeneity. CD19\u003csup\u003eKO\u003c/sup\u003e and CD70\u003csup\u003eKO\u003c/sup\u003e Raji cells were mixed at a 1:1 ratio prior to co-culturing with CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In this model, CD19 CAR T cells were unable to eliminate all the Raji tumor cells and CD19\u003csup\u003eneg\u003c/sup\u003e Raji cells expanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), whereas CD19/CD70 CAR T cells effectively eliminated all the Raji cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results were also observed in an antigenically heterogeneous Toledo lymphoma model (Fig. S6A), demonstrating enhanced tumor control of CD19/CD70 CAR T cells against multiple cancer cell lines \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the CD19/CD70 CAR T cell efficacy against antigenically heterogeneous tumors \u003cem\u003ein vivo\u003c/em\u003e, NSG mice were implanted with a 1:1 mixture of CD19\u003csup\u003eKO\u003c/sup\u003e and CD70\u003csup\u003eKO\u003c/sup\u003e Raji cells, treated with either CD19/CD70 or CD19 CAR T cells, and monitored for tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). CD19/CD70 CAR T cells potently eradicated heterogeneous Raji tumors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and expanded in peripheral blood (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), resulting in mice that remained in remission up to the end of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, and Fig. S6B). CD19 CAR T cells also expanded in peripheral blood, although significantly less than CD19/CD70 CAR T cells. In contrast to CD19/CD70 CAR T cells, CD19 CAR T cells failed to control tumor growth. Taken together, these data demonstrate the enhanced efficacy of bispecific CAR T cells and the dual-purpose function of the CD70z CAR in CD19\u003csup\u003e+\u003c/sup\u003eCD70\u003csup\u003e+\u003c/sup\u003e malignancies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCD19/CD70 CAR T cells eliminate autoreactive CD19\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eB cells, resulting in reduced immunoglobulin levels from donors with SLE\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince CD19 is a promising target for allogeneic CAR T cell therapy against B cell mediated AID\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, we evaluated the ability of CD19/CD70 CAR T cells to eliminate primary human B cells in a hematopoietic stem cell (HSC) humanized mouse model. CD34\u003csup\u003e+\u003c/sup\u003e HSCs were engrafted into NSG mice (Hu-CD34 mice), resulting in multi-lineage reconstitution of human immune cell populations. Hu-CD34 mice received a single dose of 6 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CAR\u003csup\u003e+\u003c/sup\u003e T cells or 6 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e non-transduced T cells per mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Engrafted host B and T cells and CAR\u003csup\u003e+\u003c/sup\u003e T cells were monitored in peripheral blood by flow cytometry analysis. Host B cells were completely depleted by day 7 post CAR T cell injection but started to recover and reconstitute beginning at day 20 post CAR T cell injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). No significant effects were observed on the host T cell population in this model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and CD70 expression was not observed (Fig. S7A), which is likely attributed to the lack of thymic education and proper T cell development in Hu-CD34 mice\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. CD19/CD70 CAR T cells were detectable in peripheral blood with peak expansion occurring on day 7 post CAR T cell injection, which was followed by contraction of the CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). CD19/CD70 CAR T cells were nearly undetectable in peripheral blood by 30 days post-CAR T cell injection. These data suggest that the administration of CD19/CD70 CAR T cells into mice reconstituted with a human immune system result in deep B cell depletion without prolonged B cell aplasia. Additionally, CD19/CD70 CAR T cells engrafted, expanded, and transiently depleted B cells in Hu-CD34 NSG mice without the need for prior lymphodepletion or pre-conditioning of the mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we sought to develop a humanized PBMC mouse model in which PBMCs with functional T cells can be engrafted rapidly from a variety of human donors to assess the efficacy of CD19/CD70 CAR T cells. NSG mice, aged 7 to 8 weeks, were pre-conditioned with 1 Gy irradiation and 2 x 10\u003csup\u003e7\u003c/sup\u003e PBMCs isolated from healthy human donors were adoptively transferred by intravenous (i.v.) injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In this model, adoptively transferred host B cells successfully engraft (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C) and rapidly transition from a predominantly