Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti …€¦ · and E) TFF2-CTP-Flag...
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Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer 1 Columbia University, New York, NY, 2 Albert Einstein College of Medicine, Bronx, NY, 3 Tonix Pharmaceuticals, Inc., New York, NY Woosook Kim 1 , Na Fu 1 , Phaneendra Duddempudi 2 , Zinaida Dubeykovskaya 1 , Steven Almo 2 , Chandan Guha 2 , Seth Lederman 3 , Timothy Wang 1 Abstract Conclusions Figure 1. (A) Mice (C57BL/6 WT) received azoxymethane (AOM; 10 mg/kg i.p.) followed one week later with 2.5% dextran sodium sulfate (DSS) in the drinking water for 7 days. (B-D) AOM/DSS-treated mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. (B) Gross images. Scale bars, 5mm. Tumors were more frequently observed in the distal colon. (C) Macroscopically visible tumors were counted and tumor area was measured using ImageJ Fiji. (D) H&E stains. Increased intramuco- sal immune cell infiltrates were detected at 10 weeks post-AOM. Despite remarkable responses to immune checkpoint blockade across multiple tumor types, the clinical benefit in colorectal cancer (CRC) is limited to microsatellite unstable tumors. PD-L1 expression is a negative prognostic marker in CRC but correlates with a better response to PD-1 blockade. Here, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in mouse models of CRC. We generated knockin mice that conditionally express the murine Pdl1 gene (R26-LSL-Pdl1-EGFP) and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. AOM/DSS-treated mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. AOM/DSS treatment led to a significant expansion of myeloid cells, particularly CD11b + Gr-1 + MDSCs, compared to untreated mice. Furthermore, there was a significant decrease in intratumoral CD8 + T cells, indicating attenuated anti-tumor immunity. AOM/DSS-treated PD-L1-overexpressing LysM-Cre; R26-PD-L1 mice showed markedly enhanced early colorectal tumorigenesis, with a significant increase in tumor number and size. Trefoil factor 2 (TFF2), a secreted anti-inflammatory peptide, inhibits colon tumor growth by suppressing the expansion of CD11b + Gr-1 + MDSCs. TFF2 fused with two carboxyl-terminal peptide and three Flag motifs (TFF2-CTP-Flag) prolonged the circulation time in blood but retained bioactivity. We induced tumors in R26-PD-L1 and LysM-Cre; R26-PD-L1 mice with AOM/DSS, administered fusion recombinant TFF2-CTP-Flag and/or anti-PD-1. Anti-PD-1 in combination with TFF2-CTP showed a marked reduction in tumor growth while anti-PD-1 monotherapy failed to suppress growth. Interestingly, combined treatment showed greater anti-tumor activity in PD-L1-overexpressing mice than control animals. Treatment responders showed significantly increased tumor-infiltrating CD8 + T cells and concomitantly decreased CD11b + Gr-1 + myeloid cells. These early findings suggest that TFF2 augments the response rate of CRC to PD-1 blockade, possibly through suppressing MDSC expansion, supporting the potential of TFF2-CTP in combination I-O treatment for CRC. A B Abstract control 10w post-AOM 17w post-AOM C PD-L1 overexpression in myeloid cells promotes early colorectal tumorigenesis 0 5 10 15 20 tumor number control 10w post-AOM 17w post-AOM 0 50 100 150 200 tumor area (mm 2 ) D control 10w post-AOM 17w post-AOM **** *** ** *** A DSS AOM rTFF2-CTP anti-PD-1 SAC Week 0 1 2 10 14 18 9 13 17 R26-PD-L1 LysM-Cre; R26-PD-L1 B 0 5 10 15 20 25 tumor number 0 50 100 150 200 250 tumor area (mm 2 ) 0 5 10 15 20 25 tumor number 0 50 100 150 200 250 tumor area (mm 2 ) control anti-PD-1 TFF2-CTP anti-PD-1+ TFF2-CTP non-responder responder R26-PD-L1 LysM-Cre; R26-PD-L1 0 5 10 15 20 % CD3 + CD8 + R26-PD-L1 LysM-Cre; R26-PD-L1 0 5 10 15 CD8 + : Treg ratio CD3 + CD4 + CD3 + CD8 + 0 5 10 15 20 25 % of CD45 + 0 2 4 6 % Treg 0 10 20 30 CD8 + : Treg ratio 0 20 40 60 80 % CD11b + Gr-1 + M-MDSC PMN-MDSC 0 10 20 30 40 % of CD45 + 0 5 10 15 % MQ 