Retargeting T Cells for HER2-Positive Tumor Killing by a Bispecific Fv-Fc Antibody

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

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Novel Fluorinated Spermine and Small Molecule PEI to Deliver Anti-PD-L1 and Anti-VEGF siRNA for Highly Efficient Tumor Therapy

Pharmaceutics ◽

10.3390/pharmaceutics13122058 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2058

Author(s):

Yihui Zhang ◽

Zihan Yuan ◽

Yi Jin ◽

Wenkai Zhang ◽

Wei-En Yuan

Keyword(s):

Gene Therapy ◽

T Cells ◽

Small Molecule ◽

Tumor Therapy ◽

Sirna Delivery ◽

Death Receptor ◽

Target Cells ◽

Anti Vegf ◽

Tumor Killing ◽

Death Receptor Ligand

Small interfering RNA (siRNA) can specifically silence disease gene expression. This project investigated the overexpression of programmed death receptor ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) on the surface of tumor cells. However, the main obstacle to the development of gene therapy drugs is the lack of an efficient delivery vector, which should be able to overcome multiple delivery barriers and protect siRNA to enter the target cells. Therefore, a novel fluorine-modified endogenous molecular carrier TFSPEI was constructed by linking fluorinated groups with hydrophobic and hydrophilic characteristics on the surface of PEI and spermine. The results showed that lower toxicity, higher endocytosis, and silencing efficiency were achieved. We found that the inhibition of VEGF targets can indirectly activate the immune response to promote the tumor-killing and invasion effects of T cells. The combined delivery of anti-VEGF siRNA and anti-PD-L1 siRNA could inhibit the expression of corresponding proteins, restore the anti-tumor function of T cells and inhibit the growth of neovascularization, and obtained significant anti-tumor effects. Therefore, this safe and efficient fluorinated spermine and small molecule PEI-based anti-PD-L1 and anti-VEGF siRNA delivery system is expected to provide a new strategy for gene therapy of tumors.

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A Long-Acting Pharmacological Grade Interleukin-7 Molecule Logarithmically Accelerates Ucart Proliferation, Differentiation, and Tumor Killing

Blood ◽

10.1182/blood-2018-99-117265 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2199-2199

Author(s):

Matt L Cooper ◽

Karl W. Staser ◽

Julie Ritchey ◽

Jessica Niswonger ◽

Byung Ha Lee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Effector Memory ◽

Photon Flux ◽

Interleukin 7 ◽

Tumor Killing ◽

Long Acting

Abstract Background: Chimeric antigen receptor T cell (CART) therapy is revolutionizing modern cancer therapy, with two anti-CD19 CARTs FDA-approved for relapsed/refractory B cell lymphoma/leukemia and many other CARTs for solid and liquid tumors currently undergoing clinical trials. Our group recently demonstrated multiplexed CRISPR/Cas9 gene-editing of anti-CD7 CARTs to produce CD7 and T cell receptor alpha constant (TRAC)-deleted "off-the-shelf" universal (U)CART7s that effectively kill CD7+ T cell lymphoma in vivo without causing GVHD or fratricide (Cooper et al, Leukemia, 2018). However, in current clinical practice, suboptimal CART persistence and tumor killing permit tumor cell escape and, ultimately, disease relapse. Reasoning that a pro-lymphoid growth factor could promote CART efficacy, we supplemented UCART infusion with subcutaneous injections of the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19+ lymphoma xenograft model. Methods: To create anti-CD19 universal CARTs (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC-targeted gRNA, and virally transduced an anti-CD19 scFv 3rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NOD-scid IL2Rgammanull (NSG) mice with 5x105 RamosCBR-GFP cells four days prior to UCART19 (2x106 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion. Results:RamosCBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving RamosCBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While RamosCBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of RamosCBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p<0.0001, UCART19+NT-I7 vs. UCART19). At three weeks post UCART19 infusion, bioluminescent imaging (BLI) revealed minimal tumor signal in UCART19+NT-I7 treated mice (108 vs. 1010 photon flux/s, p<0.05, UCART19+NT-I7 vs. UCART19) and near-undetectable photon flux/s at four weeks (107 vs 1011 photon flux/s, p<0.0001, UCART19+NT-I7 vs. UCART19). Quantitative 17-parameter flow cytometric analyses of the blood, bone marrow, and spleens revealed an up to ~8000-fold increase in UCART19 cells in NT-I7-treated mice four weeks post UCART19 infusion (Fig 1a). These UCART19 cells demonstrated a predominantly effector and effector memory phenotype. Discussion: CARTs engineered to express interleukin-7 and CCL19 showed increased migration to and killing of solid tumors (Adachi et al, Nature Biotechnology, 2018). However, genetically engineered potentiation strategies lack "off-switches" and may preclude additional genetic enhancements required for universal "off-the-shelf" CART development. Here, we demonstrate that a pharmacological grade long-acting interleukin-7 agonist can potentiate adoptive cellular therapies. Specifically, NT-I7 can dramatically enhance gene modified T cell proliferation, persistence and tumor killing in vivo, resulting in enhanced survival, providing a tunable clinic-ready adjuvant for reversing suboptimal CART activity in vivo. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership. Lee:NeoImmuneTech: Employment. Park:NeoImmuneTech: Employment.