na\u0026iuml;ve B cell phenotype to a more differentiated phenotype in the absence of CAR T cells (Fig. S7B-C). Furthermore, host B cells produced detectable levels of human immunoglobulin G (IgG) and M (IgM) in the serum of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), allowing us to determine the effects of CAR T cell-mediated B cell depletion on immunoglobulin production. Three days after adoptive transfer of PBMCs, mice were randomized and received a single dose of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CAR\u003csup\u003e+\u003c/sup\u003e T cells, 4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CAR\u003csup\u003e+\u003c/sup\u003e T cells, or were left untreated. CD19/CD70 CAR T cells effectively depleted host B cells at both dose levels, resulting in a significant reduction in human IgG and IgM production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). CD19/CD70 CAR T cells expanded in a dose-dependent manner in both the spleen and peripheral blood following target exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). A minor population of CD70\u003csup\u003elow\u003c/sup\u003e T cells was observed in the spleen of untreated control mice on day 10 but was absent in mice receiving CD19/CD70 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLastly, we used PBMCs from SLE donors in the model described above to test the ability for CD19 and CD19/CD70 CAR T cells to eliminate autoreactive B cells and abrogate autoantibody production. NSG mice were engrafted with PBMCs isolated from SLE donors and received a single dose of 5\u0026times; 10\u003csup\u003e6\u003c/sup\u003e CD19/CD70 CAR T cells, CD19 CAR T cells, or non-transduced control T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). As expected, CD19/CD70 and CD19 CAR T cells depleted host B cells and proliferated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-E). CD19/CD70 CAR T cells remained detectable in the spleen on day 14 post CAR T cell injection, whereas CD19 CAR T cells did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Although a reduction in immunoglobulin production was observed in mice receiving CD19 CAR T cells, CD19 CAR T cells did not significantly reduce the levels of α-dsDNA autoantibodies, IgG, and IgM detected in the serum when compared to untreated mice on day 14 post-CAR T cell injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These data suggest that CD19 CAR T cells were rejected by host T cells following activation and expansion, resulting in reduced therapeutic efficacy compared to CD19/CD70 CAR T cells. For one of the two SLE host donors tested (SLE D2), two mice receiving CD19 CAR T cells developed GvHD and were euthanized before day 14 post CAR T cell injection. Nonetheless, similar results were observed on day 7 post CAR T cell injection for both SLE host donors tested. In contrast, CD19/CD70 CAR T cells significantly reduced the production of human immunoglobulins and persisted throughout the duration of the study (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05-\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-F), likely due to the reduction of host T cells in mice dosed with CD19/CD70 CAR T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Taken together, these data highlight the ability of the CD70z CAR to enhance therapeutic efficacy through anti-rejection activity and the potential of allogeneic CD19/CD70 CAR T cells to treat AID driven by autoreactive B cells and T cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDelivering the promise of allogeneic CAR T cell therapies may require strategies to improve intrinsic CAR T cell function, including cell persistence, to achieve the desired efficacy outcomes without the risk of increased toxicity that may result from increased lymphodepletion intensity. This is particularly true in AID, where combination chemotherapy is rarely used. Our results demonstrate a simple and effective approach for boosting allogeneic CAR T cell activity and persistence by targeting both B cells and activated alloreactive T cell subsets marked by expression of CD70. We show that allogeneic CD19 CAR T cells expressing an anti-rejection CD70z CAR maintain anti-CD19 function and selectively eliminate alloreactive cells to evade rejection, while sparing CD70\u003csup\u003eneg\u003c/sup\u003e host lymphocytes. Furthermore, combination of this CD70z CAR with a CD19 CAR improves therapeutic efficacy in tumor models of antigen loss and in an AID model in which antibody titers are suppressed to a greater degree by the dual CAR T cells as compared to CD19-only CAR T cells.