scholarly journals A Novel Bispecific Antibody Targeting CD3 and Lewis Y with Potent Therapeutic Efficacy against Gastric Cancer

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 1059
Author(s):  
Jie Chen ◽  
Zhidi Pan ◽  
Lei Han ◽  
Yuexian Zhou ◽  
Huifang Zong ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cell ◽  
Therapeutic Efficacy ◽  
Bispecific Antibody ◽  
Target Cells ◽  
Lewis Y ◽  
Antibody Targeting ◽  
Anti Tumor Activity

Lewis Y antigen, a glycan highly expressed on most epithelial cancers, was targeted for cancer treatment but lacked satisfactory results in some intractable and refractory cancers. Thus, it is highly desirable to develop an effective therapy against these cancers, hopefully based on this target. In this work, we constructed a novel T cell-engaging bispecific antibody targeting Lewis Y and CD3 (m3s193 BsAb) with the IgG-[L]-scfv format. In vitro activity of m3s193 BsAb was evaluated by affinity assay to target cells, cytotoxicity assay, cytokines releasing assay, and T cells proliferation and recruiting assays. Anti-tumor activity against gastric cancer was evaluated in vivo by subcutaneous huPBMCs/tumor cells co-grafting model and huPBMCs intravenous injecting model. In vitro, m3s193 BsAb appeared to have a high binding affinity to Lewis Y positive cells and Jurkat cells. The BsAb showed stronger activity than its parent mAb in T cell recruiting, activation, proliferation, cytokine release, and cytotoxicity. In vivo, m3s193 BsAb not only demonstrated higher therapeutic efficacy in the huPBMCs/tumor co-grafting gastric carcinoma model than the parent mAb but also eliminated tumors in the model of intravenous injection with huPBMCs. Strong anti-tumor activity of m3s193 BsAb revealed that Lewis Y could be targeted in T cell-engaging BsAb for gastric cancer therapy.

The Non-Signaling Extracellular Spacer Domain of CD19-Specific Chimeric Antigen Receptors Is Decisive for in Vivo Anti-Tumor Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 951-951 ◽  
Author(s):  
Michael Hudecek ◽  
Anne Silva ◽  
Paula L. Kosasih ◽  
Yvonne Y. Chen ◽  
Cameron J. Turtle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Target Cells ◽  
Antigen Receptors ◽  
Chimeric Antigen Receptors ◽  
Car T ◽  
Anti Tumor Activity

