Therapeutic Potential of AO-176, a Next Generation Humanized CD47 Antibody, for Hematologic Malignancies

Abstract Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and over-expressed on several tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPalpha) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPalpha interaction and induces phagocytosis of hematologic and solid tumor cells, but also exhibits several unique functional properties. The first property is the ability of AO-176 to induce direct tumor cytotoxic cell death in hematologic (ex. Jurkat, Raji and Molt-4) as well as solid human tumor cell lines by a cell autonomous mechanism (not ADCC). Secondly, AO-176 exhibits preferential binding to tumor versus normal cells, including red blood cells (RBCs), T cells, endothelial cells, skeletal muscle cells and epithelial cells. A0-176 also does not affect the function of any of these primary cells when assayed ex vivo. The negligible binding of AO-176 to RBCs versus hematologic (ex. Jurkat, Raji or Molt-4) or solid tumor cells is particularly profound and different from other reported anti-CD47 antibodies. AO-176 also does not induce hemagglutination of RBCs. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. Consistent with this attribute, AO-176 was well tolerated in cynomolgus monkeys with no adverse effects in general nor with respect to RBCs which was consistent with ex vivo results. A third novel property of AO-176 is its enhanced binding to tumor cells at acidic pH. Because the microenvironment of leukemic bone marrow and solid tumors has an acidic pH, this enhanced binding of AO-176 at low pH has the potential added advantage of tumor-specific targeting. Lastly, we show that AO-176 demonstrates dose-dependent anti-tumor activity in hematologic and solid tumor xenograft models. Taken together, the unique properties and anti-tumor activity of our next generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPalpha axis targeting agents as it progresses to clinical development. Disclosures No relevant conflicts of interest to declare.

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163 Improved anti-tumor activity of next-generation TCR-engineered T cells through CD8 co-expression

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.163 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A173-A173

Author(s):

Gagan Bajwa ◽

Justin Gunesch ◽

Inbar Azoulay-Alfaguter ◽

Melinda Mata ◽

Ali Mohamed ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Solid Tumor ◽

Cd4 T Cells ◽

Tumor Response ◽

Hla Class I ◽

Next Generation ◽

Engineered T Cells ◽

Anti Tumor Activity

BackgroundSuccessful targeting of solid tumors with TCR-engineered T cells (TCR-T) will require eliciting of antigen-specific, multi-dimensional, sustained anti-tumor immune response by infused T cells while overcoming the suppressive tumor microenvironment. First-generation TCR-T approaches have demonstrated clinical efficacy in some solid cancers. However, effective treatment across several solid tumor indications may require engineered T cells with enhanced anti-tumor activity. Here, we show pre-clinical data from one of the engineering approaches currently being developed for next-generation ACTengine® TCR-T product candidates. We evaluated the impact of co-expression of different CD8 co-receptors on functionality of CD4+ and CD8+ T cells genetically modified with an HLA class I-restricted TCR and determined the depth and durability of anti-tumor response in vitro.MethodsHere, we used a PRAME-specific TCR currently being tested in the ACTengine® IMA203 clinical trial. T cells expressing either the TCR alone or co-expressing the TCR and CD8α homodimer (TCR.CD8α) or CD8αβ heterodimer (TCR.CD8αβ) were characterized for transgene expression, antigen-recognition, and functional efficacy in vitro. Comprehensive evaluation of CD4+ T cells expressing TCR.CD8α or TCR.CD8αβ was performed focusing on cytotoxic potential and the breadth of cytokine response against target-positive tumor cell lines.ResultsIntroduction of CD8α or CD8αβ enabled detection of transgenic TCR on the surface of CD4+ T cells via HLA multimer-guided flow cytometry otherwise lacking in the TCR only transduced T cells. Co-expression of either form of CD8 co-receptor endowed CD4+ T cells with the ability to recognize and kill target positive tumor cells; however, genetic modification with TCR.CD8αβ led to more pronounced CD4+ T cell activation as compared to TCR.CD8α. Most distinct differences were observed in the breadth and magnitude of cytokine responses, less in cytotoxic activity against tumor cells. T cells expressing TCR.CD8αβ showed superior induction of Th1 cytokines e.g. IFNγ, TNFα, IL-2, GM-CSF in vitro upon antigen stimulation as compared to TCR.CD8α-T cells. Additionally, TCR.CD8αβ T cells demonstrated more efficient engagement with antigen-presenting cells and consequently, modulation of cytokine response than TCR.CD8α-T cells.ConclusionsOur findings illustrate that engaging CD4+ T cells via CD8 co-expression potentiates anti-tumor activity of HLA class I restricted TCR-T cells in vitro. The pleiotropic effects mediated by activated CD4+ T cells including acquired cytotoxicity may potentially improve outcomes in solid tumor patients when applied clinically. In addition, the differential functional profile of TCR-T cells co-expressing either CD8α or CD8αβ suggests that optimizing the type of co-receptor is relevant to maximize anti-tumor response.

