Non-Stimulatory pMHC Enhance CD8 T Cell Effector Functions by Recruiting Coreceptor-Bound Lck
Under physiological conditions, CD8+ T cells need to recognize low numbers of antigenic pMHC class I complexes in the presence of a surplus of non-stimulatory, self pMHC class I on the surface of the APC. Non-stimulatory pMHC have been shown to enhance CD8+ T cell responses to low amounts of antigenic pMHC, in a phenomenon called co-agonism, but the physiological significance and molecular mechanism of this phenomenon are still poorly understood. Our data show that co-agonist pMHC class I complexes recruit CD8-bound Lck to the immune synapse to modulate CD8+ T cell signaling pathways, resulting in enhanced CD8+ T cell effector functions and proliferation, both in vitro and in vivo. Moreover, co-agonism can boost T cell proliferation through an extrinsic mechanism, with co-agonism primed CD8+ T cells enhancing Akt pathway activation and proliferation in neighboring CD8+ T cells primed with low amounts of antigen.
AbstractPhosphoantigens (PAgs) activate Vγ9Vδ2 T lymphocytes, inducing their potent and rapid response in vitro and in vivo. However, humans and nonhuman primates that receive repeated injections of PAgs progressively lose their Vγ9Vδ2 T cell response to them. To elucidate the molecular mechanisms of this in vivo desensitization, we analyzed the transcriptome of circulating Vγ9Vδ2 T cells from macaques injected with PAg. We showed that three PAg injections induced the activation of the PPARα pathway in Vγ9Vδ2 T cells. Thus, we analyzed the in vitro response of Vγ9Vδ2 T cells stimulated with a PPARα agonist. We demonstrated that in vitro PPARα pathway activation led to the inhibition of the BrHPP-induced activation and proliferation of human Vγ9Vδ2 T cells. Since the PPARα pathway is involved in the antigen-selective desensitization of human Vγ9Vδ2 T cells, the use of PPARα inhibitors could enhance cancer immunotherapy based on Vγ9Vδ2 T cells.
Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell–mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7– and IL-15–mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells.
Effective Prevention of Acute Graft-Verses-Host Disease by Targeting PKC Alpha and PKC Theta in Mice
Abstract Abstract 1898 Protein kinase C theta (PKCθ), a T cell signaling molecule, has been implicated as a therapeutic target for several autoimmune diseases as well as graft-versus-host disease (GVHD). PKCθ plays a vital role in stabilization of the immunologic synapse between T effector cells and antigen presenting cells (APC), but has been shown to be excluded from the immunologic synapse in T regulatory cells (T reg). PKCθ inhibition reduces the alloreactivity of donor T cells responsible for induction of GVHD while preserving graft-versus-leukemia (GVL) responses. The roles of PKCθ and the potential compensatory alpha isoform (PKCα) are not clearly defined with regard to alloresponses or T cell mediated responses in GVHD. In this context, we measured PKCθ and PKCα/θ gene deficient T cell activation upon TCR-ligation in vitro using [3H]-TdR incorporation and CSFE labeling assays. T cells from PKCθ and PKCα/θ gene deficient donor mice were utilized in vivo in a pre-clinical allogenic murine model of myeloablative bone marrow transplantation (BMT). The development of GVHD was monitored in recipient mice with or without injection of A20-luciferase cells to observe the progression of GVL in vivo. Combined blockade of PKCα and PKCθ causes a significant decrease in T cell proliferation compared to blocking PKCθ alone in vitro. Deficiency in PKCα and PKCθ had no effect on immune reconstitution following irradiation and BMT in vivo. Even with a high transplant load of 5×106 CD4+ and CD8+ T cells, PKCα/θ deficient (PKCα/θ−/−) T cells failed to induce acute GVHD. Our data suggest that the ability of double deficient T cells to induce GVHD was further reduced than PKCθ-deficient T cells. Additionally, a greater number and percentage of B220+ B cells and FoxP3+ T regs were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type (WT) or PKCθ−/− T cell recipients. Fewer CD4+ or CD8+ T effector cells were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type or PKCθ−/− T cell recipients. Importantly, the activity of B cells isolated from PKCα/θ−/− T cell recipient mice 120 after BMT was greater on a per cell basis, while the activity of T effector cells isolated from these mice was greatly reduced compared to WT or PKCθ−/− T cell recipients. While not absent, GVL was reduced in PKCα/θ−/− T cell recipient mice when compared to WT or PKCθ−/− T cell recipients. This work demonstrates the requirement of PKCα and θ for optimal activation and function of T cells in vitro. These experiments highlight a potential compensatory role for PKCα in the absence of PKCθ in T cell signaling and activation. Combined deficiency of PKCα and θ prevents induction of acute GVHD while improving the maintenance of splenic cellularity in PKCα/θ T cell recipient mice. Additionally, PKCα/θ dual deficient T cell transplant shifts the splenic balance toward a greater number and percentage of T reg and B cells and away from T effector cells following BMT. The reduced and sub-optimally active T effector cells isolated from PKCα/θ−/− T cell recipient mice in combination with reduced GVL stresses the importance of PKCα and θ molecules and their roles in T cell activity in the context of both GVHD and GVL. Dual deficiency of PKCα/θ is associated with a decline of T effector function that is optimal for the amelioration of GVHD, but is perhaps too reduced to substantially maintain effective GVL. Modulation of PKCα and θ signaling presents a valid avenue of investigation as a therapeutic option for GVHD. Disclosures: No relevant conflicts of interest to declare.
CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant in vivo efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses in vitro and in vivo. In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8+ T cells versus CD4+ T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4+ T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were in vitro skewing responses towards CD8+ T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4+ T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity.
Abstract Abstract 2618 Adoptive transfer of antigen-specific T cells emerged as an attractive strategy to provide cancer patients with immune cells of a desired specificity. The efficacy of such adoptive transfers has been demonstrated in clinical studies. However, enrichment and expansion of tumor-specific T cells are time-consuming and often ineffective, due to the low frequency of tumor-specific precursors in vivo. Alternatively, polyclonal T cells can be genetically modified with chimeric antigen receptors (CARs) to provide these cells with new antigen specificities. CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen fused to a signaling chain derived from a lymphocyte activating receptor. The chimeric receptor approach is able to bypass many of the mechanisms by which tumors avoid immunorecognition, such as MHC down-regulation, lack of expression of costimulatory molecules, and induction of T cell suppression. Acute myeloid leukemia (AML) is an intrinsically resistant disease and even though the majority of the patients initially respond to chemotherapy, the 3-year survival rate is still low. A promising target for immunotherapy of AML is CD33, which is absent on normal pluripotent hematopoietic stem cells and normal tissues, but is present on leukemic blasts in 85–90 % of adult and pediatric AML cases independent of the subtype of AML. Novel human CD33-specific CARs were constructed by fusing a CD33 specific scFv in series with CD3ζ chain plus an additional costimulatory sequence derived from CD28 (Fig. 1A). Both native human CD8+ and CD4+ T cells engrafted with CD33-specific CARs exhibited antigen-specific cytokine secretion, proliferation and target cell lysis (Fig. 1B, C). Moreover, AML blast derived from patients were efficiently killed by allogeneic CAR-engrafted T cells (Fig. 1D). Next, the CD33-specific scFv was fully humanized and afterwards incorporated into the CAR constructs. With this humanized CAR engrafted T cells from AML patients could be redirected against CD33+ cell lines and autologous AML blasts. Upon antigen-recognition, the modified T cells secreted high amount of inflammatory cytokines and efficiently killed the target cells.Fig. 1:Human CAR-engrafted T cells mediate effector functions against CD33+ target cells.A. Schematic representation of the CD33-specific CARs. VL: variable light chain; VH: variable heavy chain; E-Tag: linker epitope in between the VL and the VH chain.Fig. 1:. Human CAR-engrafted T cells mediate effector functions against CD33+ target cells. / A. Schematic representation of the CD33-specific CARs. VL: variable light chain; VH: variable heavy chain; E-Tag: linker epitope in between the VL and the VH chain.B. Cytotoxic effector functions of CAR engrafted human CD8+ and CD4+ T cells against the CD33+ blast line U937 were measured in a standard chromium release assay after 6h of co-incubation.B. Cytotoxic effector functions of CAR engrafted human CD8+ and CD4+ T cells against the CD33+ blast line U937 were measured in a standard chromium release assay after 6h of co-incubation.C. Killing of patient-derived AML blast by allogeneic CAR engrafted T cells was measured in a flow cytometer by exact cell count after 48h of co-cultivation. Three independent donor/patient pairings are shown.C. Killing of patient-derived AML blast by allogeneic CAR engrafted T cells was measured in a flow cytometer by exact cell count after 48h of co-cultivation. Three independent donor/patient pairings are shown. Until now, one major obstacle for an adoptive therapy of genetically modified T cells is the limited amount of T cells that can be isolated from AML patients and modified in vitro. For an efficient in vitro expansion restricted to CAR modified T cells from patients we developed a new method based on a novel CAR-mediated strategy. For this purpose, magnetic beads were coated with an antibody recognizing an epitope (Fig. 1A, E-Tag) which we included in the linker domain between the heavy and the light chain of the scFv portion of the CAR. Adding such magnetic beads to cultures of CAR modified T cells, the CAR engrafted T cells were expanded to similar extends as polyclonal T cell populations with anti-CD3/anti-CD28 coated beads. Furthermore, the antibody-coated beads can be used to isolate the CAR engrafted T cells after expansion and get rid of any contaminating non-modified cells. It may also be useful for elimination of CAR expressing T cells in vivo if necessary. The feasibility of the described method was not limited to CD33 specific CARs but was also functional for CARs equipped with scFvs of other specificities. Therefore, it might be universally applicable for the expansion and preparation of CAR modified T cell grafts in vitro before adoptive transfer back in patients. Taken together, we describe novel humanized CD33-specific CARs that (I) can be specifically expanded, (II) specifically eliminated, if necessary, and (III) may therefore become a novel potent treatment option for a cellular therapy of AML patients. Disclosures: No relevant conflicts of interest to declare.