Abstract Abstract 951 Adoptive immunotherapy with T cells engineered by gene transfer to express CD19-specific chimeric antigen receptors (CARs) has the potential to induce remissions in patients with advanced B cell malignancies. CARs are synthetic receptors with an extracellular antigen-binding domain (scFv), a spacer domain that provides separation of the scFv from the cell membrane and an intracellular signaling module, most commonly the CD3ζ chain and one or more costimulatory domains such as CD28 or 4-1BB. Several clinical trials with CD19-CAR T cells in small cohorts of patients with B cell tumors have been reported with variable results. Although most studies have used the CD19-specific FMC63 scFv as the tumor-targeting moiety, the extracellular, transmembrane and intracellular CAR domains used in each trial have been distinct, and an emerging paradigm is that including costimulation in the design of the CAR is key to achieving anti-tumor activity in vivo. In this study, we analyzed the influence of extracellular spacer domain length on the in vitro and in vivo function of CD19-CARs. We constructed a panel of four CD19-CARs comprised of the FMC63 scFv and either a long spacer derived from the IgG4-Fc Hinge-CH2-CH3 domain (229 AA) or a short Hinge domain only spacer (12 AA). Each CAR contained a signaling module of CD3ζ with CD28 (short/CD28; long/CD28) or 4-1BB (short/4-1BB; long/4-1BB). We transduced CD8+ CD45RO+ CD62L+central memory T cells of normal donors with each of the CARs, enriched transduced T cells to >90% purity by immunomagnetic selection using a tEGFR marker encoded in the CAR vector, and expanded CAR transduced T cells using a uniform culture protocol. We compared the in vitro function of T cell lines expressing each of the CD19-CARs and confirmed specific cytolytic activity against CD19+ target cells including K562/CD19, and Raji and JeKo-1 lymphoma cells. Quantitative cytokine analyses showed higher levels of IFN-γ, TNF-α, IL-2 production in T cells expressing CD19-CARs with CD28 costimulatory domain compared to the corresponding constructs with 4-1BB, consistent with prior work. T cells expressing each of the CD19-CARs proliferated in vitro after stimulation with K562/CD19 and Raji tumor cells by CFSE dye dilution, with the strongest proliferation observed in T cells expressing the CD19-CAR ‘long/CD28’, consistent with the highest levels of IL-2 production by T cells expressing this construct. We then analyzed the in vivo anti-tumor efficacy of each CD19-CAR in immunodeficient NOD/SCID/g−/− (NSG) mice engrafted with firefly luciferase transduced Raji cells. Tumor was inoculated on day 0, and once tumor was established (day 7), the mice received a single dose of 2.5×106̂ T cells expressing each CD19-CAR, a tEGFR control vector, or were left untreated. Surprisingly, only T cells expressing CD19-CARs with a short spacer domain (short/CD28 and short/4-1BB) eradicated the Raji tumors and led to long-term tumor-free survival of all mice. T cells expressing CD19-CARs with a long spacer domain (long/CD28 and long/4-1BB) did not confer a significant anti-tumor effect and all mice expired from systemic lymphoma at a similar time as control and untreated mice. The anti-tumor efficacy in vivo of T cells modified with long spacer CD19-CARs could not be improved by increasing CAR T cell dose 4 fold, or by including additional costimulatory domains into the CD19-CAR (long/CD28:4-1BB). Serial analyses in peripheral blood, bone marrow and spleen showed dramatically lower numbers of transferred T cells in mice treated with long spacer CD19-CARs compared to mice treated with short spacer CD19-CARs or control T cells. Further analysis revealed that despite strong activation in vivo as assessed by upregulation of CD69 and CD25, CD19-CARs with long extracellular spacer domain induced a high rate of activation induced T cell death in vivo. Collectively, these results demonstrate that the extracellular spacer domain that lacks intrinsic signaling function is critical in the design of effective CD19-CARs, and illustrates that tailoring spacer length is likely to be essential for designing effective CARs specific for other tumor antigens. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 203 A membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence and efficacy of CD70-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A214-A214
Author(s):  
Jian Ding ◽  
Lindsay Webb ◽  
Troy Patterson ◽  
Michelle Fleury ◽  
Adam Zieba ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Hematological Malignancies ◽  
Cell Receptor ◽  
Target Cells ◽  
Cell Therapies ◽  
Anti Tumor Activity

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies. To realize the potential of T cell therapies in solid tumors, we have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes all TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. Previously, we have described the discovery and preclinical efficacy of fratricide-resistant TRuC-T cells targeting CD70, a tumor antigen overexpressed in various solid and hematological malignancies. As a strategy to enhance T cell effector function and persistence in the hostile tumor microenvironment, we engineered anti-CD70 TRuC-T cells to co-express a membrane-bound IL15Ra-IL15 fusion protein (IL-15fu). IL-15 is a common ? chain cytokine that promotes the differentiation, maintenance, and effector function of memory CD8+ T cell subsets and confers resistance to IL-2-mediated activation induced cell death (AICD).MethodsT cells were activated by CD3/CD28 stimulation and lentivirally transduced with a T2A-containing bicistronic vector encoding the anti-CD70 CD3?-TRuC and the IL-15fu proteins; the cells were further expanded for 9 days in media containing IL-7/IL-15. Surface co-expression of the TRuC and IL-15fu proteins and the T cell memory phenotype was assessed by flow cytometry. In vitro persistence was tested in a repeated stimulation assay in which T cells were challenged by addition of fresh CD70+ target cells every four days with longitudinal assessment of T-cell expansion, phenotype, cytokine production, and cytotoxicity. In vivo, the antitumor efficacy of the anti-CD70 TRuC/IL-15fu T cells was evaluated in MHC class I/II deficient NSG mice bearing human tumor xenografts.ResultsThe anti-CD70 TRuC and IL-15fu proteins showed high transduction efficiency and robust co-expression on the surface of T cells. The IL-15fu significantly increased the proportion of naïve cells within the TRuC-T cell product, most dramatically in the CD8+ subset. In vitro, TRuC-T cells bearing the IL-15fu showed greatly enhanced expansion and persistence upon repeated stimulation with CD70+ target cells. Moreover, the IL-15fu enhanced T-cell survival and persistence under unstimulated, cytokine-free conditions. In vivo, the anti-tumor activity of CD70-targeted TRuC-T cells was significantly improved by IL-15fu in multiple tumor models and was associated with enhanced intratumoral T-cell accumulation and a preferential expansion of CD8+ T cells.ConclusionsThe addition of the IL-15fu improved the phenotype, persistence, and anti-tumor activity of CD70-targeted TRuC-T cells, potentially increasing the likelihood of clinical benefit in patients with CD70 overexpressing solid and liquid cancers.Ethics ApprovalAll animal studies were conducted by TCR2 Therapeutics staff at the Charles River Laboratories CRADL facility under a protocol approved by the Charles River Laboratories Institutional Animal Care and Use Committee.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

