IFN-gamma Secretion Research Articles

IL-12p35 and IL-12p40 functionally synergize to active NK cells

Abstract IL-12 plays critical roles in regulating the immune response toward different effector pathways. The formation of functional IL-12 requires association of the IL-12p35 subunit with the IL-12p40 subunit. IL-12p40 can be released from these hematopoietic cells as a monomer. Previous studies have reported that IL-12p40 associates with p35 extracellularly to generate IL-12 like activities on T cells, but the biological significance on NK cells remains unclear. In this study, we show that only high concentrations of IL-12p35 could activate NK cells to secrete IFN-gamma and induce cell proliferation and IL-12p40 had no effect on NK cell activation. Interestingly, IL-12p35 and p40 functionally synergized to activate NK cells by inducing IFN-gamma secretion and cell proliferation in vitro. Surprisingly, pretreatment of NK cells with IL-12p35 or p40 didn’t affect IL-12 induced IFN-gamma secretion and cell proliferation in NK cells. Furthermore, a functional ELISA binding assay revealed that IL-12p35 binds to both IL-12R beta1 and IL-12R beta2 with a low affinity, while IL-12p40 only binds to IL-12R beta1 with a high affinity. Fluorescent conjugated human IL-12, IL-12p35 and IL-12p40 proteins all significantly bound to human NK cells and IL-12p35 and p40 didn’t significantly interfere with IL-12 binding to these cells. Collectively, the data we show herein indicates that IL-12 activity can come from the collaboration between the IL-12p35 and p40 subunits and impact NK cell activation.

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

T cell redirecting and TLR7/8 agonist encapsulated liposomes targeting HER2+ tumors: Preclinical efficacy, pharmacokinetics and safety evaluations.

e14590 Background: T-cell dependent bispecific antibodies (TDBs) have shown limited success in solid tumor treatments. We hypothesized that it may be due to TDBs limited distribution in solid tumor tissues. We therefore propose to take advantage of the EPR (enhanced permeability and retention) effect of the liposome system to improve the PKPD properties of TDBs. The liposomes are named TRAFsomes (T cell Redirecting Antibody Fragment-anchored Liposomes). HF5050 is a TRAFsome containing on average 6 anti-CD3 and 10 anti-Her2 on each liposome surface and the toll-like TLR-7/8 agonist Resiquimod encapsulated inside at specific lipid to drug ratios. It preclinical efficacy, pharmacokinetics and safety evaluation studies are conducted to support the IND application. Methods: The T cell redirecting and target specific cytotoxic effect of HF5050 were evaluated in five PBMC co-cultured cancer cell models with different Her2 expression. Tumor cells derived from adenoid cystic carcinoma tumor tissues was also included. T cell redirection and activation are examined by T cell surface markers CD25/CD69 and secretion of IFN gamma, Perforin, and granzymes B in the absence and presence of cancer cells. For the incorporation of TLR7/8 agonist Resiquimod, we evaluated the effect of drug dose by varying the drug loading ratios in order to achieve a synergistic effect. For the in vivo efficacy studies, the N87 gastric cancer CDX model and an adenoid cystic carcinoma tumor PDX model were set on PBMC reconstructed humanized NCG-B2M mice. Pharmacokinetic and tissue distribution studies are carried out in tumor bearing mouse models as well. Results: HF5050 has shown to be a highly effective anti-cancer agent in both in vitro and in vivo studies. It mediates T cells-tumor cells redirection and cytotoxicity in a dose-dependent manner, especially in Her-2 highly expressing cell lines. HF5050 also effectively stimulates T cell activation and displays high efficacy in PBMC reconstruct humanized NCG-B2M tumor-bearing mice. Pharmacokinetic research confirms liposome successfully maintains drug levels in the bloodstream, and improves drug accumulation within tumor tissues. Conclusions: Overall, these data demonstrate that HF5050 processing a novel cell bridging function between T cells and tumor cells, and with the capability of activating immune cells. Based on the encouraging preclinical results, HF5050 exhibits promising development potential.

MRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth.

The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.

EFFECT OF ARTIFICIAL MITOCHONDRIAL TRANSFER FROM LINE CELLULAR HSC-3 TO LYMPHOCYTE TCD4+, AS A MECHANISM INDUCTOR OF EXHAUSTED PHENOTYPE.