Abstract The immunostimulatory properties of lenalidomide have been mostly described in vitro while in vivo studies performed in multiple myeloma or chronic lymphocytic leukemia have reported a poorly characterized T-cell activation. A better understanding of lenalidomide kinetics and mechanisms of action is mandatory to optimize its combination with other immunotherapeutic agents in particular for the treatment of non Hodgkin lymphomas. We undertook a thorough immune monitoring of patients enrolled in the French multicenter clinical trial GALEN (ClinicalTrials.gov: NCT01582776) addressing the tolerance and efficacy of the association of lenalidomide and obinutuzumab, a glycoengineered type II anti-CD20 monoclonal antibody, in relapsed/refractory B-cell lymphomas. A 1-week interval between the start of lenalidomide and the first infusion of obinutuzumab was planned, allowing an assessment of the effect of lenalidomide alone on immune-related parameters separately from the combinatorial therapy. Serial blood samples were collected in 44 patients (16 DLBCL and 28 FL) to investigate T, B, NK, and myeloid subsets. In addition, in vitro functional assays were designed to address T cell functional features (proliferation, immune synapse, activity of regulatory T cells (Treg)). In the context of the association with obinutuzumab, we first checked that CD20 expression was not affected on circulating malignant and normal B cells (p=0.43 and 0.8; respectively). Interestingly, upon 1 week of lenalidomide treatment, normal B cells, unlike malignant B cells, upregulated MHC class II (p<0.001 versus 0.16; respectively) while both increased the expression of the costimulatory molecule CD86 (p=0.001 and 0.002; respectively). More importantly, the T-cell capacity to mount a functional immune synapse with malignant B cells was restored in 5/6 relapsed/refractory patients (p<0.001) and we confirmed that this stood true for 6 FL patients at diagnosis (p<0.001). In addition, T cell proliferation was strongly increased in vivo as measured by Ki67 staining (p<0.001) but also upon TCR stimulation ex vivo (p=0.002). This immunostimulatory effect could not be ascribed to a blockade of Treg inhibitory potential by lenalidomide as effector T-cell proliferation was similarly enhanced upon in vitro Treg depletion before and after lenalidomide treatment (p=0.02). In addition, T-cell activation was associated with a reshaping of memory T-cell distribution with the central memory subset dropping in favor of effector cells (p<0.001 and 0.002 respectively). This restoration of T-cell functions was paralleled by the induction of activation markers on T cells such as HLA-DR, CD137, PD-1, and Tim-3 (p<0.001 for all markers). Finally, immune stimulation was not confined to T cells as NK cells also upregulated CD137 (p<0.001) but not PD-1 (p=0.53) expression. We also investigated the myeloid compartment including circulating MDSC and monocytes, both being putative precursors of tumor-associated macrophages. Within 1 week of lenalidomide, patients experienced a decrease of monocytes subsets count and an upregulation of the activation marker and Fcg receptor CD64 (p=0.006). Of note, preliminary experiments showed that, at least in some cases, in vitro exposure of macrophages to lenalidomide could enhance anti-CD20-mediated phagocytosis of tumor cells. Some of these immunological parameters were transiently modulated and returned to baseline levels upon lenalidomide washout but others were restored long term in particular the immune synapse score and memory T cell counts. We herein report for the first time early in vivo T cell activation by lenalidomide in relapse FL/DLBCL through a detailed phenotypic analysis strengthened by innovative functional assays. The study of T cells heterogeneity at the transcriptomic level is underway and the correlation of these immunomodulatory properties with clinical data is also currently being addressed. Our results will help build new and more relevant lenalidomide-based immunotherapeutic approaches. (This study was supported by research grants from Celgene and Roche companies) Disclosures Menard: Celgene: Consultancy; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Lamy: Roche: Consultancy, Honoraria. Morschhauser: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Servier: Consultancy; Gilead: Consultancy. Tarte: Celgene: Consultancy, Research Funding; Novimmune: Research Funding; Roche: Consultancy.