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Phase I/II Clinical Trial of the Anti-Podoplanin Monoclonal Antibody Therapy in Dogs with Malignant Melanoma

Cells ◽

10.3390/cells9112529 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2529

Author(s):

Satoshi Kamoto ◽

Masahiro Shinada ◽

Daiki Kato ◽

Sho Yoshimoto ◽

Namiko Ikeda ◽

...

Keyword(s):

Clinical Trial ◽

Clinical Trials ◽

Malignant Melanoma ◽

Adverse Effects ◽

Phase I ◽

Tumor Cells ◽

Antibody Therapy ◽

Human Tumor ◽

Preclinical Trial ◽

Canine Tumor

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed on tumor cells. PDPN is known to be linked with several aspects of tumor malignancies in certain types of human and canine tumors. Therefore, it is considered to be a novel therapeutic target. Monoclonal antibodies targeting PDPN expressed in human tumor cells showed obvious anti-tumor effects in preclinical studies using mouse models. Previously, we generated a cancer-specific mouse–dog chimeric anti-PDPN antibody, P38Bf, which specifically recognizes PDPN expressed in canine tumor cells. In this study, we investigated the safety and anti-tumor effects of P38Bf in preclinical and clinical trials. P38Bf showed dose-dependent antibody-dependent cellular cytotoxicity against canine malignant melanoma cells. In a preclinical trial with one healthy dog, P38Bf administration did not induce adverse effects over approximately 2 months. In phase I/II clinical trials of three dogs with malignant melanoma, one dog vomited, and all dogs had increased serum levels of C-reactive protein, although all adverse effects were grade 1 or 2. Severe adverse effects leading to withdrawal of the clinical trial were not observed. Furthermore, one dog had stable disease with P38Bf injections. This is the first reported clinical trial of anti-PDPN antibody therapy using spontaneously occurring canine tumor models.

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Bortezomib Augments Natural Killer Cell Targeting of Stem-Like Tumor Cells

Cancers ◽

10.3390/cancers11010085 ◽

2019 ◽

Vol 11 (1) ◽

pp. 85 ◽

Cited By ~ 9

Author(s):

Jesus Luna ◽

Steven Grossenbacher ◽

Ian Sturgill ◽

Erik Ames ◽

Sean Judge ◽

...

Keyword(s):

