munroe neutralizing tgf beta ab aacr 2009 enlarged

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Results TGFb plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFb on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFb antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas). However, TGFb antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFb in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFb on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFb significantly inhibits metastasis and anti-TGFb strategies may be effective at treating metastatic disease in the clinic. Inhibition of Metastases by a Neutralizing TGFb-specific Antibody Involves the Activity of Cytotoxic T Lymphocytes and Natural Killer Cells Kenneth Munroe 1 , Charlene Manning 1 , Tessa Green 1 , Jingzang Tao 1 , Kailene Simon 1 , John McPherson 1 , Johanne Kaplan 1 , Jan Pinkas 2 , Megan O’Brien 1 , Richard Gregory 1 , Jay Harper 1 , Scott Lonning 1 1 Genzyme Corporation, Framingham, MA; 2 ImmunoGen, Waltham, MA Abstract TGFb signaling is known to affect several aspects of primary tumor growth and metastasis by effects on both tumor cells and the stroma. As tumor progression occurs, tumor cells lose their responsiveness to the growth-inhibitory activity of TGFb. TGFb stimulates VEGF production leading to angiogenesis, and promotes epithelial to mesenchymal transition in the cancer cells. This leads to progression to a more invasive phenotype and increased access to the vasculature allowing for the extravasation of tumor cells and the establishment of distal metastases. TGFb also inhibits immunosurveillance mechanisms in the tumor by inhibiting proliferation or activation of cytolytic immune effector cells, by inducing suppressive regulatory T lymphocytes and promoting the secretion of Th2 cytokines. Figure 1 The Role of TGFb in Tumor Progression and Metastasis TGFβ Tumor Cell EMT/invasion/motility Survival Tumor promoting factors Angiogenesis Altered ECM composition Other immune cells altered cytokine production Myofibroblast Formation Immune Function Cytotoxic T cell NK cell (A) Mice with established, subcutaneous (SQ) B16-F10 melanomas implanted on the hind footpad were treated with 1D11 (50mg/kg, 3X/wk) or the isotype control, 13C4. Neutralization of TGFb with 1D11 had no effect on the growth of these SQ tumors compared to controls. (B) Established, SQ 4T1 mammary carcinoma tumors (~100mm 3 ) were also treated with 1D11 (10mg/kg, 3X/wk) or 13C4. In this model, 1D11 had essentially no effect on primary tumor growth (representative data shown) although trends of inhibition have been observed in certain studies. 0 10 20 30 40 50 60 70 0 100 200 300 400 Vehicle 13C4 1D11 Day Tumor Vol (mm 3 ) A 0 5 10 15 20 25 0 500 1000 1500 2000 Vehicle 13C4 1D11 Day Tumor Vol (mm 3 ) B Figure 2 1D11, a TGFb neutralizing antibody has nominal effects on the growth of primary tumors (A) Treatment with 1D11 inhibited pulmonary metastasis following intravenous delivery of B16-F10 melanoma cells in a dose-dependent manner (p=0.002). (B) Representative images of lungs bearing experimental B16-F10 pulmonary metastases from mice treated with either 1D11 or 13C4. 13C4 50mg/kg 1D11 50mg/kg B A vehicle 13C4 5mg/kg 20mg/kg 50mg/kg 0 10 20 30 40 50 60 1D11 Number of lung mets p=0.002 Figure 3 1D11 inhibits metastasis in an experimental model of melanoma metastasis Figure 4 Inhibition of metastasis following neutralization of TGFb in two models of breast cancer metastasis Tx Lytic Bone Lesions Adrenal Gland Kidney Vehicle 4/7 3/7 5/7 13C4 5/8 6/8 6/8 1D11 0/8 3/8 2/8 *p < 0.02 B 0 10 20 30 40 50 0 25 50 75 100 Vehicle 13C4 1D11 Days Percent survival A Vehicle 13C4 1D11 0 10 20 30 40 p<0.01 p<0.05 Number of lung mets C GOALS Determine the effect of neutralizing TGF b with 1D11, a pan- neutralizing TGFb antibody, on primary tumor growth and metastasis in preclinical, syngeneic models of melanoma and breast cancer Elucidate the mechanism of action of 1D11 in these tumor models B16-F10 melanoma cells were injected into the plantar region of the hind footpad such that SQ primary tumors developed (A) and metastasis to the draining lymph node (popliteal lymph node) occurs spontaneously (B). (C) Chronic treatment with 1D11 resulted in significant reduction of metastasis to the lymph node compared to controls. Figure 5 Neutralization of TGFb with 1D11 inhibits spontaneous metastasis from primary melanomas to the draining lymph node A B vehicle 13C4 1D11 0 25 50 75 