Abstract Abstract 2044 Introduction: The specificity of T cells can be redirected using retroviral T cell receptor (TCR) gene transfer. This has the potential to generate tumour specific T cells that can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8 T cells. However the transfer of antigen specific CD8 T cells in the absence of antigen specific CD4 T cells leads to impaired anti-tumour responses and impaired memory development in vivo. Class I restricted TCR can be used to transduce CD4 T cells for use in adoptive transfer. The majority of class I restricted TCRs are CD8 dependent and thus require co-transduction of CD8 to be fully functional in CD4 T cells. CD4 T cells transduced with the class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Dbclass I molecules) produce IL-2 and proliferate in vitro in response to class II negative tumour cells expressing NP peptide but these cells were not able to generate an IFN-γ response. In vivo, F5-TCR CD4 T cells could provide help for F5-TCR CD8 T cell mediated tumour eradication. These F5-TCR CD4 T cells persisted in vivo for up to 90 days post tumour regression and were able to re-expand following tumour challenge. In order to improve the function of class I restricted TCR expressing CD4 T cells, we co-transduced a vector containing all 4 chains of the CD3 complex. High surface expression of TCR has been shown to correlate with increased responsiveness to specific antigen. When additional TCR is introduced into a T cell, the introduced T cell must compete with the endogenous TCR for binding to CD3. The amount of CD3 within the cell will thus be rate limiting for the level of surface expression of the introduced TCR. Method: The retroviral vectors pMP71-F5α-2A-F5β (F5-TCR) and pMP71-CD3-ζ-2A-ε-2A-δ-2A-γ-IRES-GFP (CD3) were used for retroviral transduction. CD4 splenocytes obtained from C57BL/6 mice were activated with CD3/CD28 magnetic beads for 24 hours prior to transduction with either F5-TCR alone or F5-TCR and CD3. 5 days post transduction, transduced T cells were stimulated with C57BL/6 splenocytes loaded with NP (relevant) peptide or WT1 (irrelevant) peptide and cytokine production was measured by ELISA and intracellular cytokine staining and proliferation by [3H] thymidine incorporation. For in vivo tumour challenge, C57BL/6 recipient mice were irradiated with 5.5Gy and injected subcutaneously with 1 × 106 EL4-NP-luciferase cells (a lymphoma cell line stably transfected with NP peptide and luciferase) on day 0. On day 1, mice received 1 × 106 F5-TCR CD3 CD4 T cells or 1 × 106 F5-TCR CD4 T cells or 1 × 106 Mock Transduced T cells. Tumour area was measured by calipers and by bioluminescence imaging. For T cell trafficking experiments, the experimental set up was as above but transgenic CD4 luciferase T cells were used for adoptive transfer and EL4-NP luciferase negative cells were used for tumour challenge. Results: CD4 T cells transduced with F5-TCR and CD3 had a 5-fold higher expression of F5-TCR compared to cells transduced with F5-TCR alone. In vitro, F5-TCR CD3 CD4 T cells showed increased proliferation and increased production of IL-2 and IFN-γ in response to specific antigen compared to F5-TCR CD4 T cells. F5-TCR CD3 CD4 T cells responded to at a 2-fold lower concentration of specific peptide than F5-TCR CD4 T cells. Following adoptive transfer in murine models, F5-TCR CD3 CD4 T cells eradicated NP expressing EL4 tumours but transfer of equivalent doses of F5-TCR CD4 T cells did not lead to tumour regression. Using bioluminescence imaging, F5-TCR CD3 CD4 T cells trafficked to tumour site faster and accumulated in greater numbers than F5-TCR CD4 T cells. Following tumour challenge, there were higher numbers of F5-TCR CD3 CD4 T cells persisting in bone marrow, lymph node and peripheral blood than in mice that received F5-TCR CD4 T cells. Conclusion: Increased surface expression of class I restricted TCR in CD4 T cells leads to increased sensitivity to peptide in vitro and higher levels of proliferation and cytokine production in response to specific peptide. This translates in vivo to enhanced persistence of F5-TCR CD3 CD4 T cells and more efficient trafficking to tumour site and superior tumour protection. Therefore, the co-transduction of additional CD3 can improve the function of class I restricted TCR in CD4 T cells. Disclosures: Stauss: Cell Medica: Scientific Advisor Other.