scholarly journals Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Jennifer A. Juno ◽  
Kathleen M. Wragg ◽  
Anne B. Kristensen ◽  
Wen Shi Lee ◽  
Kevin J. Selva ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Seminal Plasma ◽  
Target Cells ◽  
Ccr5 Expression ◽  
Ccr5 Receptor ◽  
The Impact ◽  
Hiv 1

ABSTRACT Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggest that CCR5+ CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 h of SP exposure (P = 0.03). Meanwhile, NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody-dependent stimulation (median, 9.5% MIP-1α+ and/or MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three of the animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design. IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade In Vitro and In Vivo

2018 ◽  
Vol 18 (3) ◽  
pp. 632-641 ◽  
Author(s):  
Srimoyee Ghosh ◽  
Geeta Sharma ◽  
Jon Travers ◽  
Sujatha Kumar ◽  
Justin Choi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Therapeutic Antibody ◽  
T Cell Function ◽  
Antibody Targeting ◽  
Novel Therapeutic

scholarly journals Selective response to H-Y antigen by F1 female mice sensitized to F1 male cells.

1977 ◽  
Vol 146 (2) ◽  
pp. 606-610 ◽  
Author(s):  
R D Gordon ◽  
L E Samelson ◽  
E Simpson
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Target Cells ◽  
Gene Products ◽  
Gene Complex ◽  
Suppressor Genes ◽  
Cytotoxic Responses ◽  
Major Histocompatibility Gene

T-cell mediated cytotoxic responses to H-Y antigen require co-recognition of H-Y and H-2 gene products. F1 mael stimulating cells and target cells express H-Y antigen in association with both parental H-2 haplotypes. However, F1 females primed in vivo and challenged in vitro with F1 male cells lyse male target cells of F1 and only one parental H-2 haplotype. Thus, (CBA X B10)F1 females sensitized to (CBA X B10)F1 male cells lyse (CBA X B10)F1 and CBA but not B10 male target cells, and (BALB/c X B10)F1 females sensitized to (BALB/c X B10)F1 male cells will lyse (BALB/c X B10)F1 and B10 but not BALB/c male target cells. It is suggested that this may represent an effect of immune response or suppressor genes mapping in the major histocompatibility gene complex which regulate responsiveness to H-Y antigen.

scholarly journals 13-Methyltetradecanoic Acid Exhibits Anti-Tumor Activity on T-Cell Lymphomas In Vitro and In Vivo by Down-Regulating p-AKT and Activating Caspase-3

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e65308 ◽  
Author(s):  
Qingqing Cai ◽  
Huiqiang Huang ◽  
Dong Qian ◽  
Kailin Chen ◽  
Junhua Luo ◽  
...  
Keyword(s):  
T Cell ◽  
Caspase 3 ◽  
T Cell Lymphomas ◽  
Anti Tumor Activity

Peripheral Alloantigen Drives Early Dysfunction and Eventual Exhaustion of CTL Following Delayed Donor Leukocyte Infusions.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2346-2346
Author(s):  
Barry R Flutter ◽  
Farnaz Fallah-Arani ◽  
Clare Bennett ◽  
Janani Sivakumaran ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
T Cell ◽  
Significant Risk ◽  
T Cell Response ◽  
Target Cells ◽  
Peripheral Tissues ◽  
Hematopoietic System ◽  
Cd8 Cells ◽  
Ifn Γ

Abstract T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

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