scholarly journals Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models

2021 ◽  
Vol 12 ◽  
Author(s):  
Alexander A. Pieper ◽  
Luke M. Zangl ◽  
Dan V. Speigelman ◽  
Arika S. Feils ◽  
Anna Hoefges ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Response ◽  
Tumor Regression ◽  
Quantitative Polymerase Chain Reaction ◽  
Response To Treatment ◽  
Tumor Models ◽  
Cpg Oligodeoxynucleotides

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

scholarly journals 598 Local radiation in combination with CpG and anti-OX40 induces enhanced T cell activation and proliferation

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A628-A628
Author(s):  
Dan Spiegelman ◽  
Alexander Pieper ◽  
Luke Zangl ◽  
Arika Feils ◽  
Anna Hoefges ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Regression ◽  
Tumor Model ◽  
Cd8 T Cell ◽  
External Beam ◽  
In Situ Vaccine

BackgroundWe, and others, have previously shown that the in-situ vaccine of hypomethylated CG-enriched oligodeoxynucleotide (CpG) with agonist anti-OX40 antibody (OX40) is effective at curing mice in the A20 lymphoma model [1–4]. In separate preclinical models where CpG+OX40 fails to cause tumor regression, radiation therapy (RT) prior to the in-situ vaccine enhances the anti-tumor effect of CpG+OX40 [4]. We investigated the immune response, and specifically the activity of T cells, following treatment with RT+CpG+OX40 in the B78 melanoma model where CpG+OX40 typically fails to cause tumor regression.MethodsC57BL/6 mice were inoculated with 2x106 B78 melanoma cells on the right flank and allowed to grow until the average tumor size was ~150mm3. In two independent experiments, mice were randomized (n=4–5 per group per experiment) and treated with one of the following: 1) PBS, 2) CpG+OX40, 3) RT, 4) RT+CpG+OX40. 12 Gy external beam RT was dosed to the flank tumor on day 0 and intratumoral CpG (50µg)+OX40 (20 µg) were given on days 5, 7, and 9 after RT. Spleens and tumor draining lymph nodes (TDLNs) were harvested on day 12. T cell activation and proliferation were assessed via flow cytometry.ResultsCompared to all other groups in the study, mice treated with RT+CpG+OX40 demonstrated significantly elevated levels of CD4+ and CD8+ T cell activation in the TDLNs, as measured by interferon gamma expression. Similar trends of CD4+ and CD8+ T cell activation were measured in the spleens. Splenic CD8+ T cells from RT+CpG+OX40 treated mice demonstrated significantly elevated levels of proliferation over PBS and RT, as measured by Ki67.ConclusionsIn B78 melanoma, a weakly immunologic tumor model, combining RT with the in-situ vaccine CpG+OX40 enhances the activity of T cells, evidenced by significantly increased CD4+ and CD8+ T cell activation in the TDLN and spleen and elevated CD8+ T cell proliferation in the spleen.ReferencesHouot, R. and Levy, R. T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy. Blood, 2009. 113(15):3546–52.Marabelle, A., et al. Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J Clin Invest, 2013. 123(6):2447–63.Sagiv-Barfi, I., et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med, 2018. 10(426).Zangl, LM. Et al. External Beam Radiotherapy Required for Tumor Regression When Using CpG-Oligodeoxynucleotide and Anti-OX40 in an Immunologically Cold Tumor Model. Red Journal. 2019. 105:S88.

Prostate Cancer Immunology: Biology, Therapeutics, and Challenges

2005 ◽  
Vol 23 (32) ◽  
pp. 8262-8269 ◽  
Author(s):  
W. Scott Webster ◽  
Eric J. Small ◽  
Brian I. Rini ◽  
Eugene D. Kwon
Keyword(s):  
Prostate Cancer ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Advanced Prostate Cancer ◽  
Cell Activation ◽  
Tumor Regression ◽  
Prostate Cancer Treatment ◽  
Mechanistic Basis ◽  
Antigen Presenting