Stem Cell ◽

Natural Killer ◽

Tumor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Human Tumor ◽

Growth Delay ◽

Cell Targeting ◽

Immunodeficient Mice

Tumor cells harboring stem-like/cancer stem cell (CSC) properties have been identified and isolated from numerous hematological and solid malignancies. These stem-like tumor cells can persist following conventional cytoreductive therapies, such as chemotherapy and radiotherapy, thereby repopulating the tumor and seeding relapse and/or metastasis. We have previously shown that natural killer (NK) cells preferentially target stem-like tumor cells via non- major histocompatibility complex (MHC) restricted mechanisms. Here, we demonstrated that the proteasome inhibitor, bortezomib, augments NK cell targeting of stem cell-like tumor cells against multiple solid human tumor-derived cancer lines and primary tissue samples. Mechanistically, this was mediated by the upregulation of cell surface NK ligands MHC class I chain-related protein A and B (MICA and MICB) on aldehyde dehydrogenases (ALDH)-positive CSCs. The increased expression of MICA and MICB on CSC targets thereby enhanced NK cell mediated killing in vitro and ex vivo from both human primary tumor and patient-derived xenograft samples. In vivo, the combination of bortezomib and allogeneic NK cell adoptive transfer in immunodeficient mice led to increased elimination of CSCs as well as tumor growth delay of orthotopic glioblastoma tumors. Taken together, our data support the combination bortezomib and NK transfer as a strategy for both CSC targeting and potentially improved outcomes in clinical cancer patients.

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CMV-Specific T-Cells Are More Effective Than WT-1-Specific T-Cells in Eradicating Clonogenic Tumor Cells Co-Expressing CMVpp65 and WT-1 in-Vivo and Both Types of T-Cells Are Functionally Augmented in the Presence of IL-15/IL-15Rα

Blood ◽

10.1182/blood.v118.21.646.646 ◽

2011 ◽

Vol 118 (21) ◽

pp. 646-646 ◽

Cited By ~ 1

Author(s):

Aisha Hasan ◽

Dana Bakalar ◽

Annamalai Selvakumar ◽

Gloria C Koo ◽

Ekaterina Doubrovina ◽

...

Keyword(s):

