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Basic science

Basic Science

Basic science

8P - The characterization of tumors associated with the antitumor activity of lenvatinib plus anti-PD-1 antibody combination therapy in a mouse syngeneic model panel

Presentation Number
8P
Speakers
  • Yoichi Ozawa (Tsukuba, Japan)

Abstract

Background

Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor and fibroblast growth factor receptors. We also reported that lenvatinib has immunomodulatory activity that contributes to the antitumor activity of lenvatinib and enhances the antitumor activity in combination treatment with anti-PD-1 antibody (anti-PD-1). In this study, we investigated the characters of tumors associated with the antitumor activity of the lenvatinib plus anti-PD-1 combination treatment.

Methods

We evaluated the antitumor activities of lenvatinib (10 mg/kg, Q.D.), anti-PD-1 (200mg/head, twice weekly), and their combination in 12 mouse syngeneic tumor models. To define the characteristics of tumors, we conducted RNA-seq analysis to examine T cell inflamed GEP (GEP) score using mouse tumors before treatment. We also stained CD31 in the same tumors and calculated micro-vessel density (MVD) score by dividing CD31 positive blood vessels number by tumor area. Then, the correlation analysis among gene expression levels of GEP, IHC-based MVD score and the antitumor activity of each administration was investigated.

Results

We confirmed that lenvatinib plus anti-PD-1 inhibited tumor growth more than each single treatment in this mouse syngeneic model panel. Regarding the relationship between tumor characters and antitumor activities of each treatment, we found that antitumor activities of lenvatinib were correlated with MVD scores of pretreatment tumors. MVD-high group was significantly more sensitive to lenvatinib than MVD-low group. On the other hand, antitumor activities of anti-PD-1 were correlated with GEP score. Lenvatinib plus anti-PD-1 combination demonstrated significant enhancement of antitumor activity compared with each single agent treatment in MVD-low / GEP-high tumors.

Conclusions

In this study, we found that MVD combined with GEP might be the important predictive biomarkers reflecting the antitumor activities of lenvatinib monotherapy and lenvatinib plus anti-PD-1 combination therapy.

Legal entity responsible for the study

Eisai Co., Ltd.

Funding

Eisai Co. Ltd., Tokyo, Japan, and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Disclosure

Y. Ozawa, M. Kuronishi, Y. Kato: Financial Interests, Personal, Full or part-time Employment: Eisai Co., Ltd.

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Basic science

9P - Click Activated Protodrugs Against Cancer (CAPAC) platform enhances the safety, pharmacokinetics, and antitumor efficacy of cancer therapies in vivo

Presentation Number
9P
Speakers
  • Sangeetha Srinivasan (San Francisco, United States of America)

Abstract

Background

The Click Activated Protodrugs Against Cancer (CAPACTM) platform aims to beat cancer without poisoning the body by activating powerful cancer therapies at the tumor site(s). CAPAC’s mechanism of activation is based on click chemistry and is therefore agnostic to tumor characteristics, biomarker expression, or other biological factors that vary across patients. This allows the CAPAC platform to be readily applicable to diverse tumor types. The lead candidate, SQ3370, is being evaluated in a phase I study in patients with advanced solid tumors (NCT04106492). We describe the safety, pharmacokinetics (PK), and therapeutic benefits of SQ3370, the lead candidate of the CAPAC Platform, in vivo.

Methods

SQ3370 consists of 2 components, SQL70 biopolymer, and SQP33 protodrug. First, SQL70, a tetrazine-modified sodium hyaluronate biopolymer, is injected at the tumor site. Then, SQP33, a trans-cyclooctene (TCO)-modified protodrug of Doxorubicin (Dox) is given systemically as 5 daily doses. SQP33 has attenuated toxicity and is converted to active Dox by SQL70 at the tumor site through an efficient covalent reaction between tetrazine and TCO moieties.