\u003c/p\u003e \u003cp\u003eAn additional critical factor that may limit clinical responses to allogeneic CAR T cells is target antigen loss or downregulation, which is emerging as a major mechanism of resistance in malignancies treated with CD19 CAR T cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Given the modular nature of this platform, the enhanced tumor control and potential to prevent antigen escape conferred by the CD70z CAR may well extend to other tumor types characterized by stable or induced expression of CD70. CD70 is expressed in various hematologic malignancies including 71% percent of diffuse large B-cell lymphomas\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and is an attractive immunotherapeutic target due to its limited expression in normal tissues.\u003c/p\u003e \u003cp\u003eCD70 is also expressed on stimulated plasmacytoid dendritic cells (pDCs), which play an important role in the pathogenesis of AID, including SLE. pDCs can be stimulated by DNA-containing immune complexes, a hallmark of SLE, to produce IFN-α and IL-6. pDC-derived IFN-α and IL-6 subsequently drive autoreactive B cell proliferation and the accumulation of autoantibody producing cells\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Stimulated pDCs can also induce antibody production by direct contact with B cells through a CD70-CD27 interaction\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The CD70z CAR could therefore provide additional benefit to AID patients through elimination of CD70\u003csup\u003e+\u003c/sup\u003e pDCs.\u003c/p\u003e \u003cp\u003eCD70 may also be transiently expressed on virus-specific T cells, and it is therefore possible that endogenous T cell responses may be dampened in patients receiving CD70 CAR T cells. However, data to date remains equivocal on this point. For example, human cytomegalovirus-specific T cells have been reported to have low levels of CD70 expression following cognate antigen stimulation and expansion\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Importantly, allogeneic T cells expressing a chimeric receptor specific for the activation marker CD70 (Fig. S8), 4-1BB or OX40 did not completely reduce the cytotoxic activity of virus-specific T cells\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. These findings suggest that targeting activation-induced markers on T cells may not necessarily abrogate productive antiviral T cell responses. Some clinical studies evaluating CD70 CAR T cells and anti-CD70 antibodies have reported overall adequate safety profiles with no increased risk of infection\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Importantly, treatment with CAR T cells conveys a risk of infection\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, including from viral reactivation, and adequate viral prophylaxis and patient monitoring will be required.\u003c/p\u003e \u003cp\u003eWhile the main purpose of the anti-rejection CD70z CAR approach is to improve persistence of allogeneic CAR T cells by eliminating alloreactive host lymphocytes, engagement of the CD70z CAR with its target on alloreactive or autoreactive host immune cells may also stimulate CAR T cell activation and expansion. Studies have shown that providing CAR T cells with immediate target stimulation by locoregional delivery can enhance CAR T cell activity, avoiding anergy prior to encountering target\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Therefore, the presence of a CD70z CAR would not only preserve allogeneic CAR T cells but may contribute to expanding them through target-mediated activation, creating a primed population of cells capable of seeking out and destroying cancer cells or autoreactive cells. Preliminary data from clinical studies in renal cell carcinoma patients indicate that allogeneic CD70 CAR T cells undergo peripheral expansion at levels not previously seen with other allogeneic CAR T cell therapies, presumably due to target mediated expansion and the avoidance of allorejection\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, providing an early clinical validation of the findings described in this study.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of lentiviral and adeno-associated virus vectors encoding anti-CD70 and anti-CD19 CARs\u003c/h2\u003e \u003cp\u003eThe pLVX lentiviral vector (LVV) was employed for cloning and expression of the CD70 CAR or CD70 CAR variants. An adeno-associated virus (AAV) plasmid containing two inverted terminal repeats was modified to incorporate two homology arms, each 300\u0026ndash;400 bp in length corresponding to the genomic integration site at the TRAC locus, which flank the nuclease target site. The AAV cDNA sequences corresponding to the CD19 CAR or CD19/CD70 dual CAR were cloned downstream of the human Pgk-1 promoter and are followed by the bovine growth hormone polyA signal. All CD70 CAR constructs contained an scFv specific for CD70\u003csup\u003e28\u003c/sup\u003e followed by an extracellular spacer or \u0026ldquo;hinge\u0026rdquo; (H) and transmembrane (TM) domain derived from either human CD8α or human CD28, and the intracellular domains of 4-1BB (bb) and CD3ζ (z), or the CD3ζ domain only for constructs without a costimulatory domain. Some variants contained short mimotopes derived from either CD20 (R mimotope) and/or CD34 (Q mimotope)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e that were inserted in the extracellular domain of the CD70 CAR to increase the length of the spacer. The CD19 CAR contained the FMC63 scFv, CD8α hinge and transmembrane domain, and cytoplasmic domains derived from 4-1BB and CD3ζ.