100 % Metastatic Incidence C (A) TAMs located at the invasive front of 4T1 primary tumors are CD68 (red) and CD206 (green) positive suggesting that these are M2 macrophages. (B) CD68 + TAMs found at center of the 4T1 primary tumors are negative for CD206 immunostaining, suggesting that these represent M1 macrophages. M2 TAMs are observed at the invasive front of 4T1 primary tumors following the establishment of the tumor and persists as the tumor volume increases (A) 95mm 3 , day 7(B) 248mm 3 , day 7 (C) 524mm 3 , day 14 (D) 1020mm 3 , day 18 (E) 1468mm 3 , day 18 (F) 1928mm 3 , day 18. Figure 8 M2 tumor-associated macrophages (TAMs) are shown to localize primarily to the tumor periphery while classically-activated M1 macrophages are observed in the tumor core. Red = CD68 Green = CD206 Blue = DAPI A B A B C D E F Red = CD68 Green = CD206 Blue = DAPI Figure 9 M2 macrophages are observed at the invasive front of 4T1 primary tumors during all stages of tumor growth. tumors Neutralization of TGF b with 1D11 inhibits metastasis in two independent tumor model systems: B16 murine melanoma and 4T1 murine mammary carcinoma. 1D11 had nominal effects on the growth of primary tumor growth. CTL activity appears to play a major role in the ability of 1D11 to inhibit both experimental and spontaneous metastasis. The role of NK cell activity is less clear and may depend on the mode of metastasis. M2 macrophages are recruited to 4T1 tumors during all stages of tumor growth. As tumor progression continues, M2 macrophages become marginated to the invasive front of growing tumors. Conclusions We thank the staff at Genzyme’s DCM, as well as Doug Matthews, Sheen Zhang, Peter Piepenhagen, Brian DelGiudice and Bob Thomas for their assistance in this project. Acknowledgements To assess the role of NK cell activity in the efficacy of 1D11, tumor models were conducted in wild type mice (WT) and in mice treated chronically with anti-asialo GM1 to deplete NK cells (NK-). (A) Representative images of lungs from wildtype and NK-deficient mice bearing experimental B16-F10 melanoma metastases treated with either 1D11 or 13C4. The number and size of metastases was increased in NK-deficient mice. (B) 1D11 is able to inhibit the development of experimental pulmonary metastases in NK-deficient mice. (C) The ability of 1D11 to inhibit spontaneous metastasis to the draining lymph node is dependent on NK cell activity. Figure 6 Differential roles of natural killer (NK) cells in the ability of 1D11 to inhibit metastasis via the circulation versus metastasis through the lymphatics NK-Competent Vehicle 13C4 1D11 NK-Deficient A B C Vehicle WT 13C4 WT 1D11 WT Vehicle NK - 13C4 NK- 1D11 NK- 0 25 50 75 100 % Metastatic Incidence Vehicle NK- 13C4 NK- 1D11 NK- 0 50 100 150 200 p=0.018 p=0.011 Number of lung mets (A) Representative images of lungs bearing experimental B16-F10 melanoma metastases from CTL-competent mice and b2-migroglobulin knockout mice (b2M -/- ) lacking active CTL. The number and size of metastases was increased in b2M -/- mice. (B) Treatment with 1D11 had no effect on inhibiting the number of pulmonary metastases in b2M -/- mice. (C) The number of spontaneous metastases to the draining lymph node is reduced in 1D11-treated CTL-competent, but not in b2M -/- mice. Figure 7 Cytotoxic T lymphocyte (CTL) activity is required for 1D11 to inhibit metastasis via the circulation or the lymphatics Wild-type β 2M -/- Vehicle 13C4 1D11 A B C Vehicle WT 13C4 WT 1D11 WT -/- 2M β Vehicle -/- 2M β 13C4 -/- 2M β 1D11 0 25 50 75 100 % Metastatic Incidence -/- 2M β Vehicle -/- 2M β 13C4 -/- 2M β 1D11 0 50 100 150 200 p=0.732 p=0.691 Number of lung mets Determine the effects of neutralizing TGF b with 1D11 on Th1 and Th2 responses in primary tumors and metastases. Characterize the role of neutralizing TGF b with 1D11 on the polarization of macrophages in the tumor microenvironment. Determine if neutralization of TGF b can enhance the efficacy of chemotherapeutics against both primary tumors and metastasis. Future Directions (A) Treatment with 1D11 significantly enhanced the survival of mice receiving intracardiac injections of 4T1 cells (* p<0.02) compared to controls. (B) Neutralization of TGFb with 1D11 significantly inhibited metastasis to the bone and other organs following intracardiac injection of 4T1 cells. (C) Treatment with 1D11 resulted in a significant decrease in spontaneous metastasis to the lungs in mice bearing SQ 4T1 tumors compared to controls (p<0.05).