Results

In canines, the highest non-severely toxic dose of SQ3370 was found to be 8.95 times higher when compared to the conventional dose of Dox. There were minimal and reversible systemic adverse events, with no evidence of cardiotoxicity. The PK analysis in dogs showed that SQL70 efficiently captures SQP33 from circulation and releases active Dox throughout the treatment period. In the absence of SQL70, SQP33 was found to be stable and did not spontaneously activate. In mice, at least 50% of the SQL70 remains at the injection site for 2-4 weeks, and plasma levels of SQP33 and Dox are similar across SQL70 injection locations. SQ3370 elicited dose-dependent anti-tumor responses in three syngeneic dual tumor models.

Conclusions

SQ3370 enables higher concentrations of the active drug at the tumor site and minimizes systemic adverse effects associated with conventional chemotherapy. The CAPAC Platform represents a new therapeutic modality to treat solid tumors by using a drug with known efficacy, such as Dox, and expanding its pharmacological capabilities.

Legal entity responsible for the study

Shasqi.

Funding

Shasqi.

Disclosure

S. Srinivasan, N.A. Yee, A. Mahmoodi, M. Zakharian, M.W. Saville: Financial Interests, Personal, Full or part-time Employment: Shasqi; Financial Interests, Personal, Stocks/Shares: Shasqi. J.M. Mejía Oneto: Financial Interests, Personal, Member of the Board of Directors: Shasqi; Financial Interests, Personal, Stocks/Shares: Shasqi.

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Basic science

10P - DS-6000a, a novel CDH6-targeting antibody-drug conjugate with a novel DNA topoisomerase I inhibitor DXd, demonstrates potent antitumor activity in preclinical models

Presentation Number
10P
Speakers
  • Hirokazu Suzuki (Shinagawa-ku, Japan)

Abstract

Background

Human Cadherin 6 (CDH6) is a single transmembrane protein consisting of 790 amino acids classified into the type 2 cadherin family. Human CDH6 is specifically expressed in the brain and kidneys during the development phase and has been reported to systemically decrease CDH6 expression in the adult body. CDH6 expression is increased specifically in renal cell carcinoma (RCC) and ovarian cancer (OVC). Therefore, CDH6 could be an attractive target for cancer therapy. We created DS-6000a, a CDH6-targeting antibody-drug conjugate (ADC) using an enzymatically cleavable tetrapeptide-based linker, and a high drug-to-antibody ratio (DAR 7 to 8) with a novel DNA topoisomerase I inhibitor (DXd). In this study, the pharmacological activity and the mechanism of action of DS-6000a were evaluated in preclinical in vitro and in vivo models.

Methods

CDH6 expression was assessed by immunohistochemistry and FCM analysis. Induction of DNA damage and apoptosis to tumor cells by DXd released from DS-6000a were assessed by western blot. In vitro cell growth inhibitory and in vivo antitumor activities of DS-6000a were evaluated using CDH6-high and -low RCC and OVC cell lines, xenograft mouse models and patient derived xenograft (PDX) models.

Results

CDH6 is highly expressed in RCC and OVC patient samples. DS-6000a demonstrated in vitro cell growth inhibitory activity in CDH6-high tumor cells, but not in CDH6-low tumor cells. DNA damage and apoptosis were induced in CDH6-high tumor cells after the in vitro treatment with DXd and DS-6000a, but not with isotype control IgG ADC. DS-6000a exhibited strong antitumor activity with tumor regression in CDH6-high cell lines in mouse xenograft models. DS-6000a also showed high efficacy against PDX models.

Conclusions

Based on these preclinical results, DS-6000a could provide a valuable therapy with a potential benefit in CDH6-expressing cancers at the clinical setting.

Clinical trial identification

NCT04707248. First Posted: January 13, 2021.

Editorial acknowledgement

Hirokazu Suzuki, Shotaro Nagase, Chiemi Saito, Motoko Nagata, Yuki Kaneda, Kokichi Honda, Takashi Nakada, Riki Goto, Yusuke Myobatake, Yuki Abe, and Toshinori Agatsuma

Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan; E-mail: suzuki.hirokazu.yc@daiichisankyo.co.jp

Legal entity responsible for the study

Hirokazu Suzuki.

Funding

Has not received any funding.

Disclosure

H. Suzuki, S. Nagase, C. Saito, M. Nagata, Y. Kaneda, K. Honda, Y. Nishiya, T. Honda, T. Nakada, R. Goto, T. Ishizaka, Y. Myobatake, Y.Abe, T. Agatsuma: Financial Interests, Personal, Full Employment: Daiichi Sankyo Co. Ltd., Tokyo, Japan.