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell and viruses\u003c/h3\u003e\n\u003cp\u003ePBMCs were sourced from Stemcell Technologies and T cells were isolated using the human pan T Cell isolation kit (Miltenyi Biotec). To generate Raji and Toledo knockout cell lines, Cas9 ribonucleoproteins (RNPs) were prepared by incubating 60 ng of Cas9 (IDT) with 35 pmol sgRNA (Synthego) in Neon electroporation buffer (Invitrogen). Luciferase-GFP-expressing Raji cells were mixed with Cas9 RNPs and electroporated with a Neon transfection kit and device (Invitrogen). Transfected cells were expanded in RPMI 1640 medium (Invitrogen) containing 10% FBS (GE Healthcare) and KO cells were isolated by FACS. Cell sorting was performed on a BD FACS Aria Fusion sorter. To produce LVV, HEK293T cells were transfected with lentiviral transfer vectors, psPAX2, and pMD.2G (\u0026Eacute;cole Polytechnique F\u0026eacute;d\u0026eacute;rale de Lausanne, Switzerland) using Lipofectamine 2000 (Thermo Fisher Scientific). 24 hours after transfection, the medium was replaced with X-Vivo 15 medium (Lonza), supplemented with 10% FBS. Supernatant was then harvested and passed through a 0.45 \u0026micro;M filter 24 hours later. All AAV vectors used in this study were produced using a triple transfection with AAV-MAX system according to the manufacturer's protocol (Thermo Fisher). Briefly, the pRC6, pHelper, and transgene plasmids were mixed at the molar ratio of 1:1:1 for gene transfection. After 72 hours, cells were lysed with the AAV-MAX lysis buffer for 2 hours and cell debris were removed by centrifugation and filtration with 0.2 \u0026micro;M filter (Nalgene). The clarified lysate was loaded on a HiTrap AVB column (Cytiva) and column-bounded AAV particles were eluted with 0.1 M NaOAc, 0.5 M NaCl, pH 3.0. The elution solution was immediately neutralized with 1M Tris-HCl, pH 8.8, further concentrated, and exchanged to PBS. The titers of the concentrated AAVs were determined by qPCR (Takara).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCAR T cell production\u003c/h2\u003e \u003cp\u003eThe CD70 CAR T cells used in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e were produced with LVV as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To generate CAR T cells via site-specific integration, human T cells were first activated with T cell TransAct reagent (Miltenyi) as recommended by the manufacturer protocol and cultured in X-Vivo15 medium supplemented with 5% human AB serum (Gemini Bio-Products) and 100 IU/mL IL-2 (Miltenyi Biotech). Two days post-activation, cells were washed in PBS and electroporated using the AMAXA 4D nucleofector electroporation apparatus with 15 \u0026micro;M of RNP, TRAC crRNA combined with ABR-001 nuclease, for every 3\u0026ndash;12 x 10\u003csup\u003e6\u003c/sup\u003e cells. ABR-001 gene-editing technology was developed by Arbor Biotechnologies, Inc\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Following electroporation, T cells were transduced with AAV at an MOI of 15,000 and incubated at 30\u0026deg;C overnight. Cells were then returned to 37\u0026deg;C and were supplemented with IL-2 every 2\u0026ndash;3 days. At day 14 post-activation, TCRα/β depletion was performed using EasySep\u0026trade; human TCRα/β depletion kit (STEMCELL Technologies) as instructed by the manufacturer\u0026rsquo;s protocol. T cells were cryopreserved in 90% FBS/10% DMSO or CS5 freezing media using rate-controlled freezing chambers and stored in liquid nitrogen vapor phase.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytotoxicity and cytokine-release assay\u003c/h3\u003e\n\u003cp\u003eFor short-term cytotoxicity assays, CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells or with primary target cells at defined E:T ratios for 24 hours in RPMI 1640 medium supplemented with 10% FBS. Target cell killing was determined using Bright-glo reagent (Promega) or flow cytometric analysis. For cytokine-release assays, CAR T cells were co-cultured with Raji target cells at an E:T ratio of 1:4. At 24 hours, cell culture supernatant was assayed for cytokines using the MSD U-PLEX platform (Meso Scale Diagnostics). For long-term cytotoxicity assays, CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells at an initial E:T ratio of 8:1 for 48 hours in RPMI 1640 medium supplemented with 10% FBS. Half of each well was then removed to measure target cell killing as determined using Bright-glo reagent (Promega). The remaining cells of each well were passaged on to fresh Raji cells and this process was repeated every 2\u0026ndash;3 days until cytotoxic activity was no longer detected. The percentage killing was determined relative to target-only control wells.