0 5 10 15 % DC 0 10 20 30 monocyte to MQ ratio A R26 locus + Cre CD11b CD45 LysM-Cre; R26-PD-L1 R26-PD-L1 R26-PD-L1 LysM-Cre; R26-PD-L1 0.00 0.05 0.10 0.15 0.20 CD45 + CD11b - CD45 + CD11b + ** ** 1 l d P n o i s s e r p x e o t h d p a G DSS AOM SAC Week 0 1 2 10 0 5 10 15 tumor number 0 50 100 150 tumor area (mm 2 ) AOM/DSS-induced CRC in the murine model A 17w post-AOM control CD45 CD11b PD-L1 CD11b + Gr-1 + MDSCs markedly increase as tumors progress B *** **** *** **** *** **** control 10w post-AOM 17w post-AOM C * p = 0.053 p = 0.078 Figure 2. (A) Immunostaining for CD45, CD11b and PD-L1 on colon tissues from AOM/DSS-treated C57BL/6 WT mice. CD11b + myeloid cells and PD-L1 expression were increased as tumors progressed. (B and C) Immunopheno- typing of intratumoral myeloid cells by flow cytometry (% of CD45 + ). CD11b + Gr-1 + MDSCs and both granolulocytic (CD11b + Ly6G + ) and monocyt- ic (CD11b + Ly6G - Ly6C + ) MDSC subsets were markedly increased in tumors (B). Macrophages (MQ; CD11b Ly6C - F4/80 + ) and dendritic cells (DC; CD11c + F4/80 - ) (C). CD8 + T cells : Treg ratio is decreased as tumors develop A * ** * *** ** B CD8 + T cells CD4 + T cells NK cells Granulocytes Monocytes Dendritic cells Macrophages B cells ** *** ** **** Figure 3. (A and B) Im- munophenotyping of tu- mor-infiltrating T cells by flow cytometry (% of CD45 + ). The proportion of T cells was decreased as tumors develop; this de- crease was driven by a re- duction in CD8 + T cells (A). CD4 + CD25 + Foxp3 + regula- tory T cells (Treg) were in- creased in the late stage of tumors, leading to a greater decrease in CD8 + T cells to Treg ratio (B). (C) Dynamics of immune cell subsets during CRC de- velopment. 0 10 20 30 40 % CD3 + control 10w post-AOM 17w post-AOM 17w post-AOM control C B C D E F R26-PD-L1 LysM-Cre; R26-PD-L1 R26-PD-L1 LysM-Cre; R26-PD-L1 Figure 4. (A-C) Generation of R26-LSL-Pdl1-EGFP mice. Gene construct of R26-LSL-Pdl1-IRES-EGFP (A). Endogenous GFP expression by flow cy- tometry (B) and Pdl1 gene expression by qPCR (C) in splenic CD11b - and CD11b + cells in R26-PD-L1 and LysM-Cre; R26-PD-L1 mice. (D) Experimen- tal scheme depicting induction of CRC by AOM/DSS. (E) Gross images of colorectal tumors at 10 weeks post-AOM. Scale bars, 5 mm. (F) The tumor numbers counted and tumor area measured. Note that LysM-Cre; R26-PD-L1 mice treated with AOM/DSS showed markedly enhanced early colorectal tumorigenesis. TFF2 inhibits colorectal tumor growth by suppresing the expansion of MDSCs A C D TFF2-CTP + anti-PD-1 showed greater anti- tumor activity in PD-L1-overexpressing mice R26-PD-L1 LysM-Cre; R26-PD-L1 Figure 6. (A) R26-PD-L1 and LysM-Cre; R26-PD-L1 mice were given AOM/DSS, and treated with fusion recombinant TFF2-CTP-Flag (300 μg i.p.) and/or anti-PD-1 (RMP1-14; 200 μg i.p.) three times a week starting at the time points indicated. (B) The tumor numbers counted and tumor area mea- sured. Mice with > 50% reduction of tumor area compared to control animals were defined as responders. Note that LysM-Cre; R26-PD-L1 mice (5/5; 100%) showed higher response rates to combined treatment of TFF2-CTP and anti-PD-1 than control animals (2/5; 40%). Figure 5. (A and B) TFF2 overexpression (CD2-Tff2 mice) (A) and treatment with adenovirus Ad-Tff2 (B) conferred resistance to colon carcinogenesis through suppression of MDSCs. (C) Fusion construct Tff2-2CTP-3Flag. (D and E) TFF2-CTP-Flag prolonged the circulation time in blood (D) but re- tained bioactivity (E). Dubeykovskaya et al. 2016 Nat Commun. (A-B); 2019 Cancer Gene Ther. (C-E). B E Hypothesis Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in colorectal cancer by suppressing MDSCs Responders showed increased CD8 + T cells and decreased MDSCs in tumors R26-PD-L1 LysM-Cre; R26-PD-L1 0 20 40 60 80 100 % CD11b + Gr-1 + A B R26-PD-L1 LysM-Cre; R26-PD-L1 0 20 40 60 M-MDSC R26-PD-L1 LysM-Cre; R26-PD-L1 0 10 20 30 40 monocyte : MQ ratio Figure 7. (A) The proportion of CD3 + CD8 + T cells in CD45 + cells and a ratio of CD8 + T cells to Treg in tumors. Note that responders had more abundant tumor-infiltrating CD8 + T cells and a higher ratio of CD8 + T cells to Treg. (B) Immunophenotyping of intratumoral myeloid cells following different treat- ments. A marked reduction in MDSCs, in particular M-MDSC, was observed in responders. Responders also showed a lower ratio of monocyte to MQ. R26-PD-L1 LysM-Cre; R26-PD-L1 0 10 20 30 40 50 PMN-MDSC Anti-PD-1 monotherapy was unable to evoke anti-tumor immunity in CRC but TFF2-CTP augmented the efficacy of anti-PD-1 therapy. Anti-PD-1 in combination with TFF2-CTP showed greater anti-tumor activity in PD-L1-overexpressing mice. Responders to TFF2-CTP alone or in combination with PD-1 blockade had increased tumor-infiltrating CD8 + T cells, along with decreased MDSCs. non-responder responder control anti-PD-1 TFF2-CTP anti-PD-1+ TFF2-CTP Woosook Kim ([email protected]) +
Transcript of Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti …€¦ · and E) TFF2-CTP-Flag...
Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer
1Columbia University, New York, NY, 2Albert Einstein College of Medicine, Bronx, NY, 3Tonix Pharmaceuticals, Inc., New York, NYWoosook Kim1, Na Fu1, Phaneendra Duddempudi2, Zinaida Dubeykovskaya1, Steven Almo2, Chandan Guha2, Seth Lederman3, Timothy Wang1
Abstract
ConclusionsFigure 1. (A) Mice (C57BL/6 WT) received azoxymethane (AOM; 10 mg/kg i.p.) followed one week later with 2.5% dextran sodium sulfate (DSS) in the drinking water for 7 days. (B-D) AOM/DSS-treated mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. (B) Gross images. Scale bars, 5mm. Tumors were more frequently observed in the distal colon. (C) Macroscopically visible tumors were counted and tumor area was measured using ImageJ Fiji. (D) H&E stains. Increased intramuco-sal immune cell infiltrates were detected at 10 weeks post-AOM.
Despite remarkable responses to immune checkpoint blockade across multiple tumor types, the clinical benefit in colorectal cancer (CRC) is limited to microsatellite unstable tumors. PD-L1 expression is a negative prognostic marker in CRC but correlates with a better response to PD-1 blockade. Here, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in mouse models of CRC. We generated knockin mice that conditionally express the murine Pdl1 gene (R26-LSL-Pdl1-EGFP) and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. AOM/DSS-treated mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. AOM/DSS treatment led to a significant expansion of myeloid cells, particularly CD11b+Gr-1+ MDSCs, compared to untreated mice. Furthermore, there was a significant decrease in intratumoral CD8+ T cells, indicating attenuated anti-tumor immunity. AOM/DSS-treated PD-L1-overexpressing LysM-Cre; R26-PD-L1 mice showed markedly enhanced early colorectal tumorigenesis, with a significant increase in tumor number and size. Trefoil factor 2 (TFF2), a secreted anti-inflammatory peptide, inhibits colon tumor growth by suppressing the expansion of CD11b+Gr-1+ MDSCs. TFF2 fused with two carboxyl-terminal peptide and three Flag motifs (TFF2-CTP-Flag) prolonged the circulation time in blood but retained bioactivity. We induced tumors in R26-PD-L1 and LysM-Cre; R26-PD-L1 mice with AOM/DSS, administered fusion recombinant TFF2-CTP-Flag and/or anti-PD-1. Anti-PD-1 in combination with TFF2-CTP showed a marked reduction in tumor growth while anti-PD-1 monotherapy failed to suppress growth. Interestingly, combined treatment showed greater anti-tumor activity in PD-L1-overexpressing mice than control animals. Treatment responders showed significantly increased tumor-infiltrating CD8+ T cells and concomitantly decreased CD11b+Gr-1+ myeloid cells. These early findings suggest that TFF2 augments the response rate of CRC to PD-1 blockade, possibly through suppressing MDSC expansion, supporting the potential of TFF2-CTP in combination I-O treatment for CRC.