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Basic science

11P - Profiling adaptive responses of renal cell cancer to cabozantinib in order to develop rational drug combinations

Presentation Number
11P
Speakers
  • Diego Tosi (Montpellier, CEDEX 5, France)

Abstract

Background

Functional adaptive responses (i.e. due to cellular machinery modulations) could contribute to targeted treatment resistance. We hypothesize that understanding tumor cell adaptive responses induced by cabozantinib (C) in renal cell cancer (RCC) cells could provide a rational base for selecting treatment combinations and optimise drug doses and schedules.

Methods

We evaluated functional proteomic changes induced in VHL-mutated 786-O RCC cell line after in vitro exposure to low-dose C using reverse phase protein array (RPPA). A linear model analysis was performed on normalized intensity data from RPPA, in order to identify proteins and phosphoproteins undergoing significant treatment-induced changes. Then, we evaluated in vitro the efficacy of the interaction between C and drugs selected on the basis of RPPA analysis by mean of dose matrix tests.

Results

We exposed 786-O cells to HGF alone or in combination with C at 40 nM for 24 hours and then we performed RPPA analysis. Despite the low dose of C used, we observed a significant variation (i.e. with a log2 fold change for intensity of < 0.5 or > 1.5) in expression or phosphorylation of several protein targets. We observed inhibition of protein phosphorylation downstream of C targets (among which AXL, VEGFR−2, components of the PI3K/AKT/mTOR pathway and MEK1), which validates the cell model. Unexpectedly, we detected a significantly increased intensity signal for several protein targets involved in DNA repair process (RBBP8, RPA32_pS4_S8, BABAM1, BAP1, CDKN1A). We thus tested in vitro the association of C and inhibitors of the DNA repair proteins ATM, ATR and Wee1. We could observe that while the combination of C with KU60019 (an ATM inhibitor) is additive, the combinations of C with VE822 (an ATR inhibitor) or MK1775 (a Wee1 inhibitor) are synergistic in 2D and 3D cell culture.

Conclusions

Analysis of functional proteomic changes induced in vitro by C helped us to select targets for combination targeted therapy in RCC. Overall, our data suggest that cellular adaptive responses to drugs play a role in tumor resistance, and that elucidating them could help in designing drug combinations suitable for testing in the clinical setting.

Legal entity responsible for the study

Institut du Cancer de Montpellier, Université de Montpellier, Montpellier, France.

Funding

Ipsen.

Disclosure

D. Tosi: Other, Personal and Institutional, Research Grant: Ipsen; Other, Personal, Other, Travel support for scientific meetings: Ipsen. All other authors have declared no conflicts of interest.

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Basic science

12P - The design and development of novel pentaranes: Paving the way to EMT inhibition in triple-negative breast cancer cells

Presentation Number
12P
Speakers
  • Alexander M. Scherbakov (Moscow, Russian Federation)

Abstract

Background

Triple-negative cancer is characterized by a lack of expression of estrogen receptor α (ERα), progesterone receptor, and HER2/neu, which determines certain difficulties in the treatment of this molecular subtype of cancer. The development of drug candidates for triple-negative breast cancer is especially relevant. The work aims to design new type pentaranes (3-hydroxy-17(1-hydroxyalkyl(aryl))-16,17-cycloalkano)-estra-1,3,5(10)-trienes) and to evaluate their biological activity against breast cancer cells.

Methods

The compounds tested were synthesized in ZIOC from commercial reagents using multistage procedures. Docking was performed by AutoDock Vina using a “rigid/flexible” docking approach. MCF-7 and MDA-MB-231 cells are obtained from ATCC. Cell survival was assessed by the MTT test. ERα activity was analyzed by reporter analysis, protein expression was assessed using immunoblotting.

