Artificial Antigen Presenting Cells Research Articles

‘MR1 Tetramer-based Artificial Antigen Presenting Cells Expand MAIT Cells from Human Peripheral Blood that Effectively Kill Glioblastoma Cells.’

Abstract Immunotherapy for cancer treatment requires the activation of cytotoxic effector lymphocytes. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize the MHC class I-like molecule, MR1. MAIT cells play an important role in the immune response against microbial infections and can directly kill tumor cells. Although MAIT cells can be expanded ex vivo, this method is time-consuming, expensive and requires allogenic feeder layers. To overcome the limitations of conventional dendritic cell-based vaccines and ex vivo expansion of human T cells, an artificial antigen presenting cell (aAPC) approach to expand antitumor effector cells has several advantages. In this study, we explored an efficient in vitro method to amplify MR1-specific MAIT cells from human peripheral blood using aAPCs made by coating cell-sized latex beads with an antigen-loaded MR1 tetramer complex and anti-CD28 antibody. We further elucidated the cytotoxic potential of such expanded MAIT cells against three human glioblastoma (GBM) cell lines to explore their potential use as a novel immunotherapeutic tool, as the mostly lethal GBM poorly responds to conventional chemotherapy. When aAPCs were compared to the standard allogenic feeder layer-based approach for MAIT cell expansion, they were significantly more effective. Our results indicate that the aAPC-expanded MAIT cells remained functional, retained their original phenotype, secrete pro-inflammatory cytokines, and showed cytotoxicity against the GBM cell lines. Hence, MAIT cells have the potential to be a novel tool in immunotherapy approaches for the treatment of human GBM.

First-in-human phase 1 trial of induced regulatory T cells for graft-versus-host disease prophylaxis in HLA-matched siblings

Human CD4+25− T cells cultured in interleukin 2 (IL-2), rapamycin, and transforming growth factor β (TGFβ) along with anti-CD3 monoclonal antibody–loaded artificial antigen-presenting cells generate FoxP3+ induced regulatory T cells (iTregs) with potent suppressive function. We performed a phase 1, single-center, dose-escalation study to determine the safety profile of iTregs in adults with high-risk malignancy treated with reduced-intensity conditioning and mobilized peripheral blood stem cells (PBSCs) from HLA-identical sibling donors. Sixteen patients were enrolled and 14 were treated (2 productions failed to meet desired doses). One patient each received 3.0 × 106/kg, 3.0 × 107/kg, and 3.0 × 108/kg iTregs with corresponding T-conventional-to-iTreg ratios of 86:1, 8:1, and 1:2. After 3 patients received 3.0 × 108/kg in the presence of cyclosporine (CSA) and mycophenolate mofetil (MMF) with no dose-limiting toxicities, subsequent patients were to receive iTregs in the presence of sirolimus/MMF that favors Foxp3 stability based on preclinical modeling. However, 2 of 2 developed grade 3 acute graft-versus-host disease (GVHD), resulting in suspension of the sirolimus/MMF. An additional 7 patients received 3.0 × 108/kg iTregs with CSA/MMF. In the 14 patients treated with iTregs and CSA/MMF, there were no severe infusional toxicities with all achieving neutrophil recovery (median, day 13). Of 10 patients who received 3.0 × 108/kg iTregs and CSA/MMF, 7 had no aGVHD, 2 had grade 2, and 1 had grade 3. Circulating Foxp3+ iTregs were detectable through day 14. In summary, iTregs in the context of CSA/MMF can be delivered safely at doses as high as 3 × 108/kg. This trial was registered at www.clinicaltrials.gov as #NCT01634217.

Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells

Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions.

Engineering Nanoscale Artificial Antigen-Presenting Cells by Metabolic Dendritic Cell Labeling to Potentiate Cancer Immunotherapy.

Nanoscale artificial antigen-presenting cells (aAPCs) are promising to activate T cells directly for cancer immunotherapy, while feasible and flexible strategy to develop nanoscale aAPCs remains highly desirable. Metabolic glycoengineering is used to decorate chemical tags on cells which enables bioorthogonal chemical conjugation of functional molecules. Herein, we develop a nanoscale aAPC by metabolic dendritic cell (DC) labeling to mobilize T-cell based antitumor immunity. We coat azido-labeled DC membrane on imiquimod-loaded polymeric nanoparticles and sequentially modify anti-CD3ε antibody via click chemistry. The nanoscale aAPCs perform improved distribution in lymph nodes and stimulate T cells and resident APCs. Significant inhibition of tumor inoculation and growth is observed after the vaccination, which can be further improved by combining antiprogrammed cell death receptor 1 (PD1) therapy. Our results demonstrate the promising application of metabolically labeled DCs for designing nanoscale aAPCs, which provide a simple and general strategy to potentiate cancer immunotherapy.