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer. The tumor microenvironment of OSCC induces an alteration of the T lymphocyte, promoting an exhausted phenotype. The main organelle of metabolism is the mitochondrion and in recent years it has been indicated that several cells have the capacity to transfer mitochondria, including cancer cells. However, it has not been evaluated whether mitochondria transfer from cancer cells to T lymphocytes induces an exhausted phenotype in T lymphocyte. The aim of this work was to analyze the exhausted phenotype in TCD4+ lymphocytes after artificial transfer of mitochondria (MitoCeption) obtained from the oral cancer cell line HSC-3. Methods: Isolated MitoTracker-labeled mitochondria from oral cancer tumor cells were Mitocepted into CD4+ T lymphocytes. Then, surface molecule expression, proliferation and cytokine secretion mediated by tumor mitochondrial transfer were analyzed by flow cytometry. Results: The results showed that TCD4+ lymphocytes that acquired mitochondria had increased expression of 2 inhibitory proteins (TIGIT and CTLA4) and 3 proteins associated with exhausted phenotype (PD-1, PLD-1 and LAG3), compared to the control group. In addition, the mitocepted lymphocytes exhibited a significant decrease in proliferation compared to the control. For cytokine analysis, a significant decrease was observed in the mitocepted group in the secretion of IFN-gamma, TNF-alpha, IL-10 and IL-4, but not IL-17, compared to the control. Conclusions: Therefore, we concluded that the acquisition of isolated mitochondria from HSC-3 cancer cells by CD4+ T lymphocyte induces an exhausted phenotype in the TCD4+ lymphocyte. Regular Fondecyt Project 1211480

The Negative Impact of Triptolide on the Immune Function of Human Natural Killer Cells.

Triptolide (TP), a bioactive compound extracted the from traditional Chinese medicine Tripterygium wilfordii Hook F (TwHF), has been shown to be effective in treating several autoimmune diseases, and has suppressive effects in several key immune cells such as dendritic cells, T cells, and macrophages. However, it is unknown whether TP has an impact on natural killer (NK) cells. Here, we report that TP has suppressive effects on human NK cell activity and effector functions. The suppressive effects were observed in human peripheral blood mononuclear cell cultures and purified NK cells from healthy donors, as well as in purified NK cells from patients with rheumatoid arthritis. TP treatment induced downregulation of NK-activating receptor (CD54, CD69) expression and IFN-gamma secretion, in a dose-dependent manner. When exposed to K562 target cells, TP treatment induced inhibition of surface expression of CD107a and IFN-gamma synthesis in NK cells. Furthermore, TP treatment induced activation of inhibitory signaling (SHIP, JNK) and inhibition of MAPK signaling (p38). Thus, our findings demonstrate a previously unknown role for TP in NK cell functional suppression and reveal several key intracellular signaling that can be regulated by TP. Our findings also offer new insight into mechanisms of TP therapeutic treatment in autoimmune disease.

In vitro detection of suspected drug in maculopapular drug reaction to antibiotics using secreted cytokines from drug-specific activated T cells.

Maculopapular drug eruption (MPE) in reaction to antibiotics is associated with enhanced expression of T-helper (Th)1 cytokines such as interferon gamma (IFN-gamma) or Th2 cytokines such as interleukin (IL)-5. Identifying the culprit drug usually involves rechallenge, which may not be forthcoming. Memory lymphocytes remain responsive to the culprit drug long after the reaction has resolved. On reactivation in vitro, there is increased proliferation and expression of certain markers, which provides us with an opportunity to predict the causal drug. The study aimed to assess drug-specific cytokine production (IL-5 and IFN-gamma) in peripheral blood mononuclear cell (PBMC) culture supernatants to predict the causal antibiotic in cases of MPE. PBMCs of 18 patients who developed MPE to 20 suspected antibiotics (2 patients had 2 suspected antibiotic allergies each), along with 11 drug-matched healthy controls, were incubated for 5 days with the respective drugs at 2 different concentrations. Secreted cytokines were measured in the supernatants, using enzyme-linked immunosorbent assay (ELISA) for IL-5 and IFN-gamma, at 6 h of incubation, then on day 2 and day 5. Drug-specific IL-5 and IFN-gamma production could be demonstrated in 65% and 74% of the cases, respectively. Maximal secretion of IL-5 and IFN-gamma was observed on day 5 and day 2 of incubation, respectively. The cut-off delta values, defined as the difference in cytokine concentration between drug-stimulated and unstimulated samples, were 4 pg mL-1 for IL-5 and 6 pg mL-1 for IFN-gamma. The measurement of drug-specific secretion of IL-5 and IFN-gamma using ELISA is a valuable method for detecting antibiotic-induced MPE.

LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

Abstract 2889: Novel bispecific humanized BCMA-CD3 antibodies specifically target multiple myeloma cells

Abstract BCMA antigen is overexpressed in multiple myeloma cells and has been shown to be a promising target for novel cellular and antibody therapeutics. The humanized BCMA (clone 4C8A) antibody that effectively targeted multiple myeloma in a CAR (chimeric antigen receptor) format was used for designing several formats of bispecific BCMA-CD3 antibodies. Several different designs including univalent and bivalent BCMA-CD3 CrossMAB; triple chain BCMA-FAB-CD3 ScFv-Fc and tetravalent BCMA ScFv-CD3 ScFv-Fc antibodies were tested for affinity and binding with BCMA-positive cells and T cells. Real time cytotoxic activity (RTCA) and IFN-gamma secretion were also tested with multiple myeloma and CHO-BCMA target cells. All BCMA bispecific antibody designs demonstrated specific affinity with CHO-BCMA but not CHO cells in the nanoM range of 0.3-33 nM; all designs bound to T cells by FACS. The triple chain BCMA-FAB-CD3 ScFv-Fc designs had the highest Kd of binding with CHO-BCMA cells (Kd=0.3 nM) and with multiple myeloma MM1S, and H929 cells (0.1 and 0.3 nM, respectively). All antibodies with T cells specifically killed CHO-BCMA and multiple myeloma cells in a dose-dependent manner but not CHO or K562 cells, measured by RTCA assay. The bivalent BCMA-CD3 CrossMAB and triple chain BCMA FAB CD3scFv-Fc antibodies with T cells secreted IFN-gamma with EC50 =0.01-0.02 nM. In addition, the BCMA bispecific antibodies had high in vivo efficacy using a MM1S xenograft NSG mouse model. Significance: This data demonstrates high efficacy of novel hBCMA-CD3 antibodies with multiple myeloma cells and provides a basis for future pre-clinical and clinical development. Citation Format: Yanwei Huang, John Sienkiewicz, Jinying Sun, Liselle Guiang, Feng Li, Liming Yang, Lijun Wu, Vita M. Golubovskaya. Novel bispecific humanized BCMA-CD3 antibodies specifically target multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2889.

Abstract 5557: PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME

Abstract Purpose: We investigated the pharmacology of PMC-309 in ex vivo or in vivo characterization for the development of the preclinical study. Experimental procedures: Target selectivity: it was measured by SPR assay. Human VISTA expressing CHO k1 cells or CHO k1 mock cells were used in cell binding assay. Target cell analysis: human peripheral blood mononuclear cells (PBMC) containing lymphocyte and myeloid lineage cells were used for target cells analysis with flow cytometry. In vitro T cell activity: human PBMC was employed for the evaluation of T cell activity (IRB #1041107-201703-BR-002-02) and was stimulated with or w/o PMC-309 in the presence of anti-CD3/28 Antibody. In vivo study: MC38 bearing human VISTA knock-in (KI) mice were employed for the assessment of anti-tumor activity of PMC-309. The tumor infiltrated immune cells: Immune cells in the TME were evaluated by immunohistochemistry (IHC) or flow cytometry (FACS) analysis. Statistics: All data were presented as the mean ± SEM and analyzed using one-way ANOVA followed by Dunnett’s multiple comparison tests in Graph pad prism®. ***p<0.001, **p<0.01 and *p<0.05 compared to Control group. Summary of data: PMC-309 binding to VISTA expressing cells is highly selective and the selectivity is maintained even in the low pH conditions that mimic TME. PMC-309 enhances the secretion of IFN-gamma, TNF-alpha, and IL-2 in Mixed Lymphocyte Reaction (MLR) settings. In addition, PMC-309 promoted monocyte differentiation into M1 macrophage that stimulates proinflammatory cytokine secretion of T cells. For the in vivo study, PMC-309 was intravenously administrated in VISTA-KI mice. The tumor growth rate was suppressed in accompanied by a synergistic effect with an anti-PD1 antibody. The anti-tumor activity was associated with enhanced T cell activation, increased secretion of pro-inflammatory cytokines, and increased penetration of cytotoxic T cells, but lowing immune-suppressive MDSC cells into TME as demonstrated with IHC analysis. Conclusions: PMC-309 is a fully human anti-VISTA antibody that targets human VISTA specifically. PMC-309 enhanced both T cell activation and monocyte activation when monocytes were cultured with or without T cells by targeting VISTA. PMC-309 increased the number of T cell infiltration while a decrease of MDSCs in the TME. PMC-309 in combination with chemotherapy or other IO drugs could address highly medical unmet needs from patients with drug resistance to currently available IO treatment options. Citation Format: Cheon Ho Park, Sang Soon Byun, Jin Yong An, Hyerim Han, Weon Sup Lee. PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5557.