Results

A number of 16,17-cycloalkanoestratriene derivatives with a reduced binding affinity for the estrogen receptor α calculated by molecular docking have been obtained by multistage modification of estrone. The compounds showed weak activity as estrogen receptor alpha agonists on ERα-positive MCF-7 cells. The compounds inhibited the proliferation of triple-negative MDA-MB-231 breast cancer cells with micromolar IC50 values. The pentarane T120S (3-hydroxy-17-(1(S)-hydroxypropyl)-16,17-cyclohexano)-estra-1,3,5(10)-triene) was selected as a hit. Cyclin D1 expression was notably downregulated in MDA-MB-231 cells after treatments with T120S. Analysis of signalling pathways revealed that compound T120S inhibits Slug, a key transcriptional factor in the epithelial-mesenchymal transition.

Conclusions

Novel pentaranes with low estrogenic potency and high activity against triple-negative breast cancer have been obtained. The epithelial-mesenchymal transition is considered a promising target for this class of compounds. The work was supported by RFBR, project 19-03-00246.

Legal entity responsible for the study

The authors.

Funding

The Russian Foundation for Basic Research, project 19-03-00246.

Disclosure

All authors have declared no conflicts of interest.

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Basic science

13P - SY-5609, a highly potent and selective oral CDK7 inhibitor, exhibits robust antitumor activity in preclinical models of KRAS mutant cancers as a single agent and in combination with chemotherapy

Presentation Number
13P
Speakers
  • Susan H. Henry (Cambridge, United States of America)

Abstract

Background

Mutational KRAS activation drives oncogenic processes including aberrant cell cycle progression. CDK7 inhibition has been shown to target two fundamental processes in cancer: transcription and cell cycle control. SY-5609 is a CDK7 inhibitor in development in patients with solid tumors including pancreatic and lung cancers (NCT04247126). Here we report on SY-5609 preclinical activity in models of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small-cell lung cancer (NSCLC).

Methods

SY-5609 was evaluated as a single agent (SA) and in combination with chemotherapies. PDAC studies were done in RAS-mutant (7 KRAS, 1 NRAS) patient derived xenograft (PDX) models, and Panc-1 (KRAS-G12D) cells and xenografts +/- gemcitabine (Gem). NSCLC studies were done in A549 (KRAS G12S) cells and xenografts, and ST2972 (KRAS G12C) PDX tumors +/- docetaxel (Doc).

Results

In RAS-mutant PDAC PDX models derived from previously treated patients, SA SY-5609 (6mpk QD x28) induced regressions in 50% (4/8) of models and was well-tolerated (average body weight change [avg-BWC] 0%); regressions were sustained ≥2 weeks (wks) after drug discontinuation. In Panc-1 cells, SY-5609 inhibited proliferation (IC50, 0.7nM) and was synergistic with Gem. In vivo, SA SY-5609 (3 mpk QD x21) and SA Gem (100 mpk QW) each induced partial TGI; the combination induced nearly complete TGI (97%) and was well-tolerated (avg-BWC +2%). Similar combination results (94.3% TGI) were seen with a SY-5609 dosing regimen of 3 mpk QD, every other wk for 28d. In A549 cells, SY-5609 inhibited proliferation (IC50, 10nM) and was synergistic with Doc. In vivo, the combination of SY-5609 (3 mpk) and Doc (5 mpk QW) enhanced TGI. In ST2972 tumors, SA SY-5609 (3 mpk QD x21) induced near complete regressions, and with Doc (10 mpk QW) induced complete regressions with no tumor regrowth for ≥ 4 wks post drug discontinuation. Both regimens were well-tolerated (avg-BWC +3.6% to -6%).

Conclusions

SY-5609 shows robust antitumor activity in RAS-mutant PDAC and NSCLC preclinical models. Results support clinical evaluation of SY-5609 in combination with Gem in PDAC and Doc in NSCLC.

Legal entity responsible for the study

Syros Pharmaceuticals.

Funding

Syros Pharmaceuticals.

Disclosure

S.H. Henry, L. Johannessen, P. Sawant, A. Lefkovith, N. Ke, W. Dworakowski, G. Hodgson: Financial Interests, Institutional, Full or part-time Employment: Syros Pharmaceuticals.