A number of recently developed and promising approaches to antitumoral immunotherapy are being investigated as potential treatments for advanced prostate cancer. These approaches largely revolve around strategies to increase antigen-specific T-cell activation against prostate tumors as well as precise manipulations of critical co-regulatory receptors that help to maintain and prolong the activity of antigen-presenting cells and T cells that are capable of mediating tumor regression. Herein, we describe the experience with the most recent and promising approaches pertaining to prostate cancer immunotherapy. Additionally, we discuss the mechanistic basis for these approaches as well as current limitations that must still be addressed in order to propel immunotherapy into the forefront of prostate cancer treatment.

scholarly journals Development of a CD123xCD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2824-2824 ◽  
Author(s):  
François Gaudet ◽  
Jennifer F Nemeth ◽  
Ronan McDaid ◽  
Yingzhe Li ◽  
Benjamin Harman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Stock Options ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Tumor Regression

Abstract AML is a cancer of the myeloid lineage that is characterized by the accumulation of abnormal white blood cells in the bone marrow and blood. Existing therapies do not lead to cures, partially due to their inability to eliminate residual leukemic stem cells (LSCs) in the bone marrow. T-cell redirection has been shown to be an effective method of treatment for hematologic malignancies (eg, blinatumomab) and represents an attractive approach to treat AML. CD123 (α-chain of the interleukin-3 receptor) has been shown to be expressed on the surface of AML blasts and LSCs. To eradicate CD123+ cells, we developed a bispecific antibody (JNJ-63709178) using the Genmab DuoBody® technology that can bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. This antibody is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91 nM). In contrast, this antibody does not kill CD123- cell lines, demonstrating the specificity of cytotoxicity. Consistently, the degree of cell killing correlated with the level of T-cell activation (CD69 and CD25) and cytokine release (TGF-β and TNF-α). Control bispecific antibodies containing a null arm (viral epitope) paired with a CD123 arm (CD123xnull) or a CD3 arm (nullxCD3) did not induce cytotoxicity or T-cell activation in the assays tested. JNJ-63709178 had no effect on T-cell activation when incubated with T cells alone. In AML murine xenograft models, JNJ-63709178 was able to suppress tumor growth and induce tumor regression (MOLM-13 and KG-1, respectively) in the presence of human peripheral blood mononuclear cells (PBMCs) or T cells. Tumor regression correlated with the infiltration of T cells in the tumor and the expression of T-cell activation markers such as CD25, PD1 and TIM3. Furthermore, this antibody was able to induce the killing of primary CD123+ cancer cells from the blood of patients with AML without the need to supplement with fresh T cells (EC50 = 0.83 nM). These results indicate that JNJ-63709178 can potently and specifically kill CD123+ cancer cells in vitro, in vivo and ex vivo. Pharmacokinetic studies in cynomolgus monkeys support twice weekly dosing for human studies. JNJ-63709178 is currently being investigated in a Phase 1 clinical trial in relapsed and refractory AML (ClinicalTrials.gov ID: NCT02715011). Disclosures Gaudet: Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Nemeth:Janssen Pharmaceuticals R&D: Employment, Other: stock, Patents & Royalties: patent pending. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Harman:Janssen Pharmaceuticals R&D: Employment. Millar:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Teplyakov:Janssen Pharmaceuticals R&D: Employment. Wheeler:Janssen Pharmaceuticals R&D: Employment. Luo:Janssen Pharmaceuticals R&D: Employment. Tam:Janssen Pharmaceuticals R&D: Employment, Other: stocks, Research Funding. Wu:Janssen Pharmaceuticals R&D: Employment. Chen:Janssen Pharmaceuticals R&D: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Chu:Janssen Pharmaceuticals R&D: Employment. Hughes:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Babich:Janssen: Employment. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock. Singh:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment.

scholarly journals Modulation of Pulmonary Dendritic Cell Function during Mycobacterial Infection

2007 ◽  
Vol 76 (2) ◽  
pp. 671-677 ◽  
Author(s):  
Mursalin M. Anis ◽  
Scott A. Fulton ◽  
Scott M. Reba ◽  
Yi Liu ◽  
Clifford V. Harding ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Mycobacterial Infection ◽  
Lung Cells ◽  
Mhc Ii ◽  
Class Ii Mhc

