scholarly journals Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens

2021 ◽  
Vol 9 (7) ◽  
pp. e002401
Author(s):  
Anna Luise Bernhardt ◽  
Julia Zeun ◽  
Miriam Marecek ◽  
Hannah Reimann ◽  
Sascha Kretschmann ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Lines ◽  
T Cell Activation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cell Activation ◽  
T Cell Epitopes

BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity.MethodsWe created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells.ResultsBy testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire.ConclusionWe successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.

Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3742-3742
Author(s):  
LeShara M Fulton ◽  
Michael J Carlson ◽  
James Coghill ◽  
Michelle L. West ◽  
Angela Panoskaltisis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Allogeneic Stem Cell Transplantation ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Abstract 3742 CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results: B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. Conclusions: T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Type I Interferon Signaling before Hematopoietic Stem Cell Transplantation Lowers Donor T Cell Activation Via Reduced Allogenicity of Recipient Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4431-4431
Author(s):  
Erik Thiele Orberg ◽  
Julius Clemens Fischer ◽  
Sascha Göttert ◽  
Florian Bassermann ◽  
Hendrik Poeck
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Type I ◽  
Hematopoietic Stem ◽  
Conditioning Therapy

Background: Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterised. Methods: We applied selective RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Results: Using IFNAR1-deficient donor T and hematopoietic donor cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these respective cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted T-cells after allo-HSCT. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signalling. Notably, this immunosuppressive function of DCs was restricted to a scenario of genotoxic tissue damage as neither RIG-I activation and IFN-I induction in naive (non-irradiated) mice altered allogeneic T cell activation. Conclusion: Our findings uncover a hitherto unknown IFN-I- and context dependent immunosuppressive function of dendritic cells. This needs to be considered in the development of IFN-I based therapeutic approaches to modulate donor T cell activation after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Donor CTLA-4 Gene Polymorphism Associations with Acute GvHD After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2010-2010
Author(s):  
Irena Frydecka ◽  
Lidia Karabon ◽  
Edyta Pawlak-Adamska ◽  
Anna Tomkiewicz ◽  
Tomasz Wrobel ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Hematopoietic Stem

Abstract Abstract 2010 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been widely carried out as a therapy for several hematological malignances and non malignant disorders. Graft–versus -host disease is one of the major complications after allo-HSCT with main cause of morbidity and mortality. Donor T lymphocytes play the crucial role in alloimmune recognition and their ability to detect non –self antigens can lead to aGvHD. The effective recognition and activation of naïve T-cells requires two independent signals. The first, an antigen-specific signal, is sent via the T-cell receptor (TCR) on T-cells. The second signal, termed co-stimulation, is critical for allowing full activation, sustaining cell proliferation, preventing anergy and/or apoptosis, inducing differentiation to effector cells. CD28 is the primary T-cell co-stimulatory molecule. Cytotoxic T-cell antigen (CTLA-4) is a homologous molecule of CD28 which plays an inhibitory role in the early and late stages of T-cell activation. CTLA-4 ligation provides a negative signal for regulation of the cell cycle and inhibits the activity of the transcriptional factors: nuclear factor-kB (NF-kB), nuclear factor of activated T-cells (NF-AT), and activator protein 1 (AP-1). Moreover, CTLA-4 binds to CD28 ligands (CD80 and CD86) with higher affinity and avidity and in that way also inhibits T-cell activation. Since co-stimulatory and down regulatory molecules synthesis depend on the rate of gene transcription and/or translation, polymorphisms in the corresponding genes might result in abnormal expression, function as well as dysregulated trafficking of these molecules within cellular compartments. The human CTLA-4 gene is located on 2q33 which is susceptibility region for autoimmune diseases. The aim of this study is to investigate the associations between polymorphisms in CTLA-4 gene: CTLA-4c.49A>G (rs231775), CTLA-4g.319C>T (rs5742909), CTLA-4g6230G>A (CT60, rs3087243), CTLA-4g.10223G>T (Jo31, rs11571302) in donors of HSTC and occurrence of aGvH disease in recipients after allogeneic hematopoietic stem cell transplantation. Altogether 136 donors of HSCT (58- related donors, 88 haploidentical unrelated donors) were genotyped for all polymorphisms using allelic discrimination methods with the TaqManÒ SNP Genotyping Assay. In patients without aGvHD and in patients with aGvHD grade I-IV the similar distribution of alleles and genotypes for all investigated polymorphisms in donors was observed. However, we have noticed trend toward increased frequency of CT60 [G] donor allele among recipients with aGvHD I-IV (0.48 vs. 0.39, p=0.1, OR 1.49, 95% CI: 0.90–2.49) compared to recipients without aGvHD in whole group of patients. In patients transplanted from related donor also increased risk of aGvHD grade I-IV was observed for CT60 [G] donor allele (0.75 vs. 0.55, p=0.09, OR 2.11, 95%CI: 0.88–5.26). In contrary the frequencies of CT60 [G] donor allele in patients transplanted from unrelated donors are similar in recipients with and without aGvH symptoms. Haplotype estimation analysis indicated that donor haplotype CTLA-4c.49A>G[A], CTLA-4g.319C>T[C], CT60 [A], Jo31 [T] tended to be protective against aGvHD grade I-IV in whole studied group of patients (0.28 vs. 0.40, p=0.06, OR 0.60, 95% CI: 0.36–1.02) This association reach statistical significance in recipients of related transplantation (0.18 vs. 0.43, p=0.01, OR 0.29, 95% CI: 0.14–0.97) Our study indicated that donor CT60 polymorphism might be associated with occurrence of aGvHD, especially in recipients transplanted from HLA-identical sibling donors. Disclosures: No relevant conflicts of interest to declare.