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PD-L1 overexpression in myeloid cells promotes early colorectal tumorigenesis
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Figure 2. (A) Immunostaining for CD45, CD11b and PD-L1 on colon tissues from AOM/DSS-treated C57BL/6 WT mice. CD11b+ myeloid cells and PD-L1 expression were increased as tumors progressed. (B and C) Immunopheno-typing of intratumoral myeloid cells by flow cytometry (% of CD45+). CD11b+Gr-1+ MDSCs and both granolulocytic (CD11b+Ly6G+) and monocyt-ic (CD11b+Ly6G-Ly6C+) MDSC subsets were markedly increased in tumors (B). Macrophages (MQ; CD11b Ly6C-F4/80+) and dendritic cells (DC; CD11c+F4/80-) (C).
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Figure 3. (A and B) Im-munophenotyping of tu-mor-infiltrating T cells by flow cytometry (% of CD45+). The proportion of T cells was decreased as tumors develop; this de-crease was driven by a re-duction in CD8+ T cells (A). CD4+CD25+Foxp3+ regula-tory T cells (Treg) were in-creased in the late stage of tumors, leading to a greater decrease in CD8+ T cells to Treg ratio (B). (C) Dynamics of immune cell subsets during CRC de-velopment.
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Figure 4. (A-C) Generation of R26-LSL-Pdl1-EGFP mice. Gene construct of R26-LSL-Pdl1-IRES-EGFP (A). Endogenous GFP expression by flow cy-tometry (B) and Pdl1 gene expression by qPCR (C) in splenic CD11b- and CD11b+ cells in R26-PD-L1 and LysM-Cre; R26-PD-L1 mice. (D) Experimen-tal scheme depicting induction of CRC by AOM/DSS. (E) Gross images of colorectal tumors at 10 weeks post-AOM. Scale bars, 5 mm. (F) The tumor numbers counted and tumor area measured. Note that LysM-Cre; R26-PD-L1 mice treated with AOM/DSS showed markedly enhanced early colorectal tumorigenesis.
TFF2 inhibits colorectal tumor growth by suppresing the expansion of MDSCs
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Figure 6. (A) R26-PD-L1 and LysM-Cre; R26-PD-L1 mice were given AOM/DSS, and treated with fusion recombinant TFF2-CTP-Flag (300 µg i.p.) and/or anti-PD-1 (RMP1-14; 200 µg i.p.) three times a week starting at the time points indicated. (B) The tumor numbers counted and tumor area mea-sured. Mice with > 50% reduction of tumor area compared to control animals were defined as responders. Note that LysM-Cre; R26-PD-L1 mice (5/5; 100%) showed higher response rates to combined treatment of TFF2-CTP and anti-PD-1 than control animals (2/5; 40%).
Figure 5. (A and B) TFF2 overexpression (CD2-Tff2 mice) (A) and treatment with adenovirus Ad-Tff2 (B) conferred resistance to colon carcinogenesis through suppression of MDSCs. (C) Fusion construct Tff2-2CTP-3Flag. (D and E) TFF2-CTP-Flag prolonged the circulation time in blood (D) but re-tained bioactivity (E). Dubeykovskaya et al. 2016 Nat Commun. (A-B); 2019 Cancer Gene Ther. (C-E).
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HypothesisStabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in colorectal cancer by suppressing MDSCs
Responders showed increased CD8+ T cells and decreased MDSCs in tumors
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Figure 7. (A) The proportion of CD3+CD8+ T cells in CD45+ cells and a ratio of CD8+ T cells to Treg in tumors. Note that responders had more abundant tumor-infiltrating CD8+ T cells and a higher ratio of CD8+ T cells to Treg. (B) Immunophenotyping of intratumoral myeloid cells following different treat-ments. A marked reduction in MDSCs, in particular M-MDSC, was observed in responders. Responders also showed a lower ratio of monocyte to MQ.
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Anti-PD-1 monotherapy was unable to evoke anti-tumor immunity in CRC but TFF2-CTP augmented the efficacy of anti-PD-1 therapy. Anti-PD-1 in combination with TFF2-CTP showed greater anti-tumor activity in PD-L1-overexpressing mice. Responders to TFF2-CTP alone or in combination with PD-1 blockadehad increased tumor-infiltrating CD8+ T cells, along with decreased MDSCs.
non-responderresponder
controlanti-PD-1
TFF2-CTPanti-PD-1+TFF2-CTP
Woosook Kim ([email protected])
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