A cocktail of antibodies to multiple tumor-specific neoantigens increases binding and inhibits tumor growth

Abstract Background: Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity resulting in variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. Methods: Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. Results: Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. Conclusions: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple does were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.

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Comparative benefits of nab-paclitaxel over gemcitabine or polysorbate-based docetaxel in experimental pancreatic cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.192 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 192-192

Author(s):

Niranjan Awasthi ◽

Katherine T Ostapoff ◽

Changhua Zhang ◽

Margaret A. Schwarz ◽

Roderich Schwarz

Keyword(s):

Pancreatic Cancer ◽

Tumor Growth ◽

Single Agent ◽

Nuclear Antigen ◽

In Vivo Model ◽

Tumor Growth Inhibition ◽

Multiple Tumor ◽

Animal Survival ◽

Experimental Pancreatic Cancer

192 Background: Gemcitabine (Gem), a standard cytotoxic therapy for pancreatic cancer, has shown limited clinical benefits. Nanoparticle albumin-bound (nab) paclitaxel (NPT), an approved treatment for breast cancer, has shown efficacy as mono- and combination therapy in multiple tumor types including pancreatic, lung and ovarian cancer. We evaluated the NPT treatment benefits compared with Gem or solvent-based taxane docetaxel (DT) in experimental pancreatic cancer. Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and animal survival studies were performed in murine xenografts. Intratumoral proliferative activity was measured using Ki67 nuclear antigen staining. Results: For AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells in vitro, Gem IC50 levels were 23.9 mM, 506 nM, 332 nM and 14.5 nM; DT IC50 levels were 30 nM, 4.6 nM, 37.5 nM and 27 nM; and NPT IC50 levels were 7.6 mM, 208 nM, 519 nM and 526 nM. NPT addition decreased Gem IC50 to 1.7 mM, 189 nM, 123 nM and 913 nM; DT addition decreased Gem IC50 to 436 nM, 470 nM, 124 nM and 0.2 nM in AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells, respectively. NPT and DT treatment increased stathmin phosphorylation and decreased tubulin expression in vitro. In a heterotopic in vivo model, net tumor growth inhibition after Gem, DT and NPT was 67, 31 and 72 percent, while intratumoral proliferative index inhibition was 41, 53 and 68 percent, respectively. In an intraperitoneal model, median animal survival was significantly longer in the NPT treatment group (41 days, p<0.002 vs. control and Gem) compared to Gem (32 days, p=0.005 vs. control), DT (32 days, p=0.005 vs. control) and controls (20 days). Animal survival in NPT-Gem and DT-Gem sequential treatment groups was 43 and 40 days, and thus not superior to NPT alone. Conclusions: Nab-paclitaxel has significantly superior antitumor activity as a single agent in experimental pancreatic cancer compared with gemcitabine or docetaxel. These findings provide a strong rationale for considering nab-paclitaxel as first-line monotherapy in patients with pancreatic cancer.

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Flavonoids kaempferol and quercetin are nuclear receptor 4A1 (NR4A1, Nur77) ligands and inhibit rhabdomyosarcoma cell and tumor growth

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02199-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Rupesh Shrestha ◽

Kumaravel Mohankumar ◽

Greg Martin ◽

Amanuel Hailemariam ◽

Syng-ook Lee ◽

...

Keyword(s):