ABSTRACT We have previously reported that during mycobacterial infection, naïve CD4+ T-cell activation is enhanced in the lungs. We investigated the role of chemokine receptor CCR7 and its ligands in the ability of CD11c+ lung dendritic cells (DCs) to activate naïve CD4+ T cells during pulmonary infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection resulted in the accumulation and maturation in the lungs of DCs that persisted as the mycobacterial burden declined. Lung DCs from infected mice expressed more major histocompatibility complex class II (MHC-II) than those from uninfected mice. CCR7 expression levels on lung DCs were comparable among uninfected and infected mice. The gene expression of the CCR7 ligand CCL19 progressively increased throughout BCG infection, and its expression was MyD88 dependent. CD11c+ lung cells from BCG-infected mice activated ovalbumin (OVA)-specific naïve CD4+ T cells more than CD11c+ lung cells from uninfected mice. Interestingly, during peak mycobacterial infection, CD11chi MHChi lung DCs had slightly decreased chemotaxis toward the CCR7 ligand CCL21 and less efficiency in activating naive CD4+ T cells than DCs from mice during late-stage infection, when few bacilli are found in the lung. These findings suggest that during BCG infection, the inflammation and sustained expression of CCL19 result in the recruitment, activation, and retention in the lung of DCs that can activate naïve CD4+ T cells in situ.

scholarly journals iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti–PD-1 therapy

2020 ◽  
Vol 12 (568) ◽  
pp. eaaz5618
Author(s):  
Frank Cichocki ◽  
Ryan Bjordahl ◽  
Svetlana Gaidarova ◽  
Sajid Mahmood ◽  
Ramzey Abujarour ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Regression ◽  
Adoptive Cell Therapy ◽  
Tumor Control ◽  
Inhibitory Receptors ◽  
Activated T Cells ◽  
Tumor Lysis

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti–PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an “off-the-shelf” source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a “cold” tumor “hot” by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

scholarly journals Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6102-6111 ◽  
Author(s):  
Madhav D. Sharma ◽  
De-Yan Hou ◽  
Yanjun Liu ◽  
Pandelakis A. Koni ◽  
Richard Metz ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Helper ◽  
Indoleamine 2,3 Dioxygenase ◽  
Draining Lymph Nodes

Abstract The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by a subset of murine plasmacytoid DCs (pDCs) in tumor-draining lymph nodes (TDLNs), where it can potently activate Foxp3+ regulatory T cells (Tregs). We now show that IDO functions as a molecular switch in TDLNs, maintaining Tregs in their normal suppressive phenotype when IDO was active, but allowing inflammation-induced conversion of Tregs to a polyfunctional T-helper phenotype similar to proinflammatory T-helper-17 (TH17) cells when IDO was blocked. In vitro, conversion of Tregs to the TH17-like phenotype was driven by antigen-activated effector T cells and required interleukin-6 (IL-6) produced by activated pDCs. IDO regulated this conversion by dominantly suppressing production of IL-6 in pDCs, in a GCN2-kinase dependent fashion. In vivo, using a model of established B16 melanoma, the combination of an IDO-inhibitor drug plus antitumor vaccine caused up-regulation of IL-6 in pDCs and in situ conversion of a majority of Tregs to the TH17 phenotype, with marked enhancement of CD8+ T-cell activation and antitumor efficacy. Thus, Tregs in TDLNs can be actively reprogrammed in situ into T-helper cells, without the need for physical depletion, and IDO serves as a key regulator of this critical conversion.

Facilitating Engraftment After MHC-Matched, Allogeneic BMT by IL-2 / Anti IL-2 Complex Treatment Requires Targeting CD25 On, and Activation in Situ of, Residual CD4 Tregs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 66-66
Author(s):  
Alwi Shatry ◽  
Michael A Gorin ◽  
Jackelin G Chirinos ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Treg Cells ◽  
Direct Result ◽  
Cd4 Cells ◽  
Specific Host