T Cells ◽

Ovarian Carcinoma ◽

Tumor Cells ◽

Tumor Antigen ◽

Tumor Antigens ◽

Human Tumor ◽

Relative Efficacy ◽

Comparative Efficacy ◽

Anti Tumor Activity

Abstract Abstract 646 Adoptively transferred virus or tumor antigen-specific T-cells have demonstrated efficacy in initial clinical trials, but while clearance of viral infection is sustained, responses to tumor-specific T-cells are usually short-lived. Therefore, current efforts are focused on distinguishing attributes of virus-specific T-cells that contribute to their persistence and formulating strategies to sustain anti-tumor effects of T-cell (TC) therapies. We have developed an in-vivo model to compare the relative efficacy of T-cells specific for a tumor antigen (WT-1) versus T-cells specific for a viral antigen (CMVpp65) using human colon carcinoma cells that express the oncofetal protein WT-1 which were transduced to co-express CMVpp65 (WT-1[+] cocapp65) as a surrogate system. Groups of 6 NOD/Scid-IL2Rgc-KO/J mice (NSG) were each subcutaneously injected with 3 × 105 WT-1 [+] cocapp65 on the R flank. Each animal was also injected with 3 × 105 cells from a WT-1[+] ovarian carcinoma cell line (SKOV3-A2) on the L shoulder to compare the efficacy of WT-1 specific T-cells (WT1-CTLs) against different WT-1 [+] tumor cell types. Expression of WT-1 protein is lower in SKOV3-A2 than in cocapp65 cells. Both tumors are HLA A0201[+] and were transduced to express a GFP-firefly luciferase gene. T-cells were administered intravenously 5 days after tumor injection to enable vascularization and tumor growth was quantitated using bioluminescence. These experiments evaluated (1) the relative capacity of CMVpp65 specific T-cells (CMV-CTLs) versus WT-1 CTLs to eradicate WT1[+] cocapp65 cells that co-express a viral and tumor antigen (2) the relative efficacy of WT-1 CTLs against 2 different HLA A0201 [+] WT-1 expressing tumors; an ovarian carcinoma and a colon carcinoma, and (3) the contribution of IL-15/15Rα complex in augmenting the efficacy of antigen specific T-cells by using intraperitoneally (i.p) injected Baf-3 cells transduced to express human IL-15/15Rα complex. The treatment groups were as follows: (1) Control – no T-cells + IL-2 (2000 U) (2) Control – no T-cells + IL-15/IL-15Rα (5 × 106 baf-3 cells) (3) WT1 CTLs + IL-2 (2000 U) (4) CMV-CTLs + IL-2 (2000 U) (5) WT1 CTLs + IL-15/IL-15Rα (5 × 106 baf-3 cells) (6) CMV-CTLs + IL-15/IL-15Rα (5 × 106 baf-3 cells). IL-2 and irradiated baf-3 cells were administered intraperitoneally twice weekly. When the doses of antigen specific interferon gamma (IFNg) [+] T-cells were equivalent in the infused CMVpp65 and WT1 specific T-cells, the CMV-CTLs induced greater, and more sustained suppression of the growth of the WT-1[+] cocapp65 cells in-vivo than the WT-1 CTLs (Fig.1). The anti-tumor activity of the WT-1 CTLs was greater against WT-1[+] cocapp65 than against the WT-1[+] ovarian carcinoma (SKOV3-A2), potentially reflecting the higher expression of WT-1 in cocapp65. The SKOV3-A2 tumor began to re-grow by 24 days post T-cell infusion approaching the size of control tumors by day 38, while the WT-1[+] cocapp65 still demonstrated slower growth through day 38. The addition of IL-15/15Rα increased the efficacy of the transferred T-cells, the difference being more pronounced for the anti-tumor activity of WT-1 CTLs. Fig. 1 Comparative Efficacy of CMV and WT-1 CTLs against Human Tumor Targets Co-expressing CMVpp65 and WT-1 Fig. 1. Comparative Efficacy of CMV and WT-1 CTLs against Human Tumor Targets Co-expressing CMVpp65 and WT-1 These studies demonstrate that equivalent doses of IFNg[+] WT-1 CTLs can also suppress WT-1[+] cocapp65 tumor xenografts, but are less effective than CMV-CTLs, and that IL-15 supplementation augments the cytotoxic activity of the CTLs in-vitro and enhances the duration of the anti-tumor effects in-vivo. This model permits side by side comparisons of the anti-tumor activity of human T-cells directed against viral and tumor antigens expressed on the same clonogenic human tumor target. Because both responses are directed against the same cells, this model could thereby facilitate identification of the distinguishing features of T-cells specific for viral or tumor antigens as well as differences in the presentation of viral and oncofetal “self” antigens by tumor cells that contribute to disparities in their anti-tumor activity and persistence in-vivo. Disclosures: No relevant conflicts of interest to declare.

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Avidity-based binding to HER2 results in selective killing of HER2-overexpressing cells by anti-HER2/CD3

Science Translational Medicine ◽

10.1126/scitranslmed.aat5775 ◽

2018 ◽

Vol 10 (463) ◽

pp. eaat5775 ◽

Cited By ~ 33

Author(s):

Dionysos Slaga ◽

Diego Ellerman ◽

T. Noelle Lombana ◽

Rajesh Vij ◽

Ji Li ◽

...

Keyword(s):

T Cell ◽

Adverse Effects ◽

Tumor Cells ◽

Solid Tumor ◽

Therapeutic Index ◽

Clinical Development ◽

Bispecific Antibodies ◽

Human Tissues ◽

High Potency ◽

Normal Human

A primary barrier to the success of T cell–recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell–dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab–immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell–directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.

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Sample Processing Obscures Cancer-Specific Alterations in Leukemic Transcriptomes

Blood ◽

10.1182/blood.v124.21.2201.2201 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2201-2201

Author(s):

Heidi Dvinge ◽

Rhonda E. Ries ◽

Janine O. Ilagan ◽

Derek L. Stirewalt ◽

Soheil Meshinchi ◽

...

