Apoptotic Cells for the Prevention of Cytokine Release Syndrome (CRS) in CAR T-Cell Therapy

Abstract Background: Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have demonstrated dramatic promise against highly refractory hematologic malignancies. Clinical responses with complete remission rates as high as 90% have been reported in children and adults with relapsed/refractory acute lymphoblastic leukemia (ALL). However, very significant toxicity has been observed and as many as 30% in average developing severe forms of CRS and possibly related neurotoxicity. CRS is occurring due to large secretion of pro-inflammatory cytokines, mainly from macrophages/monocytes, and resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly additional cytokines. To better understand the mechanisms leading to CRS and to treat or prevent it, we have developed in vitro and in vivo models of CRS with and without CAR-modified T cells. Early apoptotic cells that have been successfully tested for the prevention of acute GVHD, including in 7 ALL patients, were tested in these models for their effect on cytokines and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa cells were used alone or with co-incubation with human macrophages for in vitro experiments and intraperitoneal experiments. Raji was used in vivo for leukemia induction. LPS and IFN-γ were used to trigger additional cytokine release. CD19-specific CAR-modified cells were used (ProMab) for anti-tumor effect against CD19-bearing cells. Cytotoxicity assay was examined in vivo using 7-AAD with flow cytometry and in vitro by survival curves and analysis of tumor load in bone marrow and liver. CRS occurred spontaneously or in response to LPS and IFN-γ. Mouse IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, and IL-7, as well as 32 human cytokines were evaluated by Luminex technology using the MAPIX system analyzer (Mereck Millipore) and MILLIPLEX Analyst software (Merek Millipore). Mouse IL-6Rα, MIG (CXCL9), and TGF-β1 were evaluated by Quantikine ELISA (R&D systems). Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. The IFN-γ effect was evaluated by STAT1 phosphorylation and biological products. Human macrophages and dendritic cells were generated from monocytes. Early apoptotic cells were produced as shown in GVHD clinical trial; at least 50% of cells were annexin V-positive and less than 5% were PI-positive. Results: Apoptotic cells had no negative effect in vitro or in vivo on CAR-modified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence or absence of apoptotic cells in vitro, and comparable survival curves in vivo. On the other hand, significant downregulation (p<0.01) of pro-inflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1α, MIP-1β, was documented. IFN-γ was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1 and IFN-γ-induced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-γ effect on monocyte/macrophages/ dendritic cells without harming IFN-γ levels or CAR-T cytotoxicity. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Amor:Enlivex: Employment. Shabat:Enlivex: Employment.

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Human CD4+ CD25high Regulatory T Cells Inhibit Myeloid but Not Plasmacytoid Dendritic Cells Activation.

Blood ◽

10.1182/blood.v106.11.767.767 ◽

2005 ◽

Vol 106 (11) ◽

pp. 767-767

Author(s):

Roch Houot ◽

Ivan Perrot ◽

Isabelle Durand ◽

Eric Garcia ◽

Serge Lebecque

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Regulatory T Cells ◽

Inflammatory Cytokines ◽

Human Peripheral Blood ◽

Cell Contact ◽

Cytokines Secretion ◽

Pro Inflammatory Cytokines

Abstract CD4+CD25+ regulatory T cells (Treg) are essential negative regulators of immune responses. However, the mechanisms of immune suppression and the spectrum of cells they target remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they might also affect antigen presenting cells (APC). Here, we studied the maturation of human myeloid (mDC) and plasmacytoid (pDC) dendritic cells activated with Toll-like receptor (TLR) ligands in the presence of CD4+ CD25high regulatory T cells in vitro. T cells and DC subsets were purified from normal human peripheral blood. LPS, CpG ODN 2216 and R-848 were used to trigger the maturation of mDC, pDC or both through TLR4, TLR9 and TLR7/8 respectively. Preactivated CD4+ CD25high Treg had no effect on the maturation of pDC. Conversely, they strongly suppressed TLR-triggered mDC costimulatory molecules up-regulation, pro-inflammatory cytokines secretion and their antigen presentation capacity, as opposed to conventionnal T cells (Tconv). At a ratio of 3 Treg for 1 DC, the percentage of mDC acquiring CD80 was reduced 5 fold (from 75% to 16%) while the Mean Fluorescence Intensity was decreased by approximately 65% for CD80 and 35% for CD86 after LPS stimulation and by 50% and 20% after R-848 stimulation. Furthermore, Treg dramatically decreased the secretion of IL-12p40, TNF-α, and IL-6 by mDC (95%, 93% and 50% average inhibition respectively) after LPS activation and to a lesser but still significant extent (38%, 35%, and 38% average inhibition respectively) after R848 stimulation. Finally, we found that Treg-conditionned mDC had a reduced ability to trigger naïve T cell proliferation in a mixed leukocyte reaction. Suppression of mDC activation by Treg appeared to require cell-cell contact. Moreover, the inhibition of pro-inflammatory cytokines secretion, but not of phenotypic maturation, was almost completely restored using an anti-IL10 receptor monoclonal antibody, but not anti-TGFβ nor anti-CTLA-4 blocking antibodies. Those data suggest that Treg prevent the co-stimulatory molecules up-regulation on mDC through contact dependent mechanisms, while the modulation of cytokines secretion appears to be largely mediated by IL-10. Overall, our results provide the first evidence of a direct inhibition of human mDC but not pDC maturation by CD4+ CD25high Treg. Therefore, by restraining the maturation of mDC, human Treg may enlist those cells for the initiation and the amplification of tolerance in vivo.