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry, staining, and antibodies\u003c/h3\u003e\n\u003cp\u003eMouse peripheral blood, bone marrow, and spleen samples were lysed using ACK lysing buffer (Lonza). Samples for flow cytometric analyses were stained on ice with Fixable Viability Ghost Dye (Tanbo) before cell surface staining. An anti-idiotype antibody against the CD70 scFv was raised in Balb/c mice and was purified from hybridoma cell culture supernatants. Antibodies were labeled with R-PE (Prozyme) using sulfhydryl-maleimide reaction chemistry. The following antibodies were purchased from BD Biosciences, eBioscience, BioLegend, ACROBiosystems, or ThermoFisher: CD3 (UCHT1), CD56 (5.1H11), CD19 (SJ25C1), CD20 (2H7), CD70 (113\u0026thinsp;\u0026minus;\u0026thinsp;16), PD-1 (EH12.2H7), CD137 (4B4-1), CD25 (M-A251), CD69 (FN50), TCRαβ (IP26), CD8 (SK1), CD4 (SK3), CD62L (BDB565219), CD45RO (UCHL1), HLA-A2 (BB7.2), HLA-ABC (W6/32), CD45 (HI30), FMC63 (Y45), CD27 (O323), CD38 (HB-7), CD138 (MI15), IgD (IA6-2) and CD45.1 (A20). Cells were fixed in BD Cytofix fixation buffer (BD Biosciences) prior to analysis in a CytoFLEX LX flow cytometer. CAR T cells were enumerated using 123count eBeads counting beads\u0026trade; (Invitrogen).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMixed lymphocyte reaction (MLR) assays\u003c/h2\u003e \u003cp\u003eFor PBMC MLR assays, host PBMCs were co-cultured with TRAC\u003csup\u003eKO\u003c/sup\u003e graft T cells at a ratio of 10:1 in 200 \u0026micro;L RPMI medium supplemented with 10% FBS and 20 U/mL of recombinant human IL-2 in round-bottomed 96-well plates. For alloreactive T cell MLR assays, unedited graft donor T cells were irradiated at 30 Gy and co-cultured with host PBMCs at a ratio of 1:1 in RPMI supplemented with 10% FBS and 20 U/mL of recombinant human IL-2, IL-7, and IL-15. On day 4 of the co-culture, 50% of the cell culture medium was replaced with fresh RPMI supplemented with 10% FBS. On day 7, primed T cells were isolated using EasySep\u0026trade; human T cell isolation kit (STEMCELL Technologies) as instructed by the manufacturer\u0026rsquo;s protocol. Primed alloreactive T cells were then co-cultured with TRAC\u003csup\u003eKO\u003c/sup\u003e graft T cells at a 1:1 ratio in 200 \u0026micro;L RPMI medium supplemented with 10% FBS and 20 U/mL of recombinant human IL-2 in round-bottomed 96-well plates. If MLR co-cultures exceeded 4 days, 50% of the medium was replaced on day 4. Medium was then replaced every 2\u0026ndash;3 days after. Cells were analyzed by flow cytometry at the indicated time points. Graft CAR T and host cells were differentiated based on expression of HLA-A2 (Fig. S9).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntigen escape assays\u003c/h2\u003e \u003cp\u003eTo model antigenic tumor heterogeneity, CD19\u003csup\u003eKO\u003c/sup\u003e and CD70\u003csup\u003eKO\u003c/sup\u003e target cells were first mixed at 1:1 ratio and then co-cultured with CAR T cells at an E:T ratio of 1:1 or 1:4. Cells were cultured in RPMI medium supplemented with 10% FBS in 96-well or 24-well G-Rex plates (Wilson Wolf). On days 2, 50% of the cell culture medium was replaced with fresh RPMI supplemented with 10% FBS. At the indicated time points, 100 \u0026micro;L samples of the heterogeneous cell mixtures were removed and analyzed by flow cytometry. Target cells were enumerated using CountBright\u0026trade; absolute counting beads (Invitrogen).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVirus-specific T cell tri-culture\u003c/h2\u003e \u003cp\u003eVirus-specific T cells (VSTs) and autologous B-lymphoblastoid cell lines (BLCLs) were purchased from Charles River Laboratories. CD19/CD70 double knockout (dKO) BLCLs, herein referred to as antigen presenting cells (APCs), were generated as described above. APCs were incubated with 1 \u0026micro;g/mL of pp65 peptides (Charles River Laboratories) for 1 h at 37\u0026deg;C. Peptide-pulsed APCs were washed extensively prior to tri-culture with VSTs and CAR T cells. Surviving cells in tri-cultures were quantified by flow cytometry at 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOrthotopic mouse model\u003c/h2\u003e \u003cp\u003e All procedures performed on animals were reviewed and approved by an Institutional Animal Care and Use Committee and were conducted in accordance with established guidelines. Female NSG mice 8 to 12 weeks of age were obtained from The Jackson Laboratory. Parental, CD70\u003csup\u003eKO\u003c/sup\u003e, or a 1:1 mixture of CD19\u003csup\u003eKO\u003c/sup\u003e and CD70\u003csup\u003eKO\u003c/sup\u003e luciferase-GFP-expressing Raji cells were prepared, and 1 x 10\u003csup\u003e5\u003c/sup\u003e cells were administered to mice by i.v. injection. Tumor burden was measured using an IVIS Spectrum instrument (PerkinElmer) twice weekly. Mice were randomized based on total body bioluminescence 3 