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Basic science

14P - Preclinical evaluation of intermittent dosing regimens on antitumor and PD activity of SY-5609, a potent and selective oral CDK7 inhibitor, in ovarian cancer xenografts

Presentation Number
14P
Speakers
  • Liv Johannessen (Cambridge, MA, United States of America)

Abstract

Background

Selective CDK7 inhibition has been shown to target two fundamental processes in cancer: transcription and cell cycle control. SY-5609 is a potent and selective CDK7 inhibitor in Ph1 clinical development in patients with advanced solid tumors including ovarian cancer (NCT04247126). Here we report on the impact of intermittent SY-5609 dosing regimens on tumor growth inhibition (TGI), pharmacodynamic (PD) activity, and pharmacokinetics (PK) in a xenograft model of high grade serous ovarian cancer (HGSOC).

Methods

TGI was compared in OVCAR3 xenografts across a range of SY-5609 doses (1, 3, 6 mpk) and schedules (continuous daily [QD], 5d per wk [5/2], and 7d per wk every other wk [7/7]), and the same total daily dose was evaluated twice daily (BID) vs QD on a continuous schedule. Steady state tumor PK and PD (POLR2A and E2F1 expression) were assessed 8-72 hours after the 5th QD dose in separate mice.

Results

SY-5609 induced dose dependent TGI across all dosing regimens. Higher doses on 5/2 and 7/7 schedules led to TGI or regressions consistent with lower doses given QD. SY-5609 dose-dependent changes in tumor PD markers were sustained above baseline for up to 72 hours. TGI was enhanced when the same total daily dose was administered BID versus QD. Evaluation of SY-5609 PK during BID and QD schedules demonstrated increased antitumor activity associated with maintenance of higher trough levels of SY-5609. All regimens were well tolerated with no body weight loss.

Conclusions

SY-5609 shows robust antitumor activity in preclinical HGSOC xenografts across schedules that integrate higher doses with dosing holidays, supported by sustained PD effects in tumor tissue after dose cessation. Enhanced SY-5609 antitumor activity observed BID vs. QD (controlled for dose) supports the contribution of sustained higher levels of CDK7 inhibition between doses to antitumor activity, and informs the evaluation of intermittent dosing in patients to optimize single agent or combination SY-5609 dose and schedule selection. SY-5609 results of intermittent dosing regimens in patients with advanced solid tumors are reported separately (Sharma, ESMO 2021).

Legal entity responsible for the study

Syros Pharmaceuticals.

Funding

Syros Pharmaceuticals.

Disclosure

L. Johannessen, W. Dworakowski, P. Sawant, A. Lefkovith, A. D'Ippolito, M. Eaton, S.H. Henry, G. Hodgson: Financial Interests, Institutional, Full or part-time Employment: Syros Pharmaceuticals. N. Ke: Financial Interests, Personal and Institutional, Full or part-time Employment: Syros Pharmaceuticals.

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Basic science

15P - Modified sialoglycan by neuraminidase promotes tumor immunity against epithelial ovarian cancer

Presentation Number
15P
Speakers
  • Huang J. Mei (Chengdu, China)

Abstract

Background

Sialoglycan with α-2,3-linked/α-2,6-linked sialic acid (Sia) on the cancer cell surface protects cancer cell from immunologic recognition. Binding of α-galactose/α-N-acetylgalactosamine (Gal/GalNAc) epitope to macrophage galactose type C-lectin receptor (MGL) improves dendritic cell (DC) performance. We identified if modified sialoglycan by neuraminidase (NA) stimulates cellular immune response to EOC.

Methods

Sialoglycan on human EOC OVCAR3, A2780 and mouse ID8 cells was hydrolyzed with α2,6NA. NA-treated and NA-untreated OVCAR3 and A2780 cells were incubated with immature DC (imDC) to achieve NA-DC and nonNA-DC. Human peripheral blood lymphocytes (PBL) were stimulated with NA-DC and nonNA-DC. Gal/GalNAc and α-2,6Sia epitope of EOC cells, phenotypes of DCs and PBL were measured with flow cytometry (FCM); IFN-γ expression by PBL was determined by RT-qPCR; cytotoxicity of PBL to OVCAR3 and A2780 cells were detected by using LDH release assay. NA-treated and NA-untreated ID8 cells were vaccinated subcutaneously in the right axilla of C57 mice once per week for 3 consecutive weeks, then parental ID8 cells were inoculated in the opposite axilla. The tumor growth was observed; IFN-γ of plasm was tested by ELISA.