Engineering Nano-Therapeutics to Boost Adoptive Cell Therapy for Cancer Treatment.

Although adoptive transfer of therapeutic cells to cancer patients is demonstrated with great success and fortunately approved for the treatment of leukemia and B-cell lymphoma, potential issues, including the unclear mechanism, complicated procedures, unfavorable therapeutic efficacy for solid tumors, and side effects, still hinder its extensive applications. The explosion of nanotechnology recently has led to advanced development of novel strategies to address these challenges, facilitating the design of nano-therapeutics to improve adoptive cell therapy (ACT) for cancer treatment. In this review, the emerging nano-enabled approaches, that design multiscale artificial antigen-presenting cells for cell proliferation and stimulation in vitro, promote the transducing efficiency of tumor-targeting domains, engineer therapeutic cells for in vivo imaging, tumor infiltration, and in vivo functional sustainability, as well as generate tumoricidal T cells in vivo, are summarized. Meanwhile, the current challenges and future perspectives of the nanostrategy-based ACT for cancer treatment are also discussed in the end.

Effective Activation and Expansion of Canine Lymphocytes Using a Novel Nano-Sized Magnetic Beads Approach.

Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs via MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.

Comprehensive Analysis of CD4+ T Cell Responses to CMV pp65 Antigen Restricted by Single HLA-DR, -DQ, and -DP Allotype Within an Individual.

Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4+ T cell responses restricted by a single HLA allotype, we established a panel of artificial antigen-presenting cells (aAPCs) expressing each single HLA allele of 20 HLA-DRB1, 16 HLA-DQ, and 13 HLA-DP alleles. CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.

Biodegradable Cationic Polymer Blends for Fabrication of Enhanced Artificial Antigen Presenting Cells to Treat Melanoma

Biomimetic biomaterials are being actively explored in the context of cancer immunotherapy because of their ability to directly engage the immune system to generate antitumor responses. Unlike cellular therapies, biomaterial-based immunotherapies can be precisely engineered to exhibit defined characteristics including biodegradability, physical size, and tuned surface presentation of immunomodulatory signals. In particular, modulating the interface between the biomaterial surface and the target biological cell is key to enabling biological functions. Synthetic artificial antigen presenting cells (aAPCs) are promising as a cancer immunotherapy but are limited in clinical translation by the requirement of ex vivo cell manipulation and adoptive transfer of antigen-specific CD8+ T cells. To move toward acellular aAPC technology for in vivo use, we combine poly(lactic-co-glycolic acid) (PLGA) and cationic poly(beta-amino-ester) (PBAE) to form a biodegradable blend based on the hypothesis that therapeutic aAPCs fabricated from a cationic blend may have improved functions. PLGA/PBAE aAPCs demonstrate enhanced surface interactions with antigen-specific CD8+ T cells that increase T cell activation and expansion ex vivo, associated with significantly increased conjugation efficiency of T cell stimulatory signals to the aAPCs. Critically, these PLGA/PBAE aAPCs also expand antigen-specific cytotoxic CD8+ T cells in vivo without the need of adoptive transfer. Treatment with PLGA/PBAE aAPCs in combination with checkpoint therapy decreases tumor growth and extends survival in a B16-F10 melanoma mouse model. These results demonstrate the potential of PLGA/PBAE aAPCs as a biocompatible, directly injectable acellular therapy for cancer immunotherapy.

Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy.

Simple SummaryNatural Killer cells have shown promise to treat different malignancies. Several methods have been described to obtain fully activated NK cells for clinical use. Here, we use different cell culture media and different artificial antigen presenting cells to optimize a GMP compliant manufacturing method to obtain activated and expanded NK cells suitable for clinical use.Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

Abstract IA03: Engineering artificial antigen presenting cells, aAPC, for cancer immunotherapy: From bench to bedside

Abstract Adoptive Cellular Therapies, ACT, are promising immunotherapeutic approaches for treatment of cancer that, clinically, have had significant successes and yet many challenges remain that prevent ACT approaches from becoming front line therapy. In this presentation we will discuss the challenges associated with current approaches and show how artificial Antigen Presenting Cells, aAPC-based approaches help overcome those challenges. Thus, aAPC based approaches represent an off-shelf, scalable approach to production of clinical scale and grade T cells for the adoptive immunotherapy of cancer. Citation Format: Jonathan P. Schneck. Engineering artificial antigen presenting cells, aAPC, for cancer immunotherapy: From bench to bedside [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA03.