Improved Vaccine Design For Adoptive Immunotherapy In Hematological Malignancies Through Chemically Modified Minor Histocompatibility Antigen Epitopes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5435-5435
Author(s):  
Rimke Oostvogels ◽  
Rieuwert Hoppes ◽  
Henk Lokhorst ◽  
Robbert M Spaapen ◽  
Huib Ovaa ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Hematological Malignancies ◽  
Ex Vivo

Abstract Allogeneic stem cell transplantation (allo-SCT), alone or followed by donor lymphocyte infusion (DLI), is a potentially curative treatment for various hematological malignancies. In an HLA-matched transplantation setting, the therapeutic graft-versus-tumor (GvT) effect is mediated by donor T-cells directed at minor histocompatibility antigens (mHags), which are HLA-bound polymorphic peptides. Unfortunately, most patients don’t achieve complete response or relapse after allogeneic stem cell transplantation and thus still require additional therapies. Immunotherapy aimed at hematopoietically restricted mHags could theoretically provide an ideal method to augment the GvT effect, without causing GvHD. The most relevant mHags for immunotherapy are those antigens that are only expressed on hematopoietic tissue, are presented by frequent HLA molecules and display an equally balanced population frequency. UTA2-1 and HA-1 are two of these most broadly applicable mHags identified up until now and are therefore included in on-going clinical trials of mHag-peptide loaded dendritic cell vaccination in patients with various hematological malignancies. Another method for mHag-based immunotherapy could be adoptive transfer of ex vivo cultured mHag-specific cytotoxic T lymphocytes (CTL). However, initial results of both methods, also from preclinical models and trials in patients with solid tumors, postulate the necessity for improved strategies for efficient ex vivo and in vivo induction of tumour specific CTLs. We here show for the HLA-A*02 restricted epitopes UTA2-1 and HA-1 that their MHC binding and consequent T cell reactivity can be improved through the incorporation of certain newly designed non-proteogenic amino acids at crucial MHC anchoring positions. With this novel approach we designed superior altered peptide ligands (APLs) for both epitopes, of which the best modifications not only increased MHC binding and stability, but also improved recognition by antigen specific T cells. Most importantly, these optimised peptides gave rise to superior antitumor T cell responses in vitro and in vivo in comparison to the native epitope, as they induced significantly enhanced proliferation of peptide-specific T cells with retained cytotoxic potential against malignant targets expressing the natural UTA2-1 antigen. Hence, these APLs designed with non-proteogenic amino acids with enhanced MHC-affinity and immunogenicity may improve the therapeutic outcome of mHag-based vaccination strategies, or can be utilized for ex vivo antigen-specific T cell enrichment and expansion for transfer into patients with haematological malignancies. Disclosures: Lokhorst: Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Epstein Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes (CTL) Expressing an Anti-CD30 Chimeric T Cell Receptor (CTCR) for the Treatment of Hodgkin’s Disease (HD).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 745-745
Author(s):  
B. Savoldo ◽  
C. M. Rooney ◽  
H. E. Heslop ◽  
H. Abken ◽  
A. Hombach ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cell Lines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Barr Virus