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Results

TGFb plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFb on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFb antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas). However, TGFb antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFb in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFb on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFb significantly inhibits metastasis and anti-TGFb strategies may be effective at treating metastatic disease in the clinic.

Inhibition of Metastases by a Neutralizing TGFb-specific Antibody Involves the Activity of Cytotoxic T Lymphocytes and Natural Killer Cells Kenneth Munroe1, Charlene Manning1, Tessa Green1, Jingzang Tao1, Kailene Simon1, John McPherson1,

Johanne Kaplan1, Jan Pinkas2, Megan O’Brien1, Richard Gregory1, Jay Harper1, Scott Lonning1

1Genzyme Corporation, Framingham, MA; 2ImmunoGen, Waltham, MA

Abstract

TGFb signaling is known to affect several aspects of primary tumor growth and metastasis by effects on both tumor cells and the stroma. As tumor progression occurs, tumor cells lose their responsiveness to the growth-inhibitory activity of TGFb. TGFb stimulates VEGF production leading to angiogenesis, and promotes epithelial to mesenchymal transition in the cancer cells. This leads to progression to a more invasive phenotype and increased access to the vasculature allowing for the extravasation of tumor cells and the establishment of distal metastases. TGFb also inhibits immunosurveillance mechanisms in the tumor by inhibiting proliferation or activation of cytolytic immune effector cells, by inducing suppressive regulatory T lymphocytes and promoting the secretion of Th2 cytokines.

Figure 1The Role of TGFb in Tumor Progression and Metastasis

TGFβ

Tumor CellEMT/invasion/motility

SurvivalTumor promoting factors

Angiogenesis

Altered ECM composition

Other immune cellsaltered cytokine

production

Myofibroblast Formation

Immune Function

Cytotoxic T cell

NK cell

(A) Mice with established, subcutaneous (SQ) B16-F10 melanomas implanted on the hind footpad were treated with 1D11 (50mg/kg, 3X/wk) or the isotype control, 13C4. Neutralization of TGFb with 1D11 had no effect on the growth of these SQ tumors compared to controls. (B) Established, SQ 4T1 mammary carcinoma tumors (~100mm3) were also treated with 1D11 (10mg/kg, 3X/wk) or 13C4. In this model, 1D11 had essentially no effect on primary tumor growth (representative data shown) although trends of inhibition have been observed in certain studies.

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Figure 21D11, a TGFb neutralizing antibody has nominal effects on the growth of primary tumors

(A) Treatment with 1D11 inhibited pulmonary metastasis following intravenous delivery of B16-F10 melanoma cells in a dose-dependent manner (p=0.002). (B) Representative images of lungs bearing experimental B16-F10 pulmonary metastases from mice treated with either 1D11 or 13C4.

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Figure 31D11 inhibits metastasis in an experimental model of melanoma metastasis

Figure 4Inhibition of metastasis following neutralization of TGFb in two models of breast cancer metastasis

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GOALSDetermine the effect of neutralizing TGF• b with 1D11, a pan- neutralizing TGFb antibody, on primary tumor growth and metastasis in preclinical, syngeneic models of melanoma and breast cancerElucidate the mechanism of action of 1D11 in these tumor models•

B16-F10 melanoma cells were injected into the plantar region of the hind footpad such that SQ primary tumors developed (A) and metastasis to the draining lymph node (popliteal lymph node) occurs spontaneously (B). (C) Chronic treatment with 1D11 resulted in significant reduction of metastasis to the lymph node compared to controls.