Abstract PO044: RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses

Abstract The high-risk human papillomavirus (HPV) strain 16 accounts for ~ 70% of all cervical cancers and 80% of head and neck cancers associated with HPV infection., Despite available therapies, there remains a critical need for new treatment options for advanced HPV 16-associated cancers. To address this, we have genetically engineered red blood cells to create an allogeneic artificial antigen presenting cell (APC), RTX-321, that expresses an HPV E7 peptide bound to MHC I (HLA-A*02:01), 4-1BBL and IL-12 on the cell surface to mimic the biology of T cell APC interactions. RTX-321 is designed to enhance both the quantity and quality of endogenous tumor-specific T cells. In this study, we evaluated the mechanisms through which RTX-321 promotes anti-tumor immune responses, particularly its ability to engage and activate HPV 16-specific CD8+ T cells in the context of mixed immune populations in peripheral blood mononuclear cells (PBMCs) and to modulate other key immune cell types in PBMCs. Primary CD8+ T cells were engineered to express the HPV E7 TCR, resulting in HPV E7-specific CD8+ T cells (E7 TCR-T). To determine whether RTX-321’s immune modulatory activities are dependent on the presence of antigen-specific T cells, we examined the effect of RTX-321 on PMBCs admixed with E7 TCR-T as compared to PBMCs alone. RTX-321 selectively expanded HPV E7-specific CD8+ T cells from the PBMC mixture in a dose-dependent manner. RTX-321 preferentially upregulated activation markers and effector molecules, and promoted effector memory on HPV E7-specific CD8+ T cells, compared to HPV independent CD8+ T cells. In addition to these HPV antigen-specific responses, modulations on other immune cells were observed with RTX-321 treatment. RTX-321 induced activation marker upregulation on general CD8+ T cells, as well as NK cell expansion, activation and effector molecule upregulation, independent of the presence of E7 TCR-T. In PBMCs, RTX-321 increased proinflammatory cytokine and chemokine secretion, including IFNγ, TNFα and CXCL10. The addition of E7 TCR-T cells further increased the production of IFNγ and TNFα in PBMCs, suggesting its potential role in further promoting the immune response by secreting effector molecules. Overall, our data indicate that RTX-321 not only engages HPV 16 antigen-specific CD8+ T cells, but also other key immune cell populations of the adaptive and innate immune systems to promote a broad and robust anti-tumor response. An Investigational New Drug application for RTX-321 for the treatment of patients with HPV 16-positive solid tumors is planned by the end of 2020. Citation Format: Mengyao Luo, Shamael S. Dastagir, Xuqing Zhang, Andrea Schmidt, Beatriz Marques, Timothy J. Lyford, Billy Blanco, Laurence A. Turka, Thomas J. Wickham, Tiffany F. Chen. RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO044.

A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4+ T Cells.

Functional and mechanistic studies of CD4+ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (TH17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting cells (APC) for TCR activation. The protocol described here employs plate-bound antibodies and a TH17-polarizing cytokine cocktail to activate and differentiate naïve CD4+ T (Tnai) cells, reflecting a simple and robust protocol for in vitro TH17n differentiation. Using T cells that are genetically engineered with an IL-17 reporter, this protocol may enable the rapid production of a pure population of IL17-expressing CD4+ T cells for system biology studies and high-throughput functional screening.

Engineering advanced dynamic biomaterials to optimize adoptive T-cell immunotherapy

Adoptive T-cell therapy (ACT) is a promising therapeutic approach based on the concept of potent T-cell mediated immunity against the tumor. The outcome of antigen-specific T-cells responses relies on the interaction between T-cells and antigen-presenting cells, which provides signals for generating different T-cell phenotypes with different roles in tumor removal. However, such interaction is often not optimal in vivo and results in low therapeutic efficacy. To reach the full potential of the T-cell response, current research put effort into developing dynamic biomaterials as artificial antigen-presenting cells to study and regulate the T-cell activity for controlling T-cell fate. In this perspective, we provide (1) an overview of ACT and general T-cells behaviors, (2) explore the insight on how biomaterials can be used for studying and regulating T-cell behaviors, (3) and discuss conceptual gaps in knowledge for biomaterials-based immunotherapy.

Size-transformable antigen-presenting cell-mimicking nanovesicles potentiate effective cancer immunotherapy.