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Bioengineered Autologous Dendritic Cells Enhance CAR T Cell Cytotoxicity By Providing Cytokine Stimulation and Intratumoral Dendritic Cells

Blood ◽

10.1182/blood-2018-99-115296 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3693-3693 ◽

Cited By ~ 1

Author(s):

Hyung C Suh ◽

Katherine A. Pohl ◽

Christina Termini ◽

Jenny Kan ◽

John M. Timmerman ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Tnf Alpha ◽

Ifn Gamma ◽

Car T Cells ◽

Nsg Mice ◽

Car T

Abstract Background: The combination of antigen recognition, costimulatory ligands, and dendritic cells (DC)-derived cytokines (IL-12 or type I IFNs) stimulate T cells upon antigen presentation of DC. Chimeric antigen receptor (CAR) T cells induce anti-tumor cytotoxicity independent of DC by employing antigen recognition portion (single chain variable fragment)/CD3zeta and costimulatory signaling domain. However, the clinically available CARs are not engineered to provide DC-derived cytokine stimulation to T cells. This deficiency may prevent the CAR T cells from developing optimal effector functions, surviving, and forming a responsive memory T cell population. DC can enhance CAR T cell functionality by producing T cell stimulating cytokines. Intratumoral DC, marked by the expression of CD141/CLEC9A, play a critical role in recruiting T cells into the intratumoral area and inducing T cell cytotoxicity against the tumor. IRF8 is an essential transcription factor in developing intratumoral DCs. A co-stimulatory protein, 4-1BB is expressed on activated T cells and is a part of a CAR construct. 4-1BB has been suggested to stimulate IRF8 through the NF-kB signaling and could participate in the generation of intratumoral DC. Therefore, we hypothesized that autologous DCs transduced with 4-1BB CAR would enhance the efficacy of anti-CD33 CAR T cell therapy against acute myelogenous leukemia (AML) by providing DC-derived cytokines and recruiting CAR T cells in bone marrow microenvironment. Methods: We sorted bone marrow CD34+ progenitors and T cells. Cells were transduced with an anti-CD33 41BBz CAR lentivector (pCCL-HP67.6-4-1BB-CD3z). We sorted transduced T (CAR T), and CD34+ progenitors three days after transduction. While expanding transduced CAR T cells further, we induced the differentiation of transduced CD34+ cells to DC (CAR-DC) in vitro by incubating cells with Flt3L/GM-CSF/IL-4 and AML cell lysate. After an additional four days of culture, we analyzed CAR-DC using flow cytometry. We co-cultured a human AML cell line, Kasumi-1 cells with CAR T +/- CAR-DC (E/T ratio=1), or mock control, and quantified cell death in different CAR T to Kasumi-1 ratios (10, 5, and 2) using CytoTox 96 NonRadioactive Cytotoxicity Assay and Annexin V. We also utilized multiplex cytokine immunoassays to quantify cytokine production. For in vivo studies, we injected luciferase-GFP tagged Kasumi-1 cells (10X106) into NSG mice, followed by injection of CAR T (5X105) +/- CAR-DC (1.5X105) or control T cells (5X105). We monitored the NSG mice using serial bioluminescence imaging and compared the survival of each group. Results: On phenotypic analysis using flow cytometry, we found that frequencies of cells expressing CD141/CLEC9A+ were significantly higher in CAR-DC vs. control DC (35.2 +/- 4.1 % vs. 9.0 +/- 1.7 % of HLA-DR+ cells), which suggest 4-1BB activation induce CD34+ progenitors to intratumoral DCs. The cytotoxicity assay showed 63.2 +/- 0.6 % Kasumi-1 death with CAR T/CAR-DC compared to 46.5 +/- 3.5 % with CAR T cells alone. CAR T/CAR-DC also demonstrated more Annexin V positive Kasumi-1 cells compared to CAR T and control T cells (78.4 +/- 5.1 % vs 39.9 +/- 7.7 % vs 17.6 +/- 2.2 %). These cytotoxicity assays demonstrated that CAR-DC enhanced the anti-Kasumi-1 cytotoxicity of anti-CD33 CAR T cells. CAR T cells co-cultured with CAR-DC produced a two-fold higher IFN-gamma and TNF-alpha than CAR T cells alone (p<0.01). The IFN-gamma and TNF-alpha production increases in correlation with the counts of CAR T cells. However, CAR T/CAR-DC group produced a four-fold higher IL-12 throughout different E/T ratios compared to CAR T alone group (p<0.01), which suggest DCs are the major source of IL-12 production and CAR T cells produce a higher level of IFN-gamma and TNF-alpha in response to DCs. In vivo NSG mice experiments demonstrated that CAR T/CAR-DC group had increased survival (p<0.01) and decreased AML burden than CAR T alone group. Conclusions: Our data show that 1) in vitro differentiation of DCs with 4-1BB stimulation increases intratumoral CD141/CLEC9A+ DCs, 2) interaction between CAR-DC and CAR T cells enhances cytotoxic cytokine production in response to DC-derived IL-12. These combined effects resulted in improved anti-CD33 CAR T cytotoxicity in vitro and in vivo NSG AML mice model. Our findings implicate the development of a new strategy of CAR T therapy combined to CAR-DC to increase the efficacy of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