Results

α-2,6Sia (Sambucus Nigra Lectin, SNA) decreased and Gal/GalNAc (Peanut Agglutinin, PNA) increased in the NA-treated OVCAR3, A2780 and ID8 cells. MGL, CD83 and CD86 on NA-DCs increased significantly; Siglec-9 on NA-DCs and nonNA-DCs for A2780 not OVCAR3 cells decreased markedly compared to imDCs. PBL stimulated with NA-DCs had a higher expression of IFN-γ and CD69 than PBL stimulated with nonNA-DCs did. PBL stimulated with NA-DCs for A2780 but not for OVCAR3 cells showed an enhanced cytotoxicity; the cytotoxicity of PBL stimulated with NA-DCs was slightly higher than that of PBL stimulated with nonNA-DCs for OVCAR3 cells. Tumorigenesis was reduced significantly in the vaccinated mice with NA-treated ID8 cells; the plasm level of IFN-γ was significantly increased in the vaccinated mice.

Conclusions

NA modified sialoglycan structure could promote immune response to EOC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Basic science

16P - Repurposing rosiglitazone with chemotherapy in breast cancer: A sequence-specific synergy?

Presentation Number
16P
Speakers
  • Sally Al-Moualem (Damascus, Syria)

Abstract

Background

Recent years have witnessed a trend to search for the possibility of repurposing widely prescribed drugs in the field of oncology. This applies to thiazolidinediones, peroxisome proliferator-activated receptor-gamma (PPARγ) receptor agonists, the anti-diabetic drugs which showed antitumor activities and evidence for the induction of breast cancer cells differentiation. We aimed to investigate the potential of repurposing a PPARγ ligand, rosiglitazone (RGZ), in combination with either of two chemotherapeutic agents, doxorubicin (Dox) or cisplatin (Cis) for the in vitro treatment of breast cancer cell line, MCF-7.

Methods

The drug combinations were applied to the cells, then viability was measured using MTT assay and trypan blue staining, and Median-effect analysis was performed. The cell cycle was analyzed, and the pattern of cell death was determined using a flow cytometer.

Results

RGZ augmented the growth inhibition effect of Cis and Dox on MCF-7 cells. The synergism was observed only when chemotherapy preceded RGZ and not vice versa, demonstrating a sequence-specific effect. RGZ blocked Dox and Cis cytotoxic outcomes on MCF-7 cells when applied first. The cell cycle and apoptosis/necrosis patterns of cells treated with rosiglitazone followed by the chemotherapy showed similarity to those of cells treated with rosiglitazone alone and suggest that RGZ induced a cell cycle arrest in the G1 phase blocking Dox and Cis subsequent effects.

Conclusions

Combinations of RGZ with Cis showed sequence-specific synergism when RGZ follows chemotherapy unlike the early application of RGZ which blocked the chemotherapy-induced cytotoxicities. We need more experiments to achieve better combinations.

Legal entity responsible for the study

Damascus University.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Basic science

17P - PD-1 and LAG-3 immune checkpoints constitutive activators exhibit differential expression phenotypes

Presentation Number
17P
Speakers
  • Luisa Chocarro (Pamplona, Spain)

Abstract

Background

The lack of PD-1 or LAG-3 mutants with constitutive inhibitory activities prevents a systematic approach to dissect all the intracellular events regulated by PD- 1 and LAG-3 that establish a strong T cell dysfunctionality in patients with intrinsic resistance to PD-1 therapies. It is thought that PD-1 and LAG-3 form a supramolecular complex together with TCR components in order to exert their inhibitory activity over TCR signal transduction. So far, no PD-1 and LAG-3 mutants with constitutive signalling activities have been described. Here we have constructed molecules that provide an initial TCR-dependent signal in T cells that lead to a sustained inhibitory activity of PD-1 and LAG-3.