days after tumor cell injection. CAR T cells were injected immediately after thawing, 4 days after tumor cell injection. Total T cells administered were kept constant in all groups by adding non-transduced T cells from the same donor. Peripheral blood was collected into EDTA-coated tubes using submandibular or retro-orbital bleeding and 20 \u0026micro;L was used for flow cytometric analyses. Mice were euthanized when disease model-specific endpoints were exhibited, including hind-leg paralysis, ruffled fur, or 20% reduction in body weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHumanized mouse models\u003c/h2\u003e \u003cp\u003eHu-CD34 mice, aged 16 to 17 weeks, were obtained from The Jackson Laboratory. Hu-CD34 mice received a single dose of CAR T cells or non-transduced control T cells via i.v. injection. Human B and T cells and CAR\u003csup\u003e+\u003c/sup\u003e cells were monitored in peripheral blood by flow cytometry analysis weekly. For the PBMC mouse model, NSG mice aged 7 to 8 weeks were pre-conditioned with 1 Gy irradiation and 10\u0026ndash;20 x 10\u003csup\u003e6\u003c/sup\u003e human PBMCs isolated from healthy donors or SLE donors were adoptively transferred via i.v. injection. Three or seven days after PBMC transfer, mice were randomized and received a single dose of CAR T cells via i.v. injection or were left untreated. Cells from spleen and blood were analyzed by flow cytometry at indicated time points post treatment. Engrafted B and T cells and total CAR\u003csup\u003e+\u003c/sup\u003e cells were assessed and quantified using 123count eBeads. Human α-dsDNA antibody, IgG, and IgM levels in the plasma were measured by enzyme-linked immunosorbent assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The data were analyzed with an unpaired Student \u003cem\u003et\u003c/em\u003e test or one-way and two-way ANOVA followed by Dunnett\u0026rsquo;s or Tukey\u0026rsquo;s multiple comparisons test using Prism GraphPad software. Asterisks indicate statistical significance and \u003cem\u003ep\u003c/em\u003e values are denoted as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Janette Sutton and Jerick Sanchez for support with animal studies, and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYanqi Chang for the helpful suggestions and for providing support with AAV reagents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEJL and CS designed the studies and interpreted the data. KZ, ZL, MKO, AM, DN, HC, DH, DQ, and SG performed experiments. EJL, ZJR, and CS wrote the manuscript and are responsible for the integrity of the work as a whole.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors are current employees of Allogene Therapeutics, Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Correspondence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSadelain, M. CD19 CAR T Cells. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 1471 (2017). \u003c/li\u003e\n\u003cli\u003eGeorg, S. CAR T-cell therapy in autoimmune diseases. \u003cem\u003eLancet\u003c/em\u003e (2023) doi:10.1016/S0140-6736(23)01126-1.\u003c/li\u003e\n\u003cli\u003eZhao, J., Lin, Q., Song, Y. \u0026amp; Liu, D. Universal CARs, universal T cells, and universal CAR T cells. \u003cem\u003eJ Hematol Oncol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2018). \u003c/li\u003e\n\u003cli\u003eHoffmann, J.-M. et al. Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eDepil, S., Duchateau, P., Grupp, S. A., Mufti, G. \u0026amp; Poirot, L. \u0026lsquo;Off-the-shelf\u0026rsquo; allogeneic CAR T cells: development and challenges. \u003cem\u003eNat Rev Drug Discov\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 185\u0026ndash;199 (2020).\u003c/li\u003e\n\u003cli\u003eVoravit, R. et al. Phase III Study Comparing Methotrexate and Tacrolimus (Prograf, FK506) With Methotrexate and Cyclosporine for Graft-Versus-Host Disease Prophylaxis After HLA-Identical Sibling Bone Marrow Transplantation. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 2303\u0026ndash;2314 (1998). \u003c/li\u003e\n\u003cli\u003eTorikai, H. et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 5697\u0026ndash;5705 (2012). \u003c/li\u003e\n\u003cli\u003eA., K. N. et al. Clonable T Lymphocytes in T Cell-Depleted Bone Marrow Transplants Correlate With Development of Graft-v-Host Disease. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 770\u0026ndash;773 (1986).\u003c/li\u003e\n\u003cli\u003eLi, W. et al. Simultaneous editing of TCR, HLA-I/II and HLA-E resulted in enhanced universal CAR-T resistance to allo-rejection. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eWang, D., Quan, Y., Yan, Q., Morales, J. E. \u0026amp; Wetsel, R. A. Targeted Disruption of the \u0026beta;2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells. \u003cem\u003eStem Cells Transl Med\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1234\u0026ndash;1245 (2015).