Figure 5Neutralization of TGFb with 1D11 inhibits spontaneous metastasis from primary melanomas to the draining lymph node

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(A) TAMs located at the invasive front of 4T1 primary tumors are CD68 (red) and CD206 (green) positive suggesting that these are M2 macrophages. (B) CD68+ TAMs found at center of the 4T1 primary tumors are negative for CD206 immunostaining, suggesting that these represent M1 macrophages.

M2 TAMs are observed at the invasive front of 4T1 primary tumors following the establishment of the tumor and persists as the tumor volume increases (A) 95mm3, day 7(B) 248mm3, day 7 (C) 524mm3, day 14 (D) 1020mm3, day 18 (E) 1468mm3, day 18 (F) 1928mm3, day 18.

Figure 8M2 tumor-associated macrophages (TAMs) are shown to localize

primarily to the tumor periphery while classically-activated M1 macrophages are observed in the tumor core.

Red = CD68 Green = CD206 Blue = DAPI

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B

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Red = CD68 Green = CD206 Blue = DAPI

Figure 9M2 macrophages are observed at the invasive front of 4T1 primary

tumors during all stages of tumor growth. tumors

Neutralization of TGF• b with 1D11 inhibits metastasis in two independent tumor model systems: B16 murine melanoma and 4T1 murine mammary carcinoma.

1D11 had nominal effects on the growth of • primary tumor growth.

CTL activity appears to play a major role in the • ability of 1D11 to inhibit both experimental and spontaneous metastasis.

The role of NK cell activity is less clear and may • depend on the mode of metastasis.

M2 macrophages are recruited to 4T1 tumors • during all stages of tumor growth. As tumor progression continues, M2 macrophages become marginated to the invasive front of growing tumors.

Conclusions

We thank the staff at Genzyme’s DCM, as well as Doug Matthews, Sheen Zhang, Peter Piepenhagen, Brian DelGiudice and Bob Thomas for their assistance in this project.

Acknowledgements

To assess the role of NK cell activity in the efficacy of 1D11, tumor models were conducted in wild type mice (WT) and in mice treated chronically with anti-asialo GM1 to deplete NK cells (NK-). (A) Representative images of lungs from wildtype and NK-deficient mice bearing experimental B16-F10 melanoma metastases treated with either 1D11 or 13C4. The number and size of metastases was increased in NK-deficient mice. (B) 1D11 is able to inhibit the development of experimental pulmonary metastases in NK-deficient mice. (C) The ability of 1D11 to inhibit spontaneous metastasis to the draining lymph node is dependent on NK cell activity.

Figure 6Differential roles of natural killer (NK) cells in the ability of 1D11 to inhibit metastasis via the circulation versus metastasis through the lymphatics

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(A) Representative images of lungs bearing experimental B16-F10 melanoma metastases from CTL-competent mice and b2-migroglobulin knockout mice (b2M-/-) lacking active CTL. The number and size of metastases was increased in b2M-/- mice. (B) Treatment with 1D11 had no effect on inhibiting the number of pulmonary metastases in b2M-/- mice. (C) The number of spontaneous metastases to the draining lymph node is reduced in 1D11-treated CTL-competent, but not in b2M-/- mice.

Figure 7Cytotoxic T lymphocyte (CTL) activity is required for 1D11 to inhibit metastasis via the circulation or the lymphatics

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Determine the effects of neutralizing TGF• b with 1D11 on Th1 and Th2 responses in primary tumors and metastases.

Characterize the role of neutralizing TGF• b with 1D11 on the polarization of macrophages in the tumor microenvironment.

Determine if neutralization of TGF• b can enhance the efficacy of chemotherapeutics against both primary tumors and metastasis.

Future Directions

(A) Treatment with 1D11 significantly enhanced the survival of mice receiving intracardiac injections of 4T1 cells (* p<0.02) compared to controls. (B) Neutralization of TGFb with 1D11 significantly inhibited metastasis to the bone and other organs following intracardiac injection of 4T1 cells. (C) Treatment with 1D11 resulted in a significant decrease in spontaneous metastasis to the lungs in mice bearing SQ 4T1 tumors compared to controls (p<0.05).