Methods

To construct molecules with constitutive PD-1 and LAG-3 signaling activities in T cells, we replaced their immunoglobulin domains with a sequence encoding a single chain antibody that binds CD3. These fusion genes were cloned into the pDUAL lentivector expression system, which coexpress antibiotic resistance for selection of cells lines. When expressed in T cells, the engagement with CD3 will trigger a TCR signal in neighbouring T cells, and these molecules will then transmit either PD-1 or LAG-3 signals. To test their expression, human T cells were transduced with lentivectors expressing these constructs singly or in combination and their phenotypes were characterized by flow cytometry analysis.

Results

Both constructs showed differential phenotypes and expression levels within different surface and intracellular molecules compared with their WT and CD3 activator controls, such as CD3, CD4, CD27, CD28, CD69, CD62L, CD45RA, PD1, LAG3, TIM-3, CTLA-4, KIR2DL1/S1/S3/S5, IFNg, IFNa/b, IL-12/IL-13, IL-4, IL-2 and IL-17A, among others.

Conclusions

These results showed that these molecules had functional PD-1 and LAG-3 constitutive inhibitory signalling in T cells leading T cell dysfunctionality. This will allow to study the reasons behind the intrinsic resistance to PD-1 blockade.

Legal entity responsible for the study

Navarrabiomed, Instituto de Investigaciones Sanitarias de Navarra (IdiSNA), Universidad Pública de Navarra.

Funding

This work was supported and funded by the Spanish Association against Cancer (AECC, PROYE16001ESCO), Instituto de Salud Carlos III (ISCIII, FIS. PI17/02119), Biomedicine Project grant from the Department of Health of the Government of Navarre (BMED 050-2019).

Disclosure

All authors have declared no conflicts of interest.

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Basic science

18P - In vitro analysis of the combination of APR-246 and carboplatin in triple negative breast cancer (TNBC) and high grade serous ovarian cancer (HGSOC) cell lines and its impact on Aurora kinase A (AURKA)-p53 pathway

Presentation Number
18P
Speakers
  • Juan Jose Martinez-Pretel (Valencia, Spain)

Abstract

Background

AURKA is a protein that regulates mitotic spindle formation and p53 is involved in cell cycle regulation. p53 mutations and AURKA overexpression is a frequent alteration in both TNBC and HGSOC cell lines which lead to carboplatin sensitivity. APR-246 is a new targeted agent that modulates abnormal p53 in mutated cells. The aim of our study was to assess the effect of the combination of carboplatin (carbo) plus APR-246 in TNBC and HGSOC cell lines.

Methods

We selected two TNBC (MDA-MB-231 and MDA-MB-436) and one HGSOC cell line (Kuramochi). Next-generation sequencing was performed in order to assess more relevant mutations. MTT experiments were performed to calculate the IC50 for APR-246 and carbo. For the combination (combo) increasing IC50 of either APR-246 or carbo was assessed in the presence of a constant concentration of each other. Western-blot (WB) and PCR analysis was performed in each line after exposure to APR-246, carbo or the combination to determine expression of AURKA and p53.

Results

The mutation profile of each line and the IC50 of APR-245, carbo and combo is shown in the table.

P53 - BRCA IC50 carbo P value IC50 carbo in Combo (APR-246 constant) IC50 APR-246 IC50 APR-246 in combo (carbo constant) P value
MDA-MB-231 Mut - wt 242.6 uM <0.0001 41.77 uM 22.37 uM 12.19 uM <0.0001
MDA-MB-436 Wt - mut 38.73 uM <0.0001 5.01 uM 18.81 uM 8.85 uM <0.0001
Kuramochi Mut - mut 35.86 uM <0.0001 18.86 uM 21.78 uM 13.97 uM <0.0001

Our results showed that IC50 of carbo and APR-246 decreased when administered in combination with constant doses of each other regardless of the subtype (TNBC vs HGSOC) and p53 mutations but more evident in the MDA-MB-231 BRCAwt cell line. PCR in MDA-MB-231 and MDA-MB-436 showed that AURKA was overexpressed by exposure to APR-246, Carbo and combo and p53 was upregulated after carbo (both lines) or combo (only 436). Kuramochi cells showed an underexpression of AURKA after combo and an upregulation of p53 after carbo or combo. Similar results were shown with WB in these cell lines.

Conclusions

Addition of APR246 to carboplatin increased apoptosis in both TNBC and HGSOC cell lines regardless of p53 mutations status in this in vitro study. AURKA and p53 expression was modified after exposure to carbo or combo.

