Method of inhibiting metastasis by anti-.alpha.6-integrin-antibodies

Polypeptides which bind to mammalian .alpha.6-integrin and inhibit metastasis.

BACKGROUND OF THE INVENTION 
Adhesion of invasive cancer cells to vascular endothelium is a critical 
first and selective step in metastasis. During the last few years a large 
number of different adhesion molecules have been discovered to be involved 
in cell migration and homing mechanisms of hemopoietic cells. Transformed 
tumor cells can abuse such mechanisms in an uncontrolled way leading to 
metastasis in tissues other than those of their origin. The antigen known 
as .alpha.6-integrin is an adhesion molecule involved in metastasis. 
Carcinoma, melanoma, and endothelial cells are known to adhere to laminin, 
an extracellular matrix molecule. Several integrin complexes, including 
.alpha.1/.beta.1, .alpha.2/.beta.1, and .alpha.6/.beta.1 help mediate this 
binding. .alpha.6/.beta.1 appears to react with laminin monospecifically. 
Three antibodies directed against the .alpha.6 integrin chain have been 
described in the literature, each produced in a different manner. Antibody 
GoH3 was produced by immunizing rats with blood platelets and recognizes 
the platelet protein called complex Ic-Ila, which was subsequently defined 
as .alpha.6/.beta.1 integrin. This antibody blocks cell-laminin 
interaction, and defines .alpha.6 integrin as a receptor for a fragment 
called E8 obtained by an elastase digest of laminin. [Sonnenberg et al., 
J. Biol. Chem. 262, 10376-10383 (1987); Hemler et al., J. Biol. Chem. 263, 
7660-7665 (1988)]. Antibody 135-13C was prepared in rats against purified 
tumor associated proteins TSP-180, which is now described as 
.alpha.6/.beta.4 integrin. This antibody has a less pronounced effect on 
cells binding to laminin. [Kennel et al., Cancer Res. 41, 3465-3470 
(1981); Kennel et al., J. Biol. Chem. 264, 15515-15521 (1989)]. Finally, 
antibody GB36 was raised in mice against microvilli preparations of human 
placenta. The antibody recognizes cell surface proteins on human carcinoma 
cells [Hsi et al., Placenta, 8, 209-217 (1987)]. It was suggested in this 
reference that the protein (.alpha.6/.beta.1 integrin) may play a role in 
maintenance of cell polarity, however no functional assays were performed. 
Now it has been surprisingly found on the basis of the specific ability of 
the monoclonal antibodies of the present invention to inhibit metastasis 
that a further molecule, namely the antigen recognized by these monoclonal 
antibodies, which is known as .alpha.6-integrin [for a review see Hemler 
in Annu. Rev. Immunol. 8, 365-400 (1990)], is probably the most important 
endothelial adhesion molecule to be involved in metastasis. 
The antibody of the present invention does not interfere with the binding 
of cells to laminin but blocks cell-cell interaction of melanoma cells 
with vascular endothelial cells. This finding strongly suggests that the 
antibody of the present invention recognizes a so far unknown binding 
domain on the .alpha.6 integrin chain-clearly different from the binding 
sites of GoH3, 135-13C or GB36, by the fact that GoH3 cross-reacts with 
skin homing lymphocytes in sheep whereas the antibody of the present 
invention does not. 
SUMMARY OF THE INVENTION 
The claimed invention is a monoclonal antibody or a functional derivative 
thereof which binds to mammalian .alpha.6-integrin, and which inhibits 
metastasis. The claimed antibody also prevents transformed cells from 
binding to endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION 
The claimed invention is a polypeptide which comprises a monoclonal 
antibody or a fragment thereof, which polypeptide is capable of binding to 
mammalian, preferably human, .alpha.6-integrin and of inhibiting 
metastasis in transformed cells. The polypeptide may in particular inhibit 
interaction between transformed cells and endothelial cells. Transformed 
cells include cells which carry .alpha.6-integrin or a ligand thereof as a 
surface molecule, such as melanomas, carcinomas, T-cell lymphomas, and 
sarcomas in particular melanomas. The polypeptide may be produced by 
conventional methods for synthesizing polypeptides and linking 
polypeptides. For example, the polypeptide may be produced by recombinant 
methods in a host cell into which has been inserted a nucleic acid 
sequence encoding the polypeptide, which sequence contains the sequence of 
the monoclonal antibody or fragment. The latter sequence may be obtained 
by sequencing the antibody using known methods. The polypeptide may be 
produced by chemically linking a selected peptide or peptides to the 
monoclonal antibody or a fragment using known methods to form covalent or 
other bonds between peptides and the antibody, or using known linker 
molecules to link peptides to the antibody. 
It is also an object of the present invention to provide a monoclonal 
antibody, or a functional derivative thereof, or a fragment thereof, 
especially an Fab-fragment. Both the monoclonal antibody and the fragment 
are characterized by binding to mammalian, e.g. mouse or preferably human 
.alpha.6-integrin [anti-.alpha.6i-mAb] and by inhibiting metastasis of 
transformed cells, in particular by inhibiting the interaction of the 
transformed cells with the vascular endothelium. By transformed cells is 
meant those cells which are characterized by carrying .alpha.6-integrins 
as surface molecules, e.g. melanomas, carcinomas, T cell lymphomas or 
sarcomas, with melanomas and carcinomas, especially melanomas, and those 
cells which carry a ligand of .alpha.6-integrins as a surface molecule. A 
functional derivative of the claimed monoclonal antibody includes both 
fragments of the antibody, and larger molecular entities which incorporate 
the antibody o a fragment of the antibody, for example labelled antibody, 
or antibody combined with another molecule such as a toxin. Such 
derivatives may be produced by conventional methods. 
It is also an object of the present invention to provide hybridoma cell 
lines secreting such monoclonal antibodies and the monoclonal antibodies 
secreted by such cell lines. Specifically, hybridoma cell lines secreting 
monoclonal antibodies binding to .alpha.6-integrin, preferably human 
.alpha.6-integrin, and inhibiting metastasis in transformed cells, in 
particular by inhibiting their interaction with endothelial cells. 
Transformed cells am as described above. 
The hybridoma cell line "F3C34" obtained in accordance with the teaching of 
the present invention has been deposited on OCT. 17, 1991 at the Deutsche 
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM). F3C34 has the 
accession number ACC2023. It is furthermore an object of the present 
invention to provide such monoclonal antibodies or fragments thereof as 
therapeutic agents, especially for the treatment of cancer by the 
inhibition of metastasis. 
Antibodies binding to .alpha.6-integrins can be prepared by using short 
peptides with amino acid sequences derived from the known amino acid 
sequence of human .alpha.6-integrin [Hogervorst et al., Eurp. J. Biochem. 
199, 425-433 (1991)] as starting antigen. Such peptides can be prepared by 
methods of chemical peptide and protein synthesis known in the art, e.g. 
by partial or total liquid or solid phase synthesis as described e.g. by 
Gross and Meyenhofer in "The Peptides" Vols. 1-9, Academic Press, Inc., 
Harcourt Brace Jovanovich, Publs., San Diego (1979-1987) or by Fields and 
Nobel, Int. J. Pept. Prot. Res. 35, 161-214 (1990). 
However, antibodies raised in such a way do not necessarily react with the 
native .alpha.6-integrins nor show the specific properties of those of the 
present invention. Therefore antibodies of the present invention can be 
prepared by conventional methods starting from .alpha.6-integrin positive 
cells, e.g. endothelial cells or transformed cells of endothelial origin, 
whereby an eEnd2-cell line is specifically preferred. 
By injection of such an antigen into a non-human mammal, e.g. mouse, 
rabbit, rat or sheep, polyclonal antibodies can be obtained by methods 
known in the art from the serum of the mammal. Monoclonal antibodies can 
be prepared by known methods, for example, by recovering antibody- 
producing cells from such an immunized mammal and immortalizing said cells 
obtained in conventional fashion, such as fusion with myeloma cells e.g. 
PAl mouse myeloma cells, SP2/0- or SP2/0-Ag 14-cells [ATCC No. CRL 1581; 
ATCC No. CRL 8287] [for a general guideline for producing antibodies see 
e.g. "Antibodies--A Laboratory Manual" edt. by Harlow, E. and Lane, D., 
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, 1988] 
or as described in detail in Example I. 
Supernatants of such hybridoma cultures have then to be screened for those 
secreting antibodies of the present invention by an assay determining the 
metastasis inhibiting properties of the antibodies of the present 
invention, e.g. an assay as described in detail in Example I. It is 
therefore also an object of the present invention to provide a process for 
the preparation of anti-.alpha.6i-mAbs and fragments thereof according to 
methods known in the art whereby hybridoma supernatants obtained are 
screened by the ability of the antibodies contained in such supernatants 
to inhibit metastasis and compounds obtained by such a process. 
Anti-.alpha.6i-mAbs can be purified from hybridoma supernatants by 
conventional chromatographic procedures, for example, by 
ion-exchange-chromatography, affinity chromatography on protein G, using 
anti-immunoglobulin-antibodies or the antigen or a part thereof bound to a 
solid support, HPLC or the like. 
For the production of large quantities of anti-.alpha.6i-mAbs in accordance 
with methods well-known in the art hybridomas secreting the desired 
antibody can be injected intraperitoneally into mice which have been 
pretreated with for example pristane before injection to produce ascites 
tumors in the mice. Up to around 100 mg of a monoclonal antibody can be 
produced by such ascites tumors in one mouse. Antibodies can be purified 
for example from ascites fluid produced by such tumors using methods 
described above. 
Antibodies of the present invention can be characterized according to their 
subclass by known methods, such as Ouchterlony immunodiffusion. The 
antibody secreted by hybridoma cell line "F3C34" is of the igG2a subtype. 
Furthermore antibodies of the present invention can be characterized by 
their ability to inhibit metastasis of transformed cells, especially the 
interaction of the transformed cells with the vascular endothelium. Such 
activity can be determined by assays known in the art, e.g. in vitro by an 
assay such as described in Example I or in vivo by an assay such as 
described in Example II. Any transformed cell line carrying 
.alpha.6-integrin as a surface molecule, such as melanomas, e.g. B16 cells 
[Vollmers et al., Cell 40, 547-557 (1985)], carcinomas, e.g. KLN205 [ATCC 
CRL 1453] or CMT-93 [ATCC CCL 223] or MM45T [ATCC CRL 6420], T cell 
lymphomas, e.g. BW 5147 [ATCC TIB 48] or sarcomas, e.g. MM46T [ATCC CRL 
6420] is useful in these assays. Useful in an in vitro assay is a suitable 
tissue material to which such cells and the antibodies of the present 
invention bind. An in vivo assay uses any type of animal in which such 
transformed cell lines lead to metastasis and to which the antibodies of 
the present invention crossreact. Metastasis can be determined by 
injecting such cells into the animal and detecting resulting migration and 
tumors. Mice are particularly useful in this regard. Inhibition of the 
interaction of such transformed cell lines with the vascular endothelium 
can be determined by assays known in the art, e.g. by an in vitro adhesion 
assay on monolayers of endothelial cells or cells of endothelial origin or 
as described in Example I. This is shown in FIG. 2 where arrows point to 
the position of the transformed cells binding to the vascular endothelium. 
Finally, antibodies of the present invention can be characterized by 
ability to block cell migration in an assay as described in Example V, or 
by failure to influence cell growth, in an assay as described in Example 
VI. 
Furthermore antibodies of the present invention cross-react with human 
.alpha.6-integrin, as demonstrated by the detection of .alpha.6-integrin 
on the apical surface of human endothelial cells (see Example III) which 
correlates with in vivo staining of mouse .alpha.6-integrin on the apical 
surface of mouse blood vessel endothelium (see Example IV). 
Anti-.alpha.6i-mAbs can be modified for various uses by methods known in 
the art or active fragments thereof can be generated as described for 
example in Example II. (Harlow & Lane s.a.). Fragments can be generated, 
by well-known methods for example, by enzymatic digestion of antibodies 
with papain, pepsin or the like. Specifically, papain cleavage of an 
antibody produces Fab fragments. Pepsin cleavage can be used to produce an 
F(ab).sub.2 fragment. In addition the antibodies of the present invention 
can be modified, for example by the addition of a polyethyleneglycol 
subunit as known in the art and described in U.S. Pat. No. 4,179,337, or 
coupled, for example, to a fluorescent dye, a colour-producing substance 
such as an enzyme [enzyme linked immunosorbent assay (ELISA)] or a 
radioactive substance [radioimmunoassay (RIA)] in accordance with methods 
well-known in the art and used in such assay systems as known in the art. 
Furthermore such antibodies can be "humanized" according to methods known 
in the art, and disclosed in the case of an antibody specific for a 
subunit of the human interleukin 2 receptor in International Patent 
Application Publication No. WO 90/7861. Accordingly functional derivatives 
as described above are also an object of the present invention. 
Monoclonal antibodies of the present invention may be used as therapeutic 
agents, especially in the treatment of cancer e.g. by inhibiting 
metastasis especially in the case of secondary metastasis during surgery. 
The antibodies can be used, if desired, in combination with other 
pharmaceutically active substances, preferably monoclonal antibodies or 
peptides against different adhesion molecules present on vascular 
endothelium or the metastatic cell, with conventionally used 
pharmaceutically acceptable solid or liquid carrier materials. Dosage and 
dose rates may be chosen by analogy to dosage and dose rates of currently 
used antibodies in clinical treatment of various diseases. 
Accordingly it is also an object of the present invention to provide 
anti-.alpha.6i-mAbs which can be clinically used. Furthermore it is an 
object of the present invention to provide a pharmaceutical composition 
which contains one or more anti-.alpha.6i-mAbs or fragments thereof, if 
desired, in combination with additional pharmaceutically active substances 
and/or non-toxic, inert, therapeutically compatible carrier materials. The 
preparation of such pharmaceutical compositions can be achieved in 
accordance with methodology known to one skilled in the art. 
Furthermore monoclonal antibodies of the present invention can be used as a 
marker in the diagnosis of illnesses, especially cancer. It is well known 
in the art that for such purposes monoclonal antibodies can be coupled, 
for example, to a fluorescent dye, a colour producing substance such as 
enzyme [enzyme linked immunosorbent assay (ELISA)] or a radioactive 
substance [radioimmunoassay (RIA)] in accordance with methods well-known 
in the art and used in such conventional assay systems as described, e.g. 
in Harlow & Lane (s.a). 
The following Examples are provided to illustrate the invention without 
limiting it. 
EXAMPLE I 
Preparation of anti-.alpha.6i-mAbs 
Confluent endothelial cells (eEnd2-cell line) from a 150 cm.sup.2 culture 
flask were irradiated with 10,000 rad and harvested with cell scrapers 
(Costar Data Packaging). These cells were then washed with Dulbecco's 
phosphate buffered saline (DPBS) (Gibco BRL), mixed 1:1 with complete 
Freund's adjuvant for a final volume of 300 .mu.l, and injected 
subcutaneously into the dorsal surface of the hind foot of a 2-mouth-old 
PVG rat [BRL, Fullinsdorf, CH]. Injections with cells in DPBS only were 
repeated after 7 and 14 days. At day 17, the draining popliteal lymph node 
was dissected from the rat. The tissue was enzymatically digested using 
the following enzyme stock solutions: 150 mg/ml protease type IX (P-6141; 
Sigma Chemical Co., Buchs, CH); 8 mg/ml collagenase CLS 4 (Worthington 
Biochemical Corp., Freehold, N.J., USA); 10 mg/ml DNAse I (Sigma Chemical 
Co.). The enzyme solutions were mixed to a final volume of 2 ml (0.5 ml 
collagenase, 0.1 ml protease, 0.1 ml DNAse, 1.3 ml IMDM [Iscove's modified 
MEM; Gibco BRL, Gaithersburg, Md., USA]. A lymph node was opened by two 
slight crosscuts using a 25-gauge needle. Stroma were then digested at 
37.degree. C. for two 30-min periods each with 1 ml enzyme cocktail. The 
cells were then carefully released into IMDM with Pasteur pipettes, washed 
in 50 ml IMDM, and counted. One part lymph node cells was then mixed with 
five parts mouse Sp2/0 myeloma cells [ATCC CRL 1581], centrifuged, and 
fused with PEG 4000 (E. Merck, Darmstadt, FRG) as described in Harlow and 
Lane (1988). The cells were then plated (100 .mu.l) into conditioned 
medium in microtiter plates (96 wells; Costar Data Packaging) at a density 
of 5.times.10.sup.4 cells/well in IMDM selection medium containing HAT 
(Gibco BRL), 10% fetal calf serum (FCS) (Boehringer Mannheim GmbH, 
Mannheim, FRG), 50 mM b-mercaptoethanol, penicillin/streptomycin, and 
glutamine. Conditioned medium was produced by culturing 10.sup.4 PVG rat 
thymocytes (100 .mu.l/well) in selection medium for three days before 
fusion. 
Screening of hybridoma supernatants for metastasis inhibiting mAbs was 
based on blocking of B16/129 melanoma cell binding to frozen sections of 
mouse lung or liver tissue. 
In situ binding was performed on freshly prepared frozen sections from 
adult mouse tissue [Woodruff et al., Annu. Rev. Immunol. 5, 201-222 
(1987)]. Organs were embedded and frozen in Tissue-Tek, O.C.T. Compound 
(Miles Inc., Elkhart, Ind., USA), sectioned to 5 .mu.m thickness, and 
mounted onto glass slides. The sections were outlined by a PapPen (SCI 
Science Services, Munich, FRG) and immediately placed into DPBS containing 
1% bovine serum albumin (BSA) for at least 10 min. Slides were dried 
around the PapPen marked area and the tissue section then loaded with 
10.sup.5 B16 melanoma cells in 200 ml DPBS containing 20% hybridoma 
supernatant of cultures prepared as described above a control antibody 
(rat IgG from Jackson Immuno Research Lab). Binding was allowed to occur 
for 40 min at 8.degree. C. on a mini-shaker (Kuhner, Basel, CH) at 50 rpm. 
Slides were then placed vertically into DPBS containing 0.5% 
glutaraldehyde and 2% formaldehyde, where non-bound cells were allowed to 
fall off. After 20 min. of fixation at room temperature and 
counterstaining in 0.25% thionine-acetate in 20% ethanol, B16 cells bound 
to the tissue section under study were counted using a Zeiss Axiophot 
light microscope. Results are given in FIG. 1 with respect to lung and 
liver tissue sections and additional tissue sections (kidney, thymus, 
spleen and heart) prepared in the same way as described above for lung and 
liver whereby left colums give mean values of bound B16 cell to tissues as 
indicated in the absence of anti-.alpha.6i-mAbs and right columns in the 
presence of anti-.alpha.6i-mAb containing hybridoma supernatants. FIG. 2 
shows lung tissue sections incubated with control supernatants (a) or 
anti-.alpha.6i-mAb containing hybridoma supernatants (b) and liver tissue 
sections incubated with control supernatants (c) or anti-.alpha.6i-mAb 
containing hybridoma supernatants (d). 
Immunoprecipitation with antibodies prepared as described above and partial 
N-terminal sequencing of the precipitated antigen showed that the antibody 
is directed to mouse .alpha.6 integrin since the first 15 amino acids 
sequenced are, with the exception of position 14 (serine instead of 
tyrosine) identical to the sequence of human .alpha.6 integrin. 
EXAMPLE II 
In vivo characterisation of anti-.alpha.6i-mAbs 
Thawed B16-129 cells [Vollmers et al. (1985)], a subline from B16-F10 mouse 
melanoma cells (passage 3) were passaged maximally 2-3 times in DMEM 
(Gibco, BRL, Paisley, UK) containing 10% fetal calf serum (FCS) 
(Boehringer Mannheim GmbH, Mannheim, FRG) quickly trypsinized, washed with 
complete medium and resuspended in PBS. Intraveneous injection of 
1.5.times.10.sup.5 cells resulted in 300-500 lesions per lung after 10 
days, injection of 7.5.times.10.sup.4 resulted in 30-50. 
For characterisation of anti-.alpha.6i-mAbs in vivo B16-129 cells were 
injected i.v. into mice together with control rat-mAb or 
anti-.alpha.6i-mAbs or Fab fragments thereof. Lung B16-129 colonies were 
counted after 10 days. Results are given in Tables I and II. 
With respect to Table I ten mice per group were injected with 
7.5.times.10.sup.4 cells each. With respect to Table II mice were injected 
with 1.5.times.10.sup.5 B16-129 cells: (a) injection of cells was made 
simultaneously with the antibodies, (b) antibodies were injected 24 hours 
before the cells and (c) melanoma cells were incubated 60 min. with 
antibodies, washed and then injected into animals. The table represents 
one out of three experiments performed, 5 mice were injected per group. 
TABLE I 
______________________________________ 
500 .mu.g anti- 
250 .mu.g Fab of 
500 .mu.g Fab of 
Con- .alpha.6i-mAb/ 
anti-.alpha.6i- 
anti-.alpha.6i- 
trol mouse mAb/mouse mAb/mouse 
______________________________________ 
Lung lesions 
37 1 (0-4) 0 0 
per mouse 
(5-113) 
______________________________________ 
TABLE II 
______________________________________ 
Reduction 
Fab of anti- 
of lung 
Type of treatment 
Control .alpha.6i-mAb 
lesions 
______________________________________ 
Simultaneous 
610 (321-993) 
147 (3-481) 
86% 
injection (a) 
Pretreatment of 
742 (321-1025) 
474 (45-1008) 
36% 
mice (b) 
Pretreatment of 
465 (208-563) 
224 (145-253) 
62% 
B16-129 mela- 
noma (c) 
______________________________________ 
Anti-.alpha.6i-mAbs were purified from hybridoma supernatant using protein 
G affinity columns (Pharmacia, Uppsala, Sweden). Fab fragments thereof 
were prepared using the kit "AvidChrom" (BioProbe International, Tustin, 
Calif., USA). As control normal rat IgG (Jackson Immuno Research 
Laboratories, West Grove, Pa., USA) was used. 
Example III 
Binding of anti-.alpha.6i-mAbS to human endothelial cells 
Binding of anti-.alpha.6i-mAbs on the apical surface of human endothelial 
cells was shown in the following manner: HUV-EC-C-cells [ATCC CRL 1730] 
were grown to confluency using methods known in the art, stabilized by 
formaldehyde fixation, and incubated with a graded dilution of a 100 
.mu.g/ml stock solution of anti-.alpha.6i-mAbs. Surface bound antibody was 
detected by biotinylated mouse anti-rat antibody (Jackson Immuno Research, 
West Grove, Pa., USA) followed by streptavidin coupled to peroxidase and 
2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS (Sigma), as a 
substrate readable at 405 nm. Results are shown in FIG. 3. 
EXAMPLE IV 
Vascular surface staining in vivo 
1 mg of protein G-SEPHAROSE purified anti-a6i-mAbs was injected into the 
tail vein of C57BI/6 mice [IFFAC-CREDO, Lyon, F]. Animals were sacrificed 
4 hrs after injection, the tissues embedded in Tissue-Tek (Miles Inc, 
Elkart, Id. 46515, USA) sectioned on a cryostat and prepared for 
immunofluorescence staining using FITC conjugated goat anti-rat IgG 
antibody (Jackson Immuno Research, West Grove, Pa., USA). FIG. 4(a) shows 
a phase contrast image and FIG. 4(b) an immunofluorescence staining of 
lung. FIG. 4(c) shows a phase contrast image and FIG. 4(d) shows a 
immunofluorescence stain of liver. Injection of 1 mg rat IgG as a control 
showed no signal. 
Example V 
Blocking of cell migration 
B16-129 melanoma cells were plated to confluency in 12 well culture cluster 
dishes (Costar) in medium containing 0.3, 3 or 10% fetal calf serum. The 
monolayer was then wounded by scratching the dish using blue pipette tips 
(Gibco BRL, Gaithersburg Md., USA), this left a sharply separated region 
without cells. Cell migration into this region was subsequently observed 
after further 24 hours culturing. There was no influence on cell migration 
when the cells were grown in either 3 or 10% serum (not shown), however in 
0.3% serum anti-ec6imAb blocked B16-129 cell migration dramatically. FIG. 
5(a) shows the migration of cells in 0.3% serum towards the center of the 
wounded region in the presence of 30 g/ml control rat IgG and (b)in 30 
.mu.g/ml anti-.alpha.6i-mAb. 
EXAMPLE VI 
Influence on cell growth 
B16-129 melanoma cells were plated in 24 well culture cluster dishes (3524, 
Costar, Cambridge, Mass., USA) at a density of 10.sup.4 cells per well in 
medium containing either 0.3, 3 or 10% fetal calf serum in the presence of 
30 .mu.g/ml anti-.alpha.6i-mAb (A) or as a control in the presence of 30 
.mu.g/ml of a rat IgG-mAb (B). Over 4 days the cells from two wells per 
condition were trypsinized and counted. The cells grew readily in 10 and 
3% serum but they were quiescent in 0.3% serum over the 4 days tested. 
Under neither of these conditions anti-.alpha.6imAb had any effect on cell 
growth. Results are given in FIG. 6 wherein filled boxes refer to 0.3% of 
fetal calf serum and (B), filled circles refer to 0.3% and (A), open boxes 
refer to 3% of serum and (B), open triangles refer to 3% serum and (A), 
filled triangles refer to 10% of serum and (B) and open circles refer to 
10% of serum and (A).