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Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor T-Cells for Tumor Killing

Bioconjugate Chemistry ◽

10.1021/acs.bioconjchem.0c00258 ◽

2020 ◽

Vol 31 (7) ◽

pp. 1775-1783 ◽

Cited By ~ 1

Author(s):

Christian Pellegrino ◽

Nicholas Favalli ◽

Michael Sandholzer ◽

Laura Volta ◽

Gabriele Bassi ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Tumor Killing ◽

Conjugation Chemistry

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Abstract 3500: T cells redirected against HER2 for the adoptive immunotherapy for HER2-positive osteosarcoma

10.1158/1538-7445.am2012-3500 ◽

2012 ◽

Cited By ~ 5

Author(s):

Nabil Ahmed ◽

Vita Brawley ◽

Oumar Diouf ◽

Peter Anderson ◽

John Hicks ◽

...

Keyword(s):

T Cells ◽

Adoptive Immunotherapy ◽

Her2 Positive

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Abstract 5585: TGF-ß insensitive PSMA-specific CD8+T cells derived from metastatic castration resistant prostate cancer (MCRPC) patients enhance the tumor killing ability

10.1158/1538-7445.am2017-5585 ◽

2017 ◽

Author(s):

Qiang Zhang ◽

Timothy Kuzel ◽

Brian Helfand ◽

Ximing J. Yang ◽

Weijun Qin ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

Cd8 T Cells ◽

Castration Resistant Prostate Cancer ◽

Castration Resistant ◽

Tumor Killing

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Immunotherapy for HER2-positive medulloblastoma: Regression of experimental tumors by transfer of HER2-redirected T cells in vivo

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.9008 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 9008-9008

Author(s):

N. M. Ahmed ◽

M. K. Ratnayake ◽

B. Savoldo ◽

L. Perlaky ◽

G. P. Dotti ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Adoptive Transfer ◽

Xenograft Model ◽

Her2 Positive ◽

Immunotherapeutic Approach ◽

Murine Xenograft Model ◽

Healthy Donors ◽

Ifn Γ

9008 Background: The long-term objective of this project is to develop an innovative HER2-targeted immunotherapeutic approach for medulloblastoma, the most common malignant brain tumor of childhood. HER2 is expressed in 40% of medulloblastomas and at present less than one third of patients with HER2-positive tumors are cured by conventional therapies. The aim of this study was to determine if T cells grafted with a HER2-specific chimeric antigen receptor (CAR) recognize and kill HER2-positive medulloblastomas. Methods: Mitogen-activated T cells from healthy donors and medulloblastoma patients were transduced with a retroviral vector encoding a HER2-specific CAR with a ζ-signaling domain (HER2T-cells). We analyzed the ability of HER2T-cells to 1) proliferate, 2) produce immunostimulatory cytokines (IFN-γ and IL-2), and 3) kill HER2-positive targets in cytoxicity assays upon exposure to HER2-positive primary medulloblastoma cells and cell lines. The in vivo function was tested in an orthotopic murine xenograft model of human medulloblastoma, which allows for serial imaging by bioluminescence. Results: HER2T-cells killed both HER2-positive primary medulloblastoma cells and cell lines in cytotoxicity assays, whereas HER2-negative tumor cells were not killed. Stimulation of HER2T-cells resulted in T-cell proliferation and secretion of IFN-γ and IL-2 in a HER2-dependent manner. No functional difference was observed between cells generated from medulloblastoma patients receiving dexamethasone and healthy donors. In vivo, the adoptive transfer of HER2T-cells resulted in sustained regression of established medulloblastomas in an orthotopic murine xenograft model as judged by bioluminescence imaging. In contrast, delivery of non-transduced T cells did not change tumor growth in comparison to untreated tumors. Conclusions: This study shows for the first time that HER2 is a target antigen for the immunotherapy of medulloblastoma. HER2-redirected T-cells not only recognized and killed HER2-positive medulloblastomas ex vivo, but also induced regression of experimental medulloblastoma in vivo. Hence, adoptive transfer of HER2-redirected T-cells may represent a promising immunotherapeutic approach for medulloblastoma. No significant financial relationships to disclose.

