Title of Invention

"ANTI- V - INTEGRIN MONOCLONAL ANTIBODY".

Abstract The invention relates to a novel monoclonal antibody, a hybridoma cell line producing said antibody, DNA sequences coding for said antibody, and amino acid sequences. The monoclonal antibody, a preferred embodiment of which is named 17E6, has the following properties: - reacting only with the V-chain of human V-integrins, - blocking the attachment to the integrin substrate of the V- integrin bearing cell, triggering reversal of established cell matrix interaction caused by V-integrins, blocking tumor development, and showing no cytotoxic activity.
Full Text 1A
- Anti-aV-lntegrin Monoclonal Antibody
TECHNICAL FIELD OF THE INVENTION
The invention relates to a novel monoclