Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

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Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor

Blood ◽

10.1182/blood.v80.2.359.359 ◽

1992 ◽

Vol 80 (2) ◽

pp. 359-366 ◽

Cited By ~ 3

Author(s):

DJ Tweardy ◽

PA Morel ◽

PL Mott ◽

EW Glazer ◽

HJ Zeh ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Colony Formation ◽

Therapeutic Potential ◽

Autocrine Growth ◽

Interleukin 3 ◽

Gm Csf ◽

Myeloid Development

Abstract Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

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In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3

Blood ◽

10.1182/blood.v71.2.375.bloodjournal712375 ◽

1988 ◽

Vol 71 (2) ◽

pp. 375-382 ◽

Cited By ~ 1

Author(s):

J Lotem ◽

L Sachs

Keyword(s):

Therapeutic Potential ◽

Regulatory Proteins ◽

Leukemic Cells ◽

Cell Multiplication ◽

Interleukin 3 ◽

Gm Csf ◽

Blast Cells ◽

Macrophage Colony

The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL- 3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.

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In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3

Blood ◽

10.1182/blood.v71.2.375.375 ◽

1988 ◽

Vol 71 (2) ◽

pp. 375-382 ◽

Cited By ~ 36

Author(s):

J Lotem ◽

L Sachs

Keyword(s):

Therapeutic Potential ◽

Regulatory Proteins ◽

Leukemic Cells ◽

Cell Multiplication ◽

Interleukin 3 ◽

Gm Csf ◽

Blast Cells ◽

Macrophage Colony

Abstract The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL- 3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.

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Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness

Blood ◽

10.1182/blood.v74.1.56.56 ◽

1989 ◽

Vol 74 (1) ◽

pp. 56-65 ◽

Cited By ~ 132

Author(s):

LS Park ◽

PE Waldron ◽

D Friend ◽

HM Sassenfeld ◽

V Price ◽

...

Keyword(s):

Growth Factor ◽

Cell Lines ◽

Specific Binding ◽

Absolute Number ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Interleukin 3 ◽

Gm Csf ◽

In Vitro Cell Lines

Abstract Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

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Growth Hormone in Vivo Potentiates the Stimulatory Effect of Insulin-Like Growth Factor-1 in Vitro on Colony Formation of Epiphyseal Chondrocytes Isolated from Hypophysectomized Rats*

Endocrinology ◽

10.1210/endo-121-3-1070 ◽

1987 ◽

Vol 121 (3) ◽

pp. 1070-1075 ◽

Cited By ~ 27

Author(s):

ANDERS LINDAHL ◽

JÖRGEN ISGAARD ◽

OLLE G. P. ISAKSSON

Keyword(s):

Growth Hormone ◽

Growth Factor ◽

Colony Formation ◽

Insulin Like Growth Factor ◽

Hypophysectomized Rats

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Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

Journal of Oncology ◽

10.1155/2009/951917 ◽

2009 ◽

Vol 2009 ◽

pp. 1-10 ◽

Cited By ~ 14

Author(s):

Helenia Ansuini ◽

Annalisa Meola ◽

Zeynep Gunes ◽

Valentina Paradisi ◽

Monica Pezzanera ◽

...

Keyword(s):

Pancreatic Cancer ◽

Monoclonal Antibodies ◽

Therapeutic Potential ◽

Tumor Cell Line ◽

Immunohistochemical Analysis ◽

Receptor Activation ◽

Rapid Identification ◽

Antibody Treatment

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

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Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness

Blood ◽

10.1182/blood.v74.1.56.bloodjournal74156 ◽

1989 ◽

Vol 74 (1) ◽

pp. 56-65 ◽

Cited By ~ 2

Author(s):

LS Park ◽

PE Waldron ◽

D Friend ◽

HM Sassenfeld ◽

V Price ◽

...

Keyword(s):

Growth Factor ◽

Cell Lines ◽

Specific Binding ◽

Absolute Number ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Interleukin 3 ◽

Gm Csf ◽

In Vitro Cell Lines

Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

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IL-6 Undergoes Transition from in vitro Autocrine Growth Factor to in vivo Growth Inhibitor of B Lymphoma Cells

Journal of Biomedical Science ◽

10.1159/000457002 ◽

1997 ◽

Vol 4 (5) ◽

pp. 201-207

Author(s):

Dae-Ho Cho ◽

Hyung-Sik Kang ◽

Jung-Jae Ma ◽

Sung-Sook Kim ◽

Hwan-Mook Kim ◽

...

Keyword(s):

Growth Factor ◽

Growth Inhibitor ◽

Autocrine Growth ◽

Lymphoma Cells ◽

B Lymphoma ◽

Autocrine Growth Factor ◽

B Lymphoma Cells ◽

In Vivo Growth

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Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro

Reproduction ◽

10.1530/reprod/119.1.85 ◽

2000 ◽

pp. 85-91 ◽

Cited By ~ 8

Author(s):

S Hasthorpe ◽

S Barbic ◽

PJ Farmer ◽

JM Hutson

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Colony Formation ◽

Inhibitory Action ◽

Transforming Growth Factor ◽

Epidermal Growth ◽

Growth Factor Beta

At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Mullerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Mullerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.

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