Abstract Abstract 66 We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. <1%). These findings demonstrate that IAC effects require host CD25+ Treg cells and we propose that facilitation of engraftment by this strategy was a direct result of Treg cell activation and expansion following engagement of IAC with CD25. To assess Treg activation, we examined these cells in our standard BALB.B à B6 HSCT model. Tregs isolated 7 days post-HCT from IAC-treated but not untreated recipients expressed readily demonstrable levels of pStat-5a expression (∼2X increase in IAC-treated vs. PBS controls). Moreover, culture of the former Tregs in the presence of rmIL-2 illustrated their heightened sensitivity to activation by this cytokine as virtually all Tregs from IAC-treated animals exhibited high levels of pStat-5a expression (3.2 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.6) compared to control mice (0.4 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.5). Following this activation, increased numbers of host CD4+ FoxP3+ Tregs were readily identified in the PB and spleen of IAC vs. PBS-treated recipients. We next directly examined functional capacity of residual host Tregs exposed to IAC post-5.5Gy TBI and-transplant. Eight days post-HSCT, host Tregs (0 – 25 × 103/well) were isolated and highly enriched populations assessed for suppression of TCONV in anti-CD3 activation assays. Tregs from IAC-treated recipients efficiently mediated suppression at least equivalent to that by normal, untreated Tregs in these assays. Additionally, these IAC treated residual B6 Tregs also effectively inhibited allogeneic MLR responses by B6 CD4+ CD25− responder cells. Therefore, the capacity of host Tregs to respond to IAC activation signals and suppress T cell activation remained intact following conditioning and HSCT. We conclude that host Treg cell activation/expansion is central to the suppression of host effector cell responses to donor hematopoietic antigens leading to the inhibition of HVG following IAC administration resulting in enhanced engraftment. Disclosures: No relevant conflicts of interest to declare.

Quiescent phenotype of tumor-specific CD8+ T cells following immunization

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 1970-1978 ◽  
Author(s):  
Vladia Monsurrò ◽  
Ena Wang ◽  
Yoshisha Yamano ◽  
Stephen A. Migueles ◽  
Monica C. Panelli ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Tumor Regression ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Proliferative Potential

Abstract In a human melanoma model of tumor antigen (TA)–based immunization, we tested the functional status of TA-specific CD8+ cytotoxic T lymphocytes. A “quiescent” phenotype lacking direct ex vivo cytotoxic and proliferative potential was identified that was further characterized by comparing its transcriptional profile to that of TA-specific T cells sensitized in vitro by exposure to the same TA and the T-cell growth factor interleukin 2 (IL-2). Quiescent circulating tumor-specific CD8+ T cells were deficient in expression of genes associated with T-cell activation, proliferation, and effector function. This quiescent status may explain the observed lack of correlation between the presence of circulating immunization-induced lymphocytes and tumor regression. In addition, the activation of TA-specific T cells by in vitro antigen recall and IL-2 suggests that a complete effector phenotype might be reinstated in vivo to fulfill the potential of anticancer vaccine protocols.

scholarly journals In situ immunization of a TLR9 agonist virus-like particle enhances anti-PD1 therapy

2020 ◽  
Vol 8 (2) ◽  
pp. e000940
Author(s):  
Yinwen Cheng ◽  
Caitlin D Lemke-Miltner ◽  
Wattawan Wongpattaraworakul ◽  
Zhaoming Wang ◽  
Carlos H F Chan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Tumor Regression ◽  
Subcutaneous Administration ◽  
Type I ◽  
Peripheral Blood Mononuclear