Immunopotentiation by linking Hsp70 T-cell epitopes to Gag-Pol-Env-Nef-Rev multiepitope construct and increased IFN-gamma secretion in infected lymphocytes

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.

A pilot study of neoadjuvant cemiplimab with platinum-doublet chemotherapy and cetuximab in patients with resectable, locally advanced head and neck squamous cell carcinoma (HNSCC).

TPS6109 Background: Definitive treatment of locally advanced HNSCC can require radical surgery and reconstruction often resulting in unacceptable functional consequences. Radiotherapy, often with concurrent chemotherapy, is administered postoperatively to achieve the best chance for cure. Induction chemotherapy has previously been shown to reduce the extent of surgical resection and need for adjuvant radiation (RT). The purpose of this trial is to evaluate if an induction regimen combining cytotoxic chemotherapy, EGFR targeting, and immune checkpoint blockade can pathologically downstage resectable HNSCC sufficiently to decrease surgical morbidity and justify omission of adjuvant RT-based therapy. Compared to standard docetaxel, cisplatin, and 5-FU (TPF), docetaxel, cisplatin, and cetuximab (TPC) has been shown to be a therapeutic alternative with a more favorable toxicity profile. Targeting PD-1 alone can induce significant pathologic responses in resectable HNSCC patients. Combining PD-1 inhibitors with cetuximab has shown promising activity in incurable HNSCC; cetuximab may optimize the tumor immune microenvionment for PD-1 therapy by stimulating IFN-gamma secretion to increase dendritic cell maturation and CD8 T cell expression of PD1. Based on this rationale, we are evaluating the novel induction regimen of platinum, docetaxel, cetuximab plus cemiplimab (anti-PD1 antibody). Methods: This is a 10-patient pilot study for locally advanced, resectable HNSCC patients for whom standard management requires adjuvant RT +/- chemotherapy. Patients will receive neoadjuvant treatment with a loading dose of cetuximab and cemiplimab followed by 3 cycles of cisplatin or carboplatin, docetaxel, cetuximab and cemiplimab followed by definitive surgical resection of the primary site +/- neck dissection(s). Post-operative RT +/- radiosensitizing agent(s) will be administered per standard of care (SOC) based on pathologic staging (rather than clinical staging at presentation). If the pathologic stage following induction and surgery is ypT0-2N0 without adverse features, adjuvant RT will not be administered and 6 months of adjuvant cemiplimab will be given. Otherwise, patients will receive SOC adjuvant RT-based treatment. The primary endpoint is safety and tolerability. Secondary endpoints include feasibility assessed by the number of patients whose definitive surgery was delayed due to toxicity and quantifying the number in whom clinical to pathologic downstaging is achieved and the planned surgery and/or need for adjuvant-RT based therapy is modified. Exploratory endpoints include evaluating the association between biomarkers in the tumor microenvironment and peripheral blood with pathologic response. 8 of 10 patients have been enrolled. Clinical trial information: NCT04722523.

Bispecific BCMA-CD3 Antibodies Block Multiple Myeloma Tumor Growth.