Abstract HD may be a suitable target for immunotherapy, and in patients with EBV-associated HD, adoptive transfer of EBV-CTL has produced disease responses. An alternative target is the CD30 molecule, which is present on the malignant cells of almost all patients with HD. CD30 is a member of the TNF superfamily and monoclonal antibodies directed to this antigen are currently under investigation in patients with relapsed HD. An alternative way to target CD30 is by the construction of T cells expressing cTcR specific for the antigen. T lymphocytes engineered to express this cTcR can specifically kill CD30+ HD cell lines {Cancer Res,1998;58:1116}. However, these chimeric molecules connect the antigen-recognition properties of CD30 antibodies with the endodomain of CD3ζ, which is insufficient to fully activate resting T cells to proliferate and release cytokines. As a consequence chimeric T cells that express these endodomains divide infrequently, lose activity and have performed poorly in-vivo. Full T cell activation requires receptor engagement to be accompanied by a sequence of co-stimulatory stimuli. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and further expanded with 3–4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4±16% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM-2=45.4±16% and Karpas-299=42.5±17%). Killing of CD30+ tumor cells was significantly inhibited by preincubation with an anti-CD30 blocking antibody (16.5±12%). Of potential concern, however, is that CD30 is expressed by activated normal T lymphocytes: expression was undetectable on resting T cells, but increased to 3–32% on day 4–7 after stimulation with LCL. Fortunately, expression dwindles to 3–6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTcR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat both EBV+ and EBV− HD.

scholarly journals Characterizing the Immunogenicity of DM-Sensitive and DM-Resistant Antigens

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3310-3310
Author(s):  
Anna Luise Bernhardt ◽  
Sascha Kretschmann ◽  
Judith Bausenwein ◽  
Heidi Balzer ◽  
Andreas Mackensen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Amino Acid ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Amino Acid Substitutions

Abstract Introduction: The separation of graft-versus-leukemia (GvL) effect from graft-versus-host-disease (GvHD) is a major objective after allogeneic stem cell transplantation. We recently described two types of endogenous HLA class II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, presentation of DM-sensitive antigens rely on co-expression of HLA-DO - the natural inhibitor of HLA-DM. Since the expression of HLA-DO is not upregulated by inflammatory cytokines and restricted to B-cells, dendritic cells and thymic epithelial cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues. Therefore, usage of CD4 T-cells directed against DM-sensitive antigens might allow separation of GvL from GvHD. However, it remains elusive whether immunogenicity and anti-tumorigenic potential of DM-sensitive and DM-resistant antigens have comparable properties in vivo. Methods: Therefore, we sought to create an in vivo system using a DM-sensitive and a DM-resistant variant of the same model antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM or HLA-DO, respectively) to allocate the two model antigens ovalbumin (OVA) and murine Y-chromosome antigen DBY to their category. Furthermore, we introduced one to three amino acid substitutions within the MHC II restricted T-cell epitopes of the two antigens and tested DM-sensitivity or DM-resistance by T-cell activation using proliferation and IFN-g secretion as read-out in vitro. Finally, we vaccinated B6 mice with the generated epitope variants and measured expansion, phenotype and reactivity of OVA- or DBY-specific CD4 T-cells in vivo. Results: By testing T-cell recognition of OVA or DBY on murine B-cell lines overexpressing H2-M and H2-O, respectively, we could show that OVA leads to a more potent T-cell activation in the presence of H2-O demonstrating its DM-sensitive character. In contrast the wildtype epitope of DBY does not rely on H2-O expression for strong T-cell activation and was therefore assessed as DM-resistant antigen. By introducing one to three amino acid substitutions within the T-cell epitope we could generate one further DM-sensitive variant of OVA but also two DM-resistant counterparts. Likewise, we designed both DM-resistant and DM-sensitive epitope variants of murine DBY. To assess T-cell receptor avidity to our epitope variants presented on natural antigen presenting cells, titration of DM-sensitive and DM-resistant variants of the same antigen on untreated splenocytes from OVA or DBY T-cell receptor transgenic mice, respectively, were performed. We observed comparable activation of the same T-cell clone activated by either variant of the epitope as measured by proliferation and IFN-g secretion. Furthermore, upon vaccination of B6 mice with either variant of the epitope we could measure comparable expansion, phenotype, and reactivity of OVA- and DBY-specific T-cells both invivo and ex vivo. Conclusion: We successfully generated DM-sensitive and DM-resistant variants of the same epitope for the two model antigens OVA and murine DBY. With this tool we could demonstrate that DM-sensitive antigens are not inferior to their DM-resistant counterpart. Therefore, targeting DM-sensitive antigens after allogenic stem cell transplantation might be an interesting tool to improve the GvL effect with only limited GvHD. Disclosures Bernhardt: DFG TRR221/project A1 (German Research Foundation): Research Funding.