Artificial antigen-presenting cells (aAPCs) can stimulate CD8+ T cell activation. While nanosized aAPCs (naAPCs) have a better safety profile than microsized (maAPCs), they generally induce a weaker T cell response. Treatment with aAPCs alone is insufficient due to the lack of autologous antigen-specific CD8+ T cells. Here, we devised a nanovaccine for antigen-specific CD8+ T cell preactivation in vivo, followed by reactivation of CD8+ T cells via size-transformable naAPCs. naAPCs can be converted to maAPCs in tumor tissue when encountering preactivated CD8+ T cells with high surface redox potential. In vivo study revealed that naAPC's combination with nanovaccine had an impressive antitumor efficacy. The methodology can also be applied to chemotherapy and photodynamic therapy. Our findings provide a generalizable approach for using size-transformable naAPCs in vivo for immunotherapy in combination with nanotechnologies that can activate CD8+ T cells.

Biomaterials to enhance antigen-specific T cell expansion for cancer immunotherapy

T cells are often referred to as the ‘guided missiles’ of our immune system because of their capacity to traffic to and accumulate at sites of infection or disease, destroy infected or mutated cells with high specificity and sensitivity, initiate systemic immune responses, sterilize infections, and produce long-lasting memory. As a result, they are a common target for a range of cancer immunotherapies. However, the myriad of challenges of expanding large numbers of T cells specific to each patient's unique tumor antigens has led researchers to develop alternative, more scalable approaches. Biomaterial platforms for expansion of antigen-specific T cells offer a path forward towards broadscale translation of personalized immunotherapies by providing “off-the-shelf”, yet modular approaches to customize the phenotype, function, and specificity of T cell responses. In this review, we discuss design considerations and progress made in the development of ex vivo and in vivo technologies for activating antigen-specific T cells, including artificial antigen presenting cells, T cell stimulating scaffolds, biomaterials-based vaccines, and artificial lymphoid organs. Ultimate translation of these platforms as a part of cancer immunotherapy regimens hinges on an in-depth understanding of T cell biology and cell-material interactions.

Large Scale Ex Vivo Expansion of Γδ T Cells Using Artificial Antigen Presenting Cells for the Treatment of Acute Myeloid Leukemia

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.

A dynamic CD2-rich compartment at the outer edge of the immunological synapse boosts and integrates signals.

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8+ tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.

Novel Survivin Peptides Screened With Computer Algorithm Induce Cytotoxic T Lymphocytes With Higher Cytotoxic Efficiency to Cancer Cells.

The identification of novel biomarkers and therapeutic targets in advanced cancer is critical for improving cancer diagnosis and therapeutics. Survivin (SV) is highly expressed predominantly in most cancer cells and tissues but is absent or undetectable in terminally differentiated normal adult tissues. Therefore, it functions as an almost universal tumor antigen. Peptides are short chains of amino acids linked by peptide bonds. To obtain novel SV decamers that are able to induce SV-specific cytotoxic T lymphocytes (CTLs) with a higher cytotoxic efficiency against cancer cells, major histocompatibility complex (MHC) peptide binding algorithms were conducted to predict nine modified SV95 decamers (from SV95–2 to SV95–10) based on the natural SV95–104 peptide sequence of ELTLGEFLKL (here defined as SV95–1). The fluorescent density of each SV95 peptide was determined by a MHC stability assay, followed by the generation of SV95-specific CTLs with each SV95 peptide (from SV95–1 to SV95–10) and human dendritic cells (DCs) loaded with Poly(lactic-co-glycolic) acid (PLGA) nanoparticles encapsulated with SV95 peptide. Finally, IFN-γ ELISpot and CytoTox 96® Non-Radioactive Cytotoxicity Assays were employed to verify their cytotoxic efficiency of the SV95-specific CTLs generated with the corresponding artificial antigen presenting cells (aAPCs) containing SV95 (SV95–1 to SV95–10) peptide. Furthermore, the cytotoxicity of the SV95 specific CTLs generated with nine mutated SV95 peptides was compared to the one generated with natural SV95–1 peptide and TIL2080 cells. The results indicated that the HLA-A2-restricted mutated SV95 epitope decamers (SV95–6 and SV95–7) showed significant higher binding ability compared to natural peptide SV95–1 in MHC stability assay. More importantly, SV95–specific CTLs with higher cytotoxicity were successfully induced with both SV95–6 and SV95–7 peptides, which significantly eliminated target cells (not only SV95–1 peptide pulsed T2 cells, but also both HLA-A2 and SV positive cancer cells) when compared to those generated with natural SV95–1 peptide and TIL2080 cells. These findings suggest that the SV95–6 and SV95–7 peptides are two novel HLA-A2-restricted CTL epitopes and may be useful for the immunotherapy for patients with survivin expressing cancer.

Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources

BackgroundAdoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care...