Simple SummaryMultiple myeloma accounts for approximately 10% of hematological cancers in the United States. It is a malignancy of plasma cells which accumulate in the bone marrow and produce a monoclonal protein. Novel treatments are needed to cure multiple myeloma patients. The goal of this report was to develop novel bispecific BCMA-CD3 antibodies targeting a B cell maturation antigen, BCMA, which is overexpressed in multiple myeloma. The data demonstrated high efficacy of BCMA-CD3 antibodies in multiple myeloma cell lines in vitro and in vivo. The data provide a basis for future clinical studies.BCMA antigen is overexpressed in multiple myeloma cells and has been shown to be a promising target for novel cellular and antibody therapeutics. The humanized BCMA (clone 4C8A) antibody that effectively targeted multiple myeloma in a CAR (chimeric antigen receptor) format was used for designing several formats of bispecific BCMA-CD3 antibodies. Several different designs of univalent and bivalent humanized BCMA-CD3 CrossMAB and BCMA-FAB-CD3 ScFv-Fc antibodies were tested for binding with BCMA-positive cells and T cells and for killing by real time cytotoxic activity and IFN-gamma secretion with CHO-BCMA target cells and with multiple myeloma MM1S and H929 cell lines. All BCMA-CD3 antibodies demonstrated specific binding by FACS to CHO-BCMA, multiple myeloma cells, and to T cells with affinity Kd in the nM range. All antibodies with T cells specifically killed CHO-BCMA and multiple myeloma cells in a dose-dependent manner. The BCMA-CD3 antibodies with T cells secreted IFN-gamma with EC50 in the nM range. In addition, three BCMA bispecific antibodies had high in vivo efficacy using an MM1S xenograft NSG mouse model. The data demonstrate the high efficacy of novel hBCMA-CD3 antibodies with multiple myeloma cells and provide a basis for future pre-clinical and clinical development.

Monoclonal antibodies from B cells of patients with anti-MDA5 antibody-positive dermatomyositis directly stimulate interferon gamma production

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare entity associated with poor prognosis and multiple immunologic abnormalities. These include the presence of autoAbs and deleterious interferon (IFN)-gamma production in the severe form of the disease. Here, we show that the autoAbs profile differs between patients, depending on disease severity, and that autoAbs from B cells of patients directly stimulate IFN-gamma production by peripheral blood cells. Serum of 29 anti-MDA5 DM patients were analyzed by indirect immunofluorescence (IIF) on Hep-2 cells, to identify patterns associated with poor outcome. Seventeen (59%) serum gave a specific cytoplasmic MDA5 pattern on Hep-2 cells, while 12 (41%) gave an unspecific pattern. Specific MDA5 pattern was associated with a higher risk to develop interstitial lung disease (p = 0.003). Monoclonal autoAbs were generated from B cell clones of two patients with extreme clinical presentation, one who developed a lethal form of the disease, and the other with a favorable outcome. Supernatants of the autoreactive B cell clones that gave an IIF cytoplasmic pattern were tested for their abilities to stimulate IFN-gamma production by peripheral blood cells. Out of 120,000 B cell clones analyzed, 12 produced monoclonal Abs that triggered direct IFN-gamma secretion by peripheral blood cells, by a monocyte-dependent mechanism. None of them was directed against the MDA5 antigen. Altogether, these findings demonstrated that autoAbs other than the highly specific anti-MDA5 Ab are direct contributors of the IFN-gamma upregulation that is linked to the severity of the disease.

Effects from Oral Administration of Recombinant Interferon-Alpha on Piglet Daily Care

The administration of interferon has improved the antiviral and immunomodulatory abilities of piglets, which is conductive to the prevention of potential diseases or delay the appearance of clinical symptoms. This study aimed to evaluate the effects from administration of recombinant interferon-alpha (IFN-α) on the daily care of piglets. The results were compared to a Chinese herbal drug that was shown to increase serum interferon levels. In addition, the administration routes of oral administration and intramuscular injection were evaluated. Forty (40) piglets with equal age and weight were randomly divided into four groups: control group (group C, without treatment), group H (treated with compound Chinese herbal), group K (administered orally with recombinant IFN-α, 1500 IU per day per piglet), and group J (administered intramuscularly with IFN-α, 4×106 IU per day per piglet). After the treatment of 15 days, both oral and intramuscular treatment of recombinant IFN-α significantly improved the secretion of IFN-gamma (IFN-γ) ( P < 0.05 ), and the effects of intramuscular pathway were faster. In addition, the expression levels of IFN-stimulated genes (MX1 and ISG15) were significantly enhanced ( P < 0.01 ), independently of IFN-α treatment time and serum IFN-γ level. Different from other studies, compound Chinese herbal showed weaker effects on interferon stimulation in piglets. The results indicated that oral administration of recombinant IFN-α improved interferon-induced response of piglets at both serum and molecular levels, which may be applied for improving autoimmunity of piglets.

Intranasal Coronavirus SARS-CoV-2 Immunization with Lipid Adjuvants Provides Systemic and Mucosal Immune Response against SARS-CoV-2 S1 Spike and Nucleocapsid Protein.

In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.