\u003c/li\u003e\n\u003cli\u003eK\u0026auml;rre, K., Ljunggren, H. G., Piontek, G. \u0026amp; Kiessling, R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e319\u003c/strong\u003e, 675\u0026ndash;8 (1986). \u003c/li\u003e\n\u003cli\u003eHans-Gustaf, L. \u0026amp; Klas, K. In search of the \u0026lsquo;missing self\u0026rsquo;: MHC molecules and NK cell recognition. \u003cem\u003eImmunol Today\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 237\u0026ndash;244 (1990).\u003c/li\u003e\n\u003cli\u003eJo, S. et al. Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eDeuse, T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 252\u0026ndash;258 (2019).\u003c/li\u003e\n\u003cli\u003eSiemaszko, J., Marzec-Przyszlak, A. \u0026amp; Bogunia-Kubik, K. NKG2D Natural Killer Cell Receptor-A Short Description and Potential Clinical Applications. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1420.\u003c/li\u003e\n\u003cli\u003eYamada, A. et al. CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo. \u003cem\u003eJ Immunol\u003c/em\u003e \u003cstrong\u003e174\u003c/strong\u003e, 1357\u0026ndash;1364 (2005). \u003c/li\u003e\n\u003cli\u003eZhao, J. et al. Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eBorst, J., Hendriks, J. \u0026amp; Xiao, Y. CD27 and CD70 in T cell and B cell activation. \u003cem\u003eCurr Opin Immunol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 275\u0026ndash;281 (2005).\u003c/li\u003e\n\u003cli\u003eTu, S. et al. CD19 and CD70 Dual-Target Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Relapsed and Refractory Primary Central Nervous System Diffuse Large B-Cell Lymphoma. \u003cem\u003eFront Oncol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2019). \u003c/li\u003e\n\u003cli\u003eRiether, C. et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1459\u0026ndash;1467 (2020). \u003c/li\u003e\n\u003cli\u003eSrour, S. et al. Abstract CT011: A phase 1 multicenter study (TRAVERSE) evaluating the safety and efficacy of ALLO-316 following conditioning regimen in pts with advanced or metastatic clear cell renal cell carcinoma (ccRCC). \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e, CT011\u0026ndash;CT011 (2023).\u003c/li\u003e\n\u003cli\u003eHan, B. K., Olsen, N. J. \u0026amp; Bottaro, A. The CD27\u0026ndash;CD70 pathway and pathogenesis of autoimmune disease. \u003cem\u003eSemin Arthritis Rheum\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 496\u0026ndash;501 (2016). \u003c/li\u003e\n\u003cli\u003eFlieswasser, T. et al. The CD70-CD27 axis in oncology: the new kids on the block. \u003cem\u003eJ Exp Clin Cancer Res\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a, P. et al. Signalling via CD70, a member of the TNF family, regulates T cell functions. \u003cem\u003eJ Leukoc Biol Suppl\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 263\u0026ndash;270 (2004).\u003c/li\u003e\n\u003cli\u003eVerma, K. et al. CD70 identifies alloreactive T cells and represents a potential target for prevention and treatment of acute GVHD. \u003cem\u003eBlood Adv\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 4900\u0026ndash;4912 (2024).\u003c/li\u003e\n\u003cli\u003ePanowski, S. et al. Investigation of Allocar TTM Targeting CD70 As a Potential Therapy for an Array of Hematological Malignancies. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 5157\u0026ndash;5157 (2019).\u003c/li\u003e\n\u003cli\u003ePoirot, L. Multiplex Genome-Edited T-cell Manufacturing Platform for \u0026ldquo;Off-the-Shelf\u0026rdquo; Adoptive T-cell Immunotherapies. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 3853\u0026ndash;3864 (2015).\u003c/li\u003e\n\u003cli\u003ePanowski, S. H. Preclinical Development and Evaluation of Allogeneic CAR T Cells Targeting CD70 for the Treatment of Renal Cell Carcinoma. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 2610\u0026ndash;2624 (2022).\u003c/li\u003e\n\u003cli\u003eDeng, W. et al. CD70‐targeting CAR‐T cells have potential activity against CD19‐negative B‐cell Lymphoma. \u003cem\u003eCancer Commun (Lond)\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 925\u0026ndash;929 (2021).\u003c/li\u003e\n\u003cli\u003eJ., J. et al. CD70: An emerging target in cancer immunotherapy. \u003cem\u003ePharmacol Ther\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 1\u0026ndash;10 (2015). \u003c/li\u003e\n\u003cli\u003eJacobs, J. et al. Unlocking the potential of CD70 as a novel immunotherapeutic target for non-small cell lung cancer. \u003cem\u003eOncotarget\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 13462\u0026ndash;13475 (2015). \u003c/li\u003e\n\u003cli\u003eSauer, T. et al. CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 318\u0026ndash;330 (2021).\u003c/li\u003e\n\u003cli\u003eWang, X. et al. Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis. \u003cem\u003eCell\u003c/em\u003e (2024) doi:10.1016/j.cell.2024.06.027.\u003c/li\u003e\n\u003cli\u003eWatanabe, Y. et al. The analysis of the functions of human B and T cells in humanized NOD/shi-scid/\u0026gamma;cnull (NOG) mice (hu-HSC NOG mice). \u003cem\u003eInt Immunol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 843\u0026ndash;858 (2009).