scholarly journals Inducible MyD88/CD40 Allows AP1903-Dependent Costimulation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1121-1121 ◽  
Author(s):  
Aaron Foster ◽  
Aruna Mahendravada ◽  
Peter Chang ◽  
Nicholas Shinners ◽  
Kevin Slawin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Cell Activation ◽  
First Generation ◽  
Employment Equity ◽  
Co Activation ◽  
Equity Ownership

Abstract Introduction: Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) has begun to show impressive clinical results. The efficacy of T cell therapy is dependent not only on tumor recognition, but also on the survival and expansion of T cells following infusion. T cells modified with CAR constructs encoding costimulatory domains such as CD28 or 4-1BB have the capacity to rapidly proliferate in vivo, but severe toxicities have been observed due to unchecked T cell activation. Thus, strategies to regulate T cell activation in vivowould allow physicians to prevent toxicities and maximize anti-tumor efficacy. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), that can be activated by a small molecule chemical inducer of dimerization, AP1903, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a biscistronic retrovirus encoding myristolated tandem AP1903 binding domains (FKBPv36), cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and truncated CD19 to identify transduced T cells (SFG-iMC.2A.ΔCD19). Control vectors without signaling elements, or with only MyD88 or cytoplasmic CD40 were also used to generate gene-modified T cell lines. iMC activation was measured by treating T cells with and without AP1903 and measuring cytokine production by ELISA, and assessing cell surface activation markers by flow cytometry. Co-activation of T cells through CD3ζ in combination with iMC was accomplished using anti-CD3 antibodies, or by co-transducing T cells with first generation CAR constructs recognizing prostate stem cell antigen or CD19 (PSCA.ζ or CD19.ζ, respectively), and coculturing T cells with PSCA+ (Capan-1) or CD19+ tumor cell lines (Raji, Daudi and Nalm-1) with and without AP1903. Efficacy of iMC-modified CAR T cells were assessed using NOD scid gamma (NSG) immune deficient mice engrafted with tumor cell lines followed by intravenous injection of T cells. The iMC costimulatory molecule was subsequently activated in vivo by intraperitoneal injection of AP1903 (5 mg/kg). Tumor burden was assessed and T cell expansion in vivowas measured by bioluminescent imaging using an IVIS instrument. Results: T cells transduced with iMC produce cytokines (e.g. IFN-γ, TNF-α, IL-6) in response to AP1903. iMC activation permits T cell survival in the absence of growth cytokines, such as IL-2, but by itself is not sufficient to induce IL-2 production or autonomous growth. Interestingly, AP1903-induction of MyD88 or cytoplasmic CD40 alone showed minimal T cell activation, suggesting potential synergy of the two signaling molecules. However, co-activation of the T cell receptor (TCR) with soluble anti-CD3 and iMC with AP1903 upregulated CD25 expression, induced IL-2 production and promoted T cell expansion. Importantly, endogenous TCR signaling could be substituted by a PSCA-specific CAR linked to the CD3 ζ endodomain (PSCA.ζ CAR), where co-activation of iMC by AP1903, and CAR by tumor cells expressing PSCA (Capan-1) induced high levels of IL-2 secretion, CD25 upregulation and rapid T cell proliferation. Similar results were achieved using T cells transduced with iMC-enabled CD19 CAR (SFG-iMC.2A.CD19.ζ) when cocultured with CD19+lymphoma cell lines. Treatment of tumor bearing immunodeficient mice with T cells modified with iMC and PSCA.ζ CAR showed enhanced antitumor efficacy when mice were administered with AP1903 dimerizer. Bioluminescence imaging also demonstrated marked proliferation and persistence of iMC-transduced T cells in response to AP1903 administration. Following AP1903 withdrawal, T cell levels declined, consistent with the requirement for costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3 ζ) domain from signal 2 costimulation (iMC) provides a unique mechanism by which T cells can be expanded only in response to both AP1903 and tumor antigen, or reduced in number by withdrawal of AP1903-induced iMC costimulation. Disclosures Foster: Bellicum Pharmaceuticals: Employment, Patents & Royalties. Mahendravada:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