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98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0098 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A109-A109

Author(s):

Jiangyue Liu ◽

Xianhui Chen ◽

Jason Karlen ◽

Alfonso Brito ◽

Tiffany Jheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Next Generation ◽

Phase 3 ◽

T Cell Therapy ◽

Cell Therapies ◽

Car T Cells ◽

Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

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Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy

Leukemia ◽

10.1038/s41375-021-01127-2 ◽

2021 ◽

Author(s):

Kinan Alhallak ◽

Jennifer Sun ◽

Katherine Wasden ◽

Nicole Guenthner ◽

Julie O’Neal ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Escape ◽

Multiple Cancer ◽

Cancer Antigen ◽

Cancer Antigens ◽

Car T ◽

Modular Platform

AbstractT-cell-based immunotherapy, such as CAR-T cells and bispecific T-cell engagers (BiTEs), has shown promising clinical outcomes in many cancers; however, these therapies have significant limitations, such as poor pharmacokinetics and the ability to target only one antigen on the cancer cells. In multiclonal diseases, these therapies confer the development of antigen-less clones, causing tumor escape and relapse. In this study, we developed nanoparticle-based bispecific T-cell engagers (nanoBiTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies (mAbs) targeting T cells, and mAbs targeting the cancer antigen. We also developed a nanoparticle that targets multiple cancer antigens by conjugating multiple mAbs against multiple cancer antigens for T-cell engagement (nanoMuTEs). NanoBiTEs and nanoMuTEs have a long half-life of about 60 h, which enables once-a-week administration instead of continuous infusion, while maintaining efficacy in vitro and in vivo. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in myeloma cells in vitro and in vivo, compared to nanoBiTEs targeting only one cancer antigen. Unlike nanoBiTEs, treatment with nanoMuTEs did not cause downregulation (or loss) of a single antigen, and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies.

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Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽

10.1038/s41375-021-01282-6 ◽

2021 ◽

Author(s):

Christos Georgiadis ◽

Jane Rasaiyaah ◽

Soragia Athina Gkazi ◽

Roland Preece ◽

Aniekan Etuk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Specific Binding ◽

Base Editing ◽

Car T Cells ◽

Dna And Rna ◽

Car T ◽

T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

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109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0109 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A121-A121

Author(s):

Nina Chu ◽

Michael Overstreet ◽

Ryan Gilbreth ◽

Lori Clarke ◽

Christina Gesse ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Dominant Negative ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.6 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A3.2-A4

Author(s):

J Grün ◽

I Piseddu ◽

C Perleberg ◽

N Röhrle ◽

S Endres ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

B Cells ◽

Regulatory T Cells ◽

Type I ◽

List Type ◽

Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

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124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.124 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A133-A133

Author(s):

Cheng-Fu Kuo ◽

Yi-Chiu Kuo ◽

Miso Park ◽

Zhen Tong ◽

Brenda Aguilar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

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323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.323 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A348-A348

Author(s):

Jessie Wang ◽

Kaixia Lian ◽

Jia Zheng ◽

Chenpan Nie ◽

Annie An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Immunity ◽

Immune Evasion ◽

Syngeneic Mouse ◽

Ifn Γ ◽

Draining Lymph Nodes ◽

Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

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Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

Frontiers in Immunology ◽

10.3389/fimmu.2021.577766 ◽

2021 ◽

Vol 12 ◽

Author(s):

Darina Ocadlikova ◽

Mariangela Lecciso ◽

Javier Martin Broto ◽

Katia Scotlandi ◽

Michele Cavo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cell Lines ◽

Effector T Cells ◽

Sarcoma Cell ◽

Ifn Γ ◽

Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

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