Abstract Abstract 3152 Alloreactive immune responses directed against malignant cells in recipients of allogeneic hematopoietic stem cell transplants are able to cure patients with hematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD). Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and possibly reducing the potential for non-specific immune suppression. We have generated ‘converted’ Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into polyclonal conventional CD4+ T cells. We used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH-TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI. In vitro, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells were hyporesponsive to stimulation and suppressed the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells significantly reduced early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, polyclonal CD4+ T cells transduced with Foxp3 and the MH-TCR caused marked suppression of allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH-TCR conferred specificity to their cognate antigens in vitro, the potent suppression in these in vivo models was independent of the cognate antigen for the transduced TCRs. This non-specific suppression was markedly reduced when class I-restricted TCRs were transduced into OT-II Rag1-/- CD4+ T cells that are transgenic for a single endogenous TCR. These findings demonstrate an important role for the endogenous TCRs in driving non-specific suppression by polyclonal CD4+ T cells transduced with Foxp3 and class I-restricted TCRs, and suggest that strategies to downregulate endogenous TCRs will be required to achieve antigen-specific suppression in TCR gene-modified regulatory T cells. Disclosures: No relevant conflicts of interest to declare.
Abstract Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of "off-the-shelf" T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. Methods. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population. Conclusions. We have successfully developed a differentiated "off-the-shelf" approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. Disclosures Zhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.
BackgroundAs cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are eliminated via negative selection. Therefore, human-derived TCRs are typically of low affinity and exhibit reduced anti-tumor activity. Affinity maturation by mutagenesis is a common tool to increase affinity but may result in reduced specificity and off-target toxicity. Using our proprietary HuTCR mouse platform, which consists of mouse lines carrying the full human TCR-a/ß loci and human HLA alleles, we have isolated naturally optimized high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them in vitro and in vivo to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development.MethodsMAGE-A1-specific TCRs were isolated from HuTCR mice immunized with the MAGE-1 antigen using scRNAseq or were synthesized based on publicly available databases of human donor-derived MAGE-A1-specific TCRs. All TCRs were re-expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional activity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by coculture with tumor cell lines with variable MAGE-A1 and/or MHC-class-I expression. Tumor rejection potential of TCRs was evaluated in vivo using a syngeneic mouse model (TNA2 mice) expressing MAGE-A1 and HLA-A*02 on syngeneic tumor cells.ResultsImmunization of HuTCR mice with the MAGE-A1 antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR functional avidities ranging from 0.3nM to 3nM. In sharp contrast, human-derived TCRs of the same epitope specificity exhibited lower functional avidity with EC50 from 3nM to 60nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cells expressing low MAGE-A1 and/or MHC-class-I. Adoptive T-cell transfer to TNA2-mice with established tumors resulted in complete rejection without relapse of tumors only in mice treated with HuTCR-mouse-derived TCR but not with human-derived or control TCRs.ConclusionsThe HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific human TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. The in vitro and in vivo comparative data presented herein highlight the HuTCR-derived MAGE-A1-specific TCR as the most favorable candidate for clinical translation and a clinical trial evaluating its safety and efficacy in a variety of solid malignancies will be initiated November 2021.Ethics ApprovalAll animal experiments were performed according to institutional and national guidelines, after approval by the responsible authority (Landesamt für Gesundheit und Soziales, Berlin). Blood collection from healthy human donors was done after prior informed consent and experiments were conducted in accordance with the ethical standards of Declaration of Helsinki.