Cell Growth ◽

Tumor Growth ◽

Nuclear Receptor ◽

Anticancer Agents ◽

Xenograft Model ◽

Boyden Chamber ◽

Tumor Growth Inhibition ◽

Western Blots ◽

Multiple Tumor ◽

Mouse Xenograft Model

Abstract Background Flavonoids exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with KD values of 3.1 and 0.93 μM, respectively. Methods The activities of kaempferol and quercetin were determined in direct binding to NR4A1 protein and in NR4A1-dependent transactivation assays in Rh30 and Rh41 rhabdomyosarcoma (RMS) cells. Flavonoid-dependent effects as inhibitors of cell growth, survival and invasion were determined in XTT and Boyden chamber assays respectively and changes in protein levels were determined by western blots. Tumor growth inhibition studies were carried out in athymic nude mice bearing Rh30 cells as xenografts. Results Kaempferol and quercetin bind NR4A1 protein and inhibit NR4A1-dependent transactivation in RMS cells. NR4A1 also regulates RMS cell growth, survival, mTOR signaling and invasion. The pro-oncogenic PAX3-FOXO1 and G9a genes are also regulated by NR4A1 and, these pathways and genes are all inhibited by kaempferol and quercetin. Moreover, at a dose of 50 mg/kg/d kaempferol and quercetin inhibited tumor growth in an athymic nude mouse xenograft model bearing Rh30 cells. Conclusion These results demonstrate the clinical potential for repurposing kaempferol and quercetin for clinical applications as precision medicine for treating RMS patients that express NR4A1 in order to increase the efficacy and decrease dosages of currently used cytotoxic drugs.

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Flavonoids Kaempferol and Quercetin are Nuclear Receptor 4A1 (NR4A1, Nur77) Ligands and inhibit Rhabdomyosarcoma Cell and Tumor Growth

10.21203/rs.3.rs-638373/v1 ◽

2021 ◽

Author(s):

Rupesh Shrestha ◽

Kumaravel Mohankumar ◽

Greg Martin ◽

Amanuel Hailemariam ◽

Syng-ook Lee ◽

...

Keyword(s):

Cell Growth ◽

Tumor Growth ◽

Nuclear Receptor ◽

Anticancer Agents ◽

Xenograft Model ◽

Boyden Chamber ◽

Tumor Growth Inhibition ◽

Western Blots ◽

Multiple Tumor ◽

Mouse Xenograft Model

Abstract BackgroundFlavonoid’s exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with KD values of 3.1 and 0.93 µM, respectively. MethodsThe activities of kaempferol and quercetin were determined in direct binding to NR4A1 and in NR4A1-dependent transactivation assays in Rh30 and Rh41 rhabdomyosarcoma (RMS) cells, flavonoid-dependent effects as inhibitors of cell growth, survival and invasion were determined in XTT, Annexin V and Boyden chamber assays respectively and changes in protein levels were determined by western blots. Tumor growth inhibition studies were carried out in athymic nude mice bearing Rh30 cells. ResultsKaempferol and quercetin bind NR4A1 protein and inhibit NR4A1-dependent transactivation in RMS cells. NR4A1 also regulates RMS cell growth, survival, and invasion and pro-oncogenic PAX3-FOX01 and G9a gene expression, mTOR signaling and gene products and, these pathways and genes are inhibited by kaempferol and quercetin. Moreover, at a dose of 50 mg/kg/d kaempferol and quercetin inhibited tumor growth in athymic nude mouse xenograft model bearing Rh30 cells. ConclusionThese results demonstrate the clinical potential for repurposing these flavonoids for clinical applications as precision medicine for treating RMS patients that express NR4A1 in order to increase the efficacy and decrease dosages of currently used cytotoxic drugs.

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Anti-Metastatic Benefits Produced by Hyperthermia and a CCL3 Derivative

Cancers ◽

10.3390/cancers11111770 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1770

Author(s):

Liqiu Ma ◽

Ryosuke Kambe ◽

Tomoko Tsuchiya ◽

Shiro Kanegasaki ◽

Akihisa Takahashi

Keyword(s):

Combination Therapy ◽

Tumor Growth ◽

Lung Metastases ◽

Single Amino Acid ◽

Tumor Growth Inhibition ◽

Adenocarcinoma Cells ◽

High Frequency Waves ◽

The Right ◽

Improve Patient ◽

Treated Site

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 μg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated after 18 days was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

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Tumor growth inhibition with bispecific antibody fragments in a syngeneic mouse melanoma model: The role of targeted T-cell costimulation via CD28

Immunology Letters ◽

10.1016/s0165-2478(97)88217-x ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 340