Legal entity responsible for the study

The authors.

Funding

Mutua Madrileña Grant Sociedad Española de Oncologia Medica Grant.

Disclosure

A. Lluch-Hernandez: Financial Interests, Personal, Advisory Board: Novartis; Financial Interests, Personal, Advisory Board: Pfizer; Financial Interests, Personal, Advisory Board: Roche; Financial Interests, Personal, Advisory Board: Eisai; Financial Interests, Personal, Advisory Board: Celgene; Financial Interests, Institutional, Research Grant: Roche; Financial Interests, Institutional, Research Grant: AstraZeneca; Financial Interests, Institutional, Research Grant: Merck; Financial Interests, Institutional, Research Grant: PharmaMar; Financial Interests, Institutional, Research Grant: Novartis; Financial Interests, Institutional, Research Grant: Boehringer Ingelheim; Financial Interests, Institutional, Research Grant: Pfizer; Financial Interests, Institutional, Research Grant: Eisai; Financial Interests, Institutional, Research Grant: Celgene; Financial Interests, Institutional, Research Grant: Pierre Fabre; Financial Interests, Personal, Other: Roche; Financial Interests, Personal, Other: Novartis. A. Cervantes: Financial Interests, Personal, Invited Speaker: MerckSerono; Financial Interests, Personal, Invited Speaker: Amgem; Financial Interests, Personal, Advisory Board: BeiGene; Financial Interests, Personal, Advisory Board: BMS; Financial Interests, Personal, Member: Annals of Oncology; Financial Interests, Personal, Member of the Board of Directors: Cancer Treat Reviews; Financial Interests, Institutional, Principal Investigator: Abbvie; Financial Interests, Institutional, Principal Investigator: Actuate Therap; Financial Interests, Institutional, Principal Investigator: Alkermes Inc; Financial Interests, Institutional, Principal Investigator: Amgem; Financial Interests, Institutional, Principal Investigator: BeiGene; Financial Interests, Institutional, Principal Investigator: Boehringer; Financial Interests, Institutional, Principal Investigator: Debiopharm; Financial Interests, Institutional, Principal Investigator: Roche; Financial Interests, Institutional, Principal Investigator: Fibrogen; Financial Interests, Institutional, Principal Investigator: Genmab; Financial Interests, Institutional, Principal Investigator: Janssen; Financial Interests, Institutional, Principal Investigator: MedImmune; Financial Interests, Institutional, Principal Investigator: Novartis; Financial Interests, Institutional, Principal Investigator: Puma Biotech; Financial Interests, Institutional, Principal Investigator: Symphogen; Financial Interests, Institutional, Principal Investigator: Tahio; Financial Interests, Institutional, Principal Investigator: Transgene; Financial Interests, Institutional, Principal Investigator: WNT Research; Non-Financial Interests, Institutional, Member of the Board of Directors: INCLIVA Biomedical Institute of Research. J.A. Perez Fidalgo: Financial Interests, Personal, Invited Speaker: GSK; Financial Interests, Personal, Invited Speaker: Clovis; Financial Interests, Personal, Invited Speaker: AstraZeneca; Financial Interests, Personal, Invited Speaker: PharmaMar; Financial Interests, Institutional, Funding: GSK; Financial Interests, Institutional, Research Grant: PharmaMar; Financial Interests, Personal, Advisory Board: Abilify pharma; Financial Interests, Personal, Advisory Board: GSK; Financial Interests, Personal, Advisory Board: Clovis; Financial Interests, Personal, Advisory Role: AstraZeneca; Financial Interests, Personal, Advisory Board: Roche; Financial Interests, Personal, Invited Speaker: Roche; Financial Interests, Institutional, Project Lead: Novartis; Financial Interests, Institutional, Principal Investigator: AstraZeneca; Financial Interests, Institutional, Principal Investigator: GSK; Financial Interests, Institutional, Principal Investigator: Immunogene; Financial Interests, Institutional, Project Lead: AstraZeneca; Financial Interests, Institutional, Principal Investigator: Karyopharm. All other authors have declared no conflicts of interest.

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