Keyword(s):

Solid Tumor ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Biological Pathways ◽

Blood Collection ◽

Sample Processing ◽

Prolonged Incubation ◽

Rna Surveillance ◽

Antisense Rnas ◽

Induced Changes

Abstract Substantial effort is currently devoted to identifying cancer-associated alterations using genomics technologies. While the genome is inherently stable over short time frames, the transcriptome is dynamic and potentially susceptible to alteration as samples move from the patient to the lab bench. Here, we show that standard blood collection procedures rapidly change the transcriptional and post-transcriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways, up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms, and inhibition of RNA surveillance. These artifacts affect almost all published leukemia genomics studies, explaining up to 40% of putative cancer-associated differential expression and alternative splicing. To determine how standard blood collection procedures affect hematopoietic transcriptomes, we collected whole blood from four healthy donors in anticoagulant blood collection tubes. We then left this blood at room temperature for defined lengths of time (0-48h) in order to mimic the variable incubation that patient samples are typically subjected to during transfer from the primary treating physician to a research center, isolated peripheral blood mononuclear cells (PBMCs), and performed deep RNA-seq (Figure A). Contrary to the common assumption that RNA degrades during incubation, we observed no evidence of decreased RNA quality. Nonetheless, rapid and dramatic changes affected virtually every level of the gene expression process. The changes were highly biased; for example, pseudogenes and antisense RNAs were preferentially up-regulated, while cassette exons were preferentially repressed (Figure B). Incubation-induced changes in the transcriptome confound the identification of true cancer-specific alterations. Many genes affected by incubation participate in biological pathways of current interest in leukemia, including cytokine production, NF-κB signaling, chromatin modification, and RNA splicing. Furthermore, incubation for as little as 4 hours, our shortest time point, introduced dramatic changes in the post-transcriptional landscape. We observed widespread isoform switches, wherein isoforms that were rare or even undetectable at 0h became the major isoform after 8 to 24h of incubation, in genes such as NOTCH2, LEF1, and PHF20 that have been previously used as leukemic biomarkers (Figure C). Perhaps most surprisingly, incubation rapidly inhibited RNA surveillance, leading to genome-wide expression of normally degraded RNAs. This highly abnormal RNA surveillance inhibition was readily detectable in all published leukemia genomics datasets that we analyzed, with the exception of TCGA, and undetectable in any lymphoma or solid tumor dataset (Figure D). Together, our data show that incubation-induced changes in the transcriptional and post-transcriptional landscapes generate substantial artifacts that complicate the interpretation of leukemia genomics studies. To facilitate the incorporation of sample processing artifacts into downstream analysis, we provide biomarkers that detect prolonged incubation of individual samples. We furthermore show that the simple expedient of keeping blood on ice drastically reduces changes to the transcriptome. Our findings have important implications for the interpretation of published and ongoing leukemia genomics studies. Figure. (A) Leukemic samples are frequently subject to an ex vivo incubation period of variable length. (B) Ex vivo incubation causes biased up-regulation of pseudogenes and antisense RNAs and repression of cassette exons. (C) Incubation causes complete isoform switches in the LEF1 and PHF20 genes. (D) RNA surveillance is inhibited after only 4h of incubation. This abnormal inhibition of RNA surveillance is visible in all analyzed leukemia genomics datasets, with the exception of TCGA, and not in any lymphoma or solid tumor. Numbers above each x axis label indicate the number of samples in each dataset. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Proteolytic Disassembly of Viral Outer Capsid Proteins Is Crucial for Reovirus-Mediated Type-I Interferon Induction in Both Reovirus-Susceptible and Reovirus-Refractory Tumor Cells

BioMed Research International ◽

10.1155/2015/468457 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Yuki Katayama ◽

Yuichi Terasawa ◽

Masashi Tachibana ◽

Hiroyuki Mizuguchi ◽

Fuminori Sakurai

Keyword(s):

Immune Responses ◽

Tumor Cells ◽

Human Tumor ◽

Innate Immune ◽

Capsid Proteins ◽

Type I ◽

Innate Immune Responses ◽

Outer Capsid ◽

Proapoptotic Gene

Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, followingin vivoapplication. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN-)βand of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-βand Noxa were significantly induced by reovirusviathe IFN-βpromoter stimulator-1 (IPS-1) signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-βand Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

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681 An ex vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess the phagocytic activity of tumor resident innate immune cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.681 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A709-A709

Author(s):

Kelly Guzman ◽

Olivia McIntosh ◽

Brittany Bunch ◽

Jacob Yarinsky ◽

Jared Ehrhart ◽

...