scholarly journals A Novel CD34-Specific T-Cell Engager Efficiently Depletes Stem Cells and Acute Myeloid Leukemia Cells in Vitro and In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2861-2861
Author(s):  
Lucas C M Arruda ◽  
Liqing Jin ◽  
Melanie Lambert ◽  
Laura Sanchez Rivera ◽  
Renato Alvez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Activation ◽  
Privately Held

Abstract ASH Abstract. Intro Acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) are poor prognosis hematological malignancies characterized by abnormal hematopoiesis and dysfunctions of the hematopoietic stem cell system. Chemotherapy remains the standard of care but is associated with side effects and often high rates of relapse. Today, less than a third of patients diagnosed with AML are cured. Bispecific T-cell engagers (BiTEs) are promising immunotherapeutic agents intended for cancer treatment. BiTEs are small molecules constructed of two single chain variable fragments (scFv) connected in tandem by a flexible linker that acts by retargeting T-cells against tumor cells. One scFv binds to CD3, while the second scFv binds to a tumor-associated antigen. This structure and specificity allow a BiTE construct to physically link a T-cell to a tumor cell, stimulating effector cell activation ultimately leading to cytokine production and tumor killing. Material BiTEs against CD34/CD3 and relevant controls were constructed by recombinant DNA technology and purified from the supernatants of transfected CHO cells following standard procedures. The scFv domain binding to CD34 is positioned N-terminally, and the scFv binding to CD3e C-terminally followed by a hexa-histidine sequence. Results By co-culturing T-cells and target AML cells for 48 h in the presence of increasing concentrations of BiTE or controls, we observed that CD34-BiTE efficiently triggered T-cell-mediated depletion of the CD34 hi and CD34 low cell lines, while negative controls killed none of the target cell lines. Next, we examined the T-cell activation and proliferation. We observed that both CD4+ and CD8+ T-cells presented high levels of CD25/CD69 expressions when the CD34+ cell lines were co-cultured with T-cells in the presence of the CD34/CD3 BiTE. No unspecific activation was found when CD34- cell line was used as target cell. Since CD34 is constitutively expressed by HSCs, the CD34-specific BiTE may deplete not only CD34 +AML blasts but also healthy HSCs. To test this, T-cells and HSCs were purified from PBSC grafts and co-cultured in the presence of either CD34/CD3 BiTE or controls. After co-culture, a significant depletion of CD34 + HSCs was observed for the CD34/CD3 BiTE. To address the potential of the anti-CD34 BiTE in vivo, we next established a human CD34 + cell line in NSG mice per intravenous injection and randomized into three different groups and started treatment the day after. Two groups of mice received two consecutive cycles of one intraperitoneal injection of freshly isolated human T-cells followed by daily intravenous injections of either BiTE or control. The mice were euthanatized at day 21 by which the AML burden was measured, and T-cells quantified. No side effects of the treatment, including after BiTE administration, was observed. There were statistically significant reductions of leukemia burden in both bone marrow and spleen in mice receiving T-cells and BiTE compared to T-cells only and control. Conclusions We show that the CD34/CD3 BiTE is able to promote T-cell activation and killing of CD34-expressing target cells with high efficacy in vitro and in vivo, supporting the translation of this drug into clinical trials. In this scenario, the treatment with CD34-targeting BiTE prior to HSCT would trigger the patient's T-cells to deplete CD34 + leukemic blasts and HSCs. As consequence, this adjuvant treatment would decrease the use of cytotoxic and cytostatic conditioning drugs before HSCT, reducing life-threatening complications such as GvHD and infections. Disclosures Arruda: Anocca: Current Employment, Research Funding. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mattsson: MattssonAB medical: Current Employment, Current holder of individual stocks in a privately-held company. Onfelt: Desumo: Current Employment, Current holder of individual stocks in a privately-held company. Uhlin: XNK therapeutics: Current Employment, Current holder of stock options in a privately-held company.

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