BackgroundCMP-001 is a novel Toll-like receptor-9 agonist that consists of an unmethylated CpG-A motif-rich G10 oligodeoxynucleotide (ODN) encapsulated in virus-like particles. In situ vaccination of CMP-001 is believed to activate local tumor-associated plasmacytoid dendritic cells (pDCs) leading to type I interferon secretion and tumor antigen presentation to T cells and systemic antitumor T cell responses. This study is designed to investigate if CMP-001 would enhance head and neck squamous cell carcinoma (HNSCC) tumor response to anti-programmed cell death protein-1 (anti-PD-1) therapy in a human papilloma virus-positive (HPV+) tumor mouse model.MethodsImmune cell activation in response to CMP-001±anti-Qβ was performed using co-cultures of peripheral blood mononuclear cells and HPV+/HPV- HNSCC cells and then analyzed by flow cytometry. In situ vaccination with CMP-001 alone and in combination with anti-PD-1 was investigated in C57BL/6 mice-bearing mEERL HNSCC tumors and analyzed for anti-Qβ development, antitumor response, survival and immune cell recruitment. The role of antitumor immune response due to CMP-001+anti-PD-1 treatment was investigated by the depletion of natural killer (NK), CD4+ T, and CD8+ T cells.ResultsResults showed that the activity of CMP-001 on immune cell (pDCs, monocytes, CD4+/CD8+ T cells and NK cells) activation depends on the presence of anti-Qβ. A 2-week ‘priming’ period after subcutaneous administration of CMP-001 was required for robust anti-Qβ development in mice. In situ vaccination of CMP-001 was superior to unencapsulated G10 CpG-A ODN at suppressing both injected and uninjected (distant) tumors. In situ vaccination of CMP-001 in combination with anti-PD-1 therapy induced durable tumor regression at injected and distant tumors and significantly prolonged mouse survival compared with anti-PD-1 therapy alone. The antitumor effect of CMP-001+anti-PD-1 was accompanied by increased interferon gamma (IFNγ)+ CD4+/CD8+ T cells compared with control-treated mice. The therapeutic and abscopal effect of CMP-001+ anti-PD-1 therapy was completely abrogated by CD8+ T cell depletion.ConclusionsThese results demonstrate that in situ vaccination with CMP-001 can induce both local and abscopal antitumor immune responses. Additionally, the antitumor efficacy of CMP-001 combined with α-PD-1 therapy warrants further study as a novel immunotherapeutic strategy for the treatment of HNSCC.

scholarly journals 235 Novel biparatopic TIM-3 antibody effectively blocks multiple inherent ligands and activates anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A251-A251
Author(s):  
Yuji Mishima ◽  
Kanto Nakajima ◽  
Mamoru Shiraishi ◽  
Haruka Matsumura ◽  
Takahiko Aramaki ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tumor Regression ◽  
Apoptotic Cell ◽  
Phage Display Libraries ◽  
Lymph Node Cells ◽  
Multiple Ligands

BackgroundT cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is a part of modules expressed on dysfunctional or exhausted T cells as well as dendritic cells and has emerged as a target for several therapeutic antibodies that are under clinical development. Co-blockade of TIM-3 and PD-1 results in tumor regression in preclinical models and improves anticancer T cell responses in patients with advanced cancers. TIM-3 has been reported to have multiple ligands including galectin-9, phosphatidylserine, CEACAM-1 and HMGB1, which bind to different regions on the extracellular domain of TIM-3. Most of the TIM-3 antibodies developed to date are intended to inhibit phosphatidylserine that binds to the pocket in TIM-3 immunoglobulin V domain. Galectin-9 binds to carbohydrate motifs on the opposite side of phosphatidylserine-binding site in immunoglobulin V domain and thereby induces cell death in TIM-3+ T cells. We report herein novel antibodies that block TIM-3 binding to multiple ligands including these two important ligands simultaneously.MethodsAnti-TIM-3 antibodies were generated by immunizing mice with a purified recombinant TIM-3 protein and TIM-3-expressing mammalian cell line. Phage display libraries were constructed using cDNAs of splenocytes and lymph node cells of the immunized mice, then subjected to the biopanning using recombinant TIM-3 proteins. After analyzing specificities and affinities to the TIM-3 protein, scFvs obtained were classified by epitope bin and inhibitory effects on TIM-3 binding to the multiple ligands. The scFvs were converted to scFv-Fc to generate biparatopic (bispecific) antibodies.ResultsAt least five classes of TIM-3 antibodies were obtained, and each class was grouped into different epitope bins and has unique inhibitory profiles for multiple ligands of TIM-3. Their biparatopic (bispecific) forms were produced from the scFv clones and subjected to the analyses of TIM-3 binding, inhibition of ligand binding, and immune activation. As expected, the biparatopic antibodies that recognize two different epitopes showed higher affinity and specificity to TIM-3 than monospecific forms. A lead biparatopic antibody that block the binding of TIM-3 to galectin-9 and phosphatidylserine showed remarkable potency on T cell activation, protection from exhaustion and apoptotic cell death of T cells as well as more potent anti-tumor efficacy.ConclusionsThis study demonstrates the successful development of a novel biparatopic antibody that blocks the binding of TIM-3 to phosphatidylserine and galectin-9 simultaneously. The antibody shows the advantages over conventional TIM-3 antibodies in reducing T cell exhaustion and potentially manipulated for the development of human monoclonal antibodies for therapeutic treatment of cancer.

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