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Phagocytic Activity ◽

Ex Vivo ◽

Cell Killing ◽

Innate Immune ◽

Immune Microenvironment ◽

Tumor Immune Microenvironment ◽

The Impact ◽

Tumor Cell Killing

BackgroundCD47 is an innate immune checkpoint receptor that is overexpressed on tumor cells and contributes to immune evasion through engagement of a myeloid-lineage inhibitory protein SIRPα. Blockade of the CD47-SIRPα interaction is proved to enhance the phagocytosis of cancer cells and to induce effective antitumor immune response. Here we developed a novel ex vivo platform using fresh patient tumor samples with intact stromal components and tumor immune microenvironment to assess the therapeutic activity of immunotherapeutic drugs targeting CD47-SIRPα signaling axis in combination with the human IgG1 αPD-L1 antibody avelumab.MethodsAll tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma using proprietary technology developed at Nilogen Oncosystems. Tumoroids prepared from each patient's tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with phrodo-labeled avelumab alone or in combination with anti-CD47 or anti-SIRPα therapeutics.ResultsMultiparameter flow analysis demonstrated tumor binding of avelumab confirming drug penetration into the intact tumor stroma that is further corroborated by high content confocal analysis. Using our confocal-based tumor cell killing assay we were able to quantify drug-induced tumor cell killing ex vivo. We further documented the impact of anti-CD47 and anti-SIRPα therapeutics on phagocytosis of dead tumor cells by tumor resident macrophages and activation of innate and adaptive effector cells by flow cytometry and confocal imaging. Additionally, pHrodo-labeled bioparticles were used to corroborate treatment-mediated changes in the phagocytic activity of tumor resident macrophages.ConclusionsIn this comprehensive study we demonstrate that the 3D-EXplore ex vivo platform can be used to assess the efficacy of therapeutic blockade of CD47/SIRPα axis on stimulation of phagocytic process within an intact tumor immune microenvironment.

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P08.01 Rationale of using the combination of anti-PD-1 antibody and anti-IL-8 antibody for the pancreatic cancer treatment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.45 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A24.2-A25

Author(s):

P Li ◽

N Rozich ◽

J Wang ◽

J Gai ◽

J Wang ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Innate Immune Response ◽