Author(s):

L Hovest

Keyword(s):

T Cell ◽

Tumor Growth ◽

Growth Inhibition ◽

Bispecific Antibody ◽

Antibody Fragments ◽

Tumor Growth Inhibition ◽

Syngeneic Mouse ◽

Mouse Melanoma Model ◽

Melanoma Model

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245 Human TLR8 knock-in mice potentiate immunotherapy responses of MC38 syngeneic tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0245 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A264-A264

Author(s):

Shanshan Qi ◽

Hongjuan Zhang ◽

Ruilin Sun ◽

Annie An ◽

Henry Li ◽

...

Keyword(s):

T Cells ◽

Tumor Growth ◽

Cancer Immunotherapy ◽

Tumor Regression ◽

Tumor Growth Rate ◽

Tumor Growth Inhibition ◽

List Type ◽

Rna Recognition ◽

Ethical Guidelines ◽

Ifn Γ

BackgroundToll-like receptors (TLRs) serve critical roles in mediating innate immune responses against many pathogens. However, they may also bind to endogenous ligands and lead to the pathogenesis of autoimmunity. Although TLR8 belongs to the same TLR family as TLR7, its role in inflammation and tumor progression is not yet fully understood due to the lack of suitable animal models. In humans, both TLR7 and TLR8 recognize single-stranded self-RNA, viral RNA, and synthetic small molecule agonists.1, 2 However, mouse Tlr8 is non-functional due to the absence of 5 amino acids necessary for RNA recognition. In order to create a mouse model with functional TLR8, we replaced exon 3 of mouse Tlr8 with human TLR8, therefore developing a hTLR8 knock-in (KI) model. Both heterozygous and homozygous hTLR8 KI mice are viable with inflammatory phenotypes, i.e. enlarged spleens and livers, and significantly higher IL-12 p40 levels under TLR8 agonist treatment. In this study, we evaluated the potential use of hTLR8 mice for cancer immunotherapy studies.MethodshTLR8 mice, together with naïve C57BL/6 mice, were inoculated with MC38 syngeneic tumor cells. Tumor bearing mice were grouped at a mean tumor volume of approximately 100 mm3 for treatment with PBS or 10 mg/kg anti-PD-1 (RMP1-14) antibody. At the efficacy endpoint, spleens and tumors were collected for flow cytometry profiling.ResultsAnti-PD-1 treatment of MC38 tumors in naïve C57BL/6 led to moderate tumor growth inhibition (TGI = 54%). Interestingly, anti-PD-1 treatment showed improved efficacy in hTLR8 mice (TGI = 79%), including 2/10 tumors with complete tumor regression. In comparison, non-treated MC38 tumor growth rate was slower in hTLR8 mice than in naïve mice. Anti-PD-1 treated hTLR8 mice also had significantly increased IFN-γ and TNF-a positive CD4+ T cells in the spleen, along with higher numbers of differentiated effector T cells. In addition, hTLR8 mice have activated dendritic cells and macrophages, acting as critical steps in initiation of the inflammatory process, with higher levels of pro-inflammatory cytokines, such as IL-6, IFN-γ, TNF-a, and IL-1β, which may promote Th1 priming and differentiation of T cells into IFN-γ or TNF-a producing cells.ConclusionshTLR8 mice offer a great tool to model cancer immunotherapy in an inflammatory/autoimmunity prone background. Moreover, hTLR8 mice can be effectively used to shift a ‘cold’ tumor phenotype to ‘hot’ tumors in a syngeneic setting.Ethics ApprovalAnimal experiments were conducted in accordance with animal welfare law, approved by local authorities, and in accordance with the ethical guidelines of CrownBio (Taicang).ReferencesKugelberg E. Making mice more human the TLR8 way. Nat Rev Immunol 2014;14:6.Guiducci C, Gong M, Cepika A-M, et al. RNA recognition by human TLR8 can lead to autoimmune inflammation. J Exp Med 2013;210:2903–2919.

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