373 Enhancement of TCR-engineered T-cells targeting MAGE-A4 antigen by co-expression of CD8α and inhibition of AKT signaling during ex vivo T-cell expansion

BackgroundAutologous Specific Peptide Enhanced Affinity Receptor (SPEAR) T-cells targeting MAGE-A4 can be effective treatment for solid tumors.1–3 To improve efficacy, we developed a next-generation SPEAR-T cell targeting MAGE-A4 co-expressing CD8α (ADP-A2M4CD8). ADP-A2M4CD8 is under investigation in the Phase 1 SURPASS trial (NCT04044859). Enhancements have also been made to the manufacturing process with an AKT inhibitor (AKTi) during ex vivo expansion to provide a greater proliferative potential and enhanced memory phenotype.4MethodsSPEAR-T cells were manufactured using a Lentiviral vector with CD8α and MAGE-A4 targeted TCR genes. AKTi was added during ex vivo expansion. T-cell attributes were evaluated, including markers of differentiation (flow cytometry), capacity for in vitro tumor lysis (Incucyte) and changes to gene expression (scRNASeq) initially assessed with the first-gen product. Post-infusion, the presence of transduced T-cells in the peripheral circulation (PCR) and levels of inflammatory cytokines in serum (MesoScale Discovery Assay [MSD]) were evaluated.ResultsAs of May 24, 2021, 18 patients with 9 different primary tumor types were evaluable. Twelve pts received product that had AKTi during manufacture. Five patients had objective responses (RECIST), and 10 had stable disease. Responses occurred at lower MAGE-A4 expression levels and lower transduced T-cell doses relative to the first-gen product targeting MAGE-A4.1 CD4 T-cells from manufactured ADP-A2M4CD8 demonstrated direct in vitro tumor cell killing similar to CD8+ T-cells (Incucyte). scRNASeq gene expression profiles of first-gen ADP-A2M4 product manufactured with AKTi revealed the AKTi-expanded T-cells had a greater proliferation or an enhanced memory phenotype; scRNASeq analyses are ongoing for the ADP-A2M4CD8 product.An increase in IL-12 levels (MSD) in serum post-infusion suggests that endogenous immune cells are being activated, further resulting in increased levels of IFN gamma (MSD) secretion relative to patients who received first-gen product. Manufacturing with AKTi resulted in T-cells with a less differentiated phenotype (flow cytometry), and post-infusion was associated with enhanced antigen-specific serum cytokine responses, increased proliferative state (i.e., elevated levels of IL-2), and higher persistence of T-cells in peripheral blood by PCR.ConclusionsSPEAR T-cells targeting MAGE-A4 expressing cancers have been enhanced by co-expressing CD8α and adding AKTi during manufacture. These enhanced products improve CD4+ T-cell killing, release more inflammatory cytokines, proliferate more robustly with an early memory phenotype, and better engage the patient‘s endogenous immune system when compared to first-gen products or next-gen manufactured without AKTi.Trial RegistrationNCT04044859ReferencesHong, et al. ASCO 2020.D’Angelo, et al. ASCO 2021.Hong, et al. SITC 2020.Mardilovich, et al. SITC 2020.

Novel CS1 CAR-T Cells and Bispecific CS1-BCMA CAR-T Cells Effectively Target Multiple Myeloma.

Multiple myeloma (MM) is a hematological cancer caused by abnormal proliferation of plasma cells in the bone marrow, and novel types of treatment are needed for this deadly disease. In this study, we aimed to develop novel CS1 CAR-T cells and bispecific CS1-BCMA CAR-T cells to specifically target multiple myeloma. We generated a new CS1 (CD319, SLAM-7) antibody, clone (7A8D5), which specifically recognized the CS1 antigen, and we applied it for the generation of CS1-CAR. CS1-CAR-T cells caused specific killing of CHO-CS1 target cells with secretion of IFN-gamma and targeted multiple myeloma cells. In addition, bispecific CS1-BCMA-41BB-CD3 CAR-T cells effectively killed CHO-CS1 and CHO-BCMA target cells, killed CS1/BCMA-positive multiple myeloma cells, and secreted IFN-gamma. Moreover, CS1-CAR-T cells and bispecific CS1-BCMA CAR-T cells effectively blocked MM1S multiple myeloma tumor growth in vivo. These data for the first time demonstrate that novel CS1 and bispecific CS1-BCMA-CAR-T cells are effective in targeting MM cells and provide a basis for future clinical trials.