Ex Vivo ◽

Myeloid Cells ◽

Innate Immune ◽

Sequencing Analysis ◽

Nuclear Rna ◽

Part Time ◽

Anti Tumor Activity

BackgroundPancreatic ductal adenocarcinoma (PDAC) does not respond to immune checkpoint inhibitors (ICI) therapy as single agent treatments including anti-PD-1 antibody. One of the mechanisms for the resistance of PDAC to ICI is now attributed to the immunosuppressive microenvironment (TME) in PDAC. Myeloid cells are thought to be the predominant immunosuppressive cells in the TME. Human interleukin-8 (IL-8) is a pro-inflammatory chemokine in the CXC family and has the capability of recruiting myeloid cells into the TME to promote tumor progression and immune escape. Therefore, several anti-IL-8 blockade antibodies were developed including HuMax-IL8 and B108-IL8, which both are fully human IgG1 kappa monoclonal antibodies. We therefore tested whether anti-IL-8 antibodies can potentiate anti-tumor activity of anti-PD-1 antibody in a humanized model of PDAC.Materials and MethodsWe reconstituted the immune system of the NGS mice with ex vivo activated human T cells and a combination of CD14+ and CD16+ myeloid cells after the mice were orthotopically implanted with human PDAC cells. 10x single nuclei RNA-Seq data processing was further performed to analyze differentially expressed genes among certain cell clusters.ResultsOur results showed that anti-PD-1 antibody alone had a minimal anti-tumor activity when mice was reconstituted with ex vivo activated T cells. Interestingly, the infusion of the combination of CD14+ and CD16+ myeloid cells together with anti-PD-1 antibody resulted in a modest anti-tumor activity. Adding either HuMAX-IL8 or B108-IL8 led to a significantly enhanced anti-tumor activity. Both CD14+ and CD16+ myeloid cells appeared to be needed for the full anti-tumor activity of IL-8 blockade because mice infused with only CD14+ myeloid cells did not respond to IL-8 blockade and mice infused with only CD16+ myeloid cells responded partially to IL-8 blockade. This result suggested that the target of IL-8 is mainly present in CD16+ myeloid cells and is likely to be granulocytes. Tumor infiltrating immune cells were isolated and demonstrated that IL-8 blockade increases CD45+CD11b+CD15+CD14- myeloid cells, which is known to comprise neutrophils and granulocytic myeloid derived suppressive cells (G-MDSC), in the tumors. Reconstitution of the mice with myeloid cells led to a decrease of CD8+ T cells in the tumors; however, IL-8 blockade brought the CD8+ T cell number back to the baseline. Consistent with an effect of IL-8 blockade on the increase of CD15+CD14- myeloid cells, single nuclear RNA sequencing analysis of the tumor tissues showed that the innate immune response and cytokine response pathways in the myeloid cell cluster were activated by IL-8 blockade.ConclusionsThis result suggested that IL-8 blockade did not simply inhibit myeloid cells as previously anticipated, but potentiated myeloid cells for the innate immune response and concomitant production of type I cytokines. Such immune responses may subsequently activate the effector T cells as the single nuclear RNA sequencing analysis demonstrated enhanced activation signals in the T cell cluster from the tumors treated by anti-IL-8 antibodies. Taken together, this study supports further testing of anti-IL-8 antibodies including B108-IL8 and HuMax-IL8 in combination with anti-PD-1 antibodies for PDAC treatment.Disclosure InformationP. Li: None. N. Rozich: None. J. Wang: None. J. Gai: None. J. Wang: None. Y. Xu: None. B. Herbst: None. R. Yu: A. Employment (full or part-time); Significant; NovaRock. S. Muth: None. N. Niu: None. K. Li: None. V. Fune: None. A. Osipov: None. C. Wolfgang: None. M. Lei: A. Employment (full or part-time); Significant; NovaRock. T. Liang: None. L. Zheng: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Bristol-Meyer Squibb, Merck, iTeos, Amgen, NovaRock, Inxmed, Halozyme. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Alphamab, Mingruzhiyao. F. Consultant/Advisory Board; Significant; Biosion, Alphamab, NovaRock, Akrevia/Xilio, Ambrx, Novagenesis, Datarevive, Snow Lake Capitals, Mingruzhiyao. Other; Significant; Aduro Biotech.

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GRP-78 Secreted by Tumor Cells Blocks the Anti-Angiogenic Activity of Bortezomib.

Blood ◽

10.1182/blood.v114.22.4805.4805 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4805-4805

Author(s):

Johann Kern ◽

Gerold Untergasser ◽

Christoph Zenzmaier ◽

Bettina Sarg ◽

Gunther Gastl ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Cell ◽

Tumor Cells ◽

Cell Lines ◽

Solid Tumor ◽

Conflicts Of Interest ◽

Exchange Chromatography ◽

Size Exclusion ◽

Tumor Xenografts

Abstract Abstract 4805 Anti-angiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane (CAM). Bortezomib's inhibitory effects on CAM vascularization were abrogated in the presence of distinct tumor xenografts thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. In fact, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib, but induced prosurvival signals by phosphorylation of ERK and inhibited p53-mediated expression of pro-apoptotic Bok and Noxa proteins in endothelial cells. From our data we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. Disclosures: No relevant conflicts of interest to declare.

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