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Effect of TCHL-based therapy on immune cell content in on-treatment, neoadjuvant-treated HER2-positive breast cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.583 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 583-583

Author(s):

Niamh M. Keegan ◽

Sinead Toomey ◽

Joanna Fay ◽

Stephen F. Madden ◽

Bruce Moran ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Immune Cell ◽

Cell Populations ◽

Breast Cancer Patients ◽

Myeloid Dendritic Cells ◽

Her2 Positive ◽

Her2 Positive Breast Cancer ◽

The Impact ◽

Positive Breast Cancer

583 Background: In the TCHL trial (NCT01485926) 78 women with HER2-positive breast cancer (BC) underwent neo-adjuvant treatment with either TCH (Docetaxel, Carboplatin, Trastuzumab) or TCHL (TCH + Lapatinib) therapy. Of the 78 patients, 24 consented to an optional on-treatment biopsy 20 days after 1 cycle of therapy. We analysed the impact of tumour infiltrating lymphocytes (TILs) on pathological complete response (pCR) and also determined the impact of TCH/TCHL therapy on immune cell modulation after 20 days of treatment. Methods: We assessed TIL and stromal lymphocytes (SL) counts using immunohistochemical staining with Haemotoxalyin+Eosin, AE1/AE3 and CD45 in formalin fixed paraffin embedded (FFPE) baseline biopsy samples and in fresh frozen (FF) biopsies taken 20-days post cycle 1 (Day-20) of TCH/TCHL. RNA libraries were generated, using the Truseq mRNA library prep kit on the Neoprep platform and sequenced on the NextSeq 500. We measured the transcriptomic profile of 8 pre and on-treatment sample pairs and then used the Microenvironment Cell Populations (MCP)-counter method to measure the abundance of 10 immune cell populations (T cells, CD8 T cells, cytotoxic lymphocytes, NK cells, B lineage, myeloid dendritic cells, neutrophils, endothelial cells and fibroblasts). Results: We found that higher baseline levels of TILs (p = 0.045) but not SL were associated with an increased likelihood of a patient achieving a pCR to TCH/L based therapy. We found in day 20 on-treatment biopsies of women that subsequently went onto have a pCR that levels of SLs but not TILs were significantly higher (p = 0.049) than in those women who did not have a pCR. Finally we found significant increases in the level of monocytes (p = 0.05) and fibroblasts (p = 0.01), but not other immune cell populations, in the day 20 on-treatment biopsies in comparison with the mutated pre-treatment biopsies. Conclusions: In our study baseline TILs but not SLs have a predictive role in the likelihood of a patient achieving a pCR. We also found that TCHL based therapy significantly altered both monocytes and fibroblasts, indicating a possible role for these immune subtypes in response to TCHL therapy.

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Preliminary biomarker and pharmacodynamic data from a phase I study of single-agent bispecific antibody T-cell engager GBR 1302 in subjects with HER2-positive cancers.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.69 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 69-69 ◽

Cited By ~ 4

Author(s):

Martin Wermke ◽

Juergen Alt ◽

John S. Kauh ◽

Jonathan Back ◽

Yacine Salhi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Peripheral Blood ◽

Bispecific Antibody ◽

Cell Populations ◽

Single Subject ◽

Her2 Positive ◽

Tnf Α ◽

Ifn Γ

69 Background: HER2 is overexpressed in many solid tumors and is a validated therapeutic target. GBR 1302 is a HER2xCD3 bispecific antibody engineered (using Glenmark’s BEAT® platform) to direct T-cells to HER2-expressing tumor cells. GBR1302-101 (NCT02829372) is an ongoing, multicenter, open-label, first-in-human study of GBR 1302 in subjects with HER2-positive cancers to evaluate the safety, tolerability, and preliminary efficacy of GBR 1302, and to elucidate the mechanism(s) by which it redirects T-cells to tumor and enhances cytolytic activity of cytotoxic T-cells. Methods: Adults with progressive HER2-positive solid tumors with no available standard or curative treatment receive intravenous GBR 1302 on Day 1 and Day 15 in 28-day treatment cycles at escalating dose levels, starting at 1 ng/kg. The first 4 cohorts consist of a single subject; subsequent cohorts enroll using a 3+3 design. The primary and secondary efficacy and safety endpoints of this trial will be reported at the end of the study. Preliminary pharmacodynamic (PD) data are reported for cellular biomarkers and cytokines as assessed by FACS and ELISA in peripheral blood. Results: Beginning at 30 ng/kg dosing of GBR 1302 (Cohort 4), numbers of peripheral blood CD3, CD4, and CD8 positive T-cell populations decreased within 6 hours of initiating administration, but recovered to levels at or above baseline by 48 hours. A parallel, transient increase was observed in peripheral blood cytokines (IL-2, IL-6, IL-10, IFN-γ, TNF-α). At doses greater than 30 ng/kg, more pronounced cytokine increases were observed, which normalized at 12 hours. At the highest dose level for which data are available (n = 8 subjects; Cohort 5), changes from baseline in cytokine expression at ~340 hours were greater by ~60-fold for IL-6, ~30-fold for IL-2, ~3-fold for IFN-γ, ~5-fold for TNF-α, and ~18-fold for IL-10. Two subjects treated at 100 ng/kg experienced Grade 1 cytokine release syndrome, evidenced by short-lived fever spikes. Dose escalation is ongoing. Conclusions: Preliminary PD data indicate changes in peripheral T-cell populations and inflammatory cytokines following GBR 1302 treatment. Clinical trial information: NCT02829372.

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