\u003c/li\u003e\n\u003cli\u003eSpiegel, J. Y. et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1419\u0026ndash;1431 (2021).\u003c/li\u003e\n\u003cli\u003eFlieswasser, T. et al. Screening a Broad Range of Solid and Haematological Tumour Types for CD70 Expression Using a Uniform IHC Methodology as Potential Patient Stratification Method. \u003cem\u003eCancers\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1611 (2019).\u003c/li\u003e\n\u003cli\u003eDavison, L. M. \u0026amp; Jorgensen, T. N. New Treatments for Systemic Lupus Erythematosus on the Horizon: Targeting Plasmacytoid Dendritic Cells to Inhibit Cytokine Production. \u003cem\u003eJ Clin Cell Immunol\u003c/em\u003e \u003cstrong\u003e08\u003c/strong\u003e, (2017).\u003c/li\u003e\n\u003cli\u003eZhang, H. et al. A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 1988\u0026ndash;1993 (2017). \u003c/li\u003e\n\u003cli\u003eShaw, J., Wang, Y.-H., Ito, T., Arima, K. \u0026amp; Liu, Y.-J. Plasmacytoid dendritic cells regulate B-cell growth and differentiation via CD70. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 3051\u0026ndash;3057 (2010).\u003c/li\u003e\n\u003cli\u003eLu, J. et al. Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults. \u003cem\u003eImmun Ageing\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eMo, F. et al. Engineered off-the-shelf therapeutic T cells resist host immune rejection. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 56\u0026ndash;63 (2021). \u003c/li\u003e\n\u003cli\u003eMo, F. et al. Engineering T cells to suppress acute GVHD and leukemia relapse after allogeneic hematopoietic stem cell transplantation. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 1194\u0026ndash;1208 (2023).\u003c/li\u003e\n\u003cli\u003eIyer, S. P. et al. S262: THE COBALT-LYM STUDY OF CTX130: A PHASE 1 DOSE ESCALATION STUDY OF CD70-TARGETED ALLOGENEIC CRISPR-CAS9\u0026ndash;ENGINEERED CAR T CELLS IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) T-CELL MALIGNANCIES. in \u003cem\u003eHemaSphere\u003c/em\u003e vol. 6 163\u0026ndash;164 (2022).\u003c/li\u003e\n\u003cli\u003eKampouri, E. et al. Infections after chimeric antigen receptor (CAR)‐T‐cell therapy for hematologic malignancies. \u003cem\u003eTranspl Infect Dis\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eAdusumilli, P. S. A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease, in Combination with the Anti\u0026ndash;PD-1 Agent Pembrolizumab. \u003cem\u003eCancer Discov\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2748\u0026ndash;2763 (2021). \u003c/li\u003e\n\u003cli\u003eDonovan, L. K. et al. Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 720\u0026ndash;731 (2020).\u003c/li\u003e\n\u003cli\u003eValton, J. et al. A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eMcGaw, C. et al. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6157466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6157466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAllogeneic chimeric antigen receptor (CAR) T cells can achieve sustained clinical benefit in B cell malignancies and autoimmune diseases. Despite the many potential advantages over autologous products, allogeneic CAR T cells carry a higher risk of rejection, which may limit persistence and therapeutic efficacy. We report the design and evaluation of an optimized CD70 CAR that prevents rejection of allogeneic CAR T cells by targeting activated alloreactive lymphocytes. Co-expression of this CD70 CAR with a CD19 CAR resulted in sustained CAR T cell persistence in the presence of alloreactive lymphocytes and prolonged antitumor activity in a CD19 antigen escape model. In vivo, CD19/CD70 dual CAR T cells resisted rejection and eliminated B cells and CD70\u003csup\u003e+\u003c/sup\u003e T cells from patients with systemic lupus erythematosus, lowering immunoglobulin production. An allogeneic CD19/CD70 dual CAR T cell therapy may therefore reduce the need for lymphodepleting conditioning regimens required prior to CAR T cell infusion.\u003c/p\u003e","manuscriptTitle":"Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-20 09:52:12","doi":"10.21203/rs.3.rs-6157466/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f07f677-985a-40d0-9760-36a95b3a8432","owner":[],"postedDate":"March 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":45819704,"name":"Health sciences/Molecular medicine"},{"id":45819705,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma"},{"id":45819706,"name":"Biological sciences/Immunology/Applied immunology/Autoimmune diseases"}],"tags":[],"updatedAt":"2026-04-03T03:20:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-20 09:52:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6157466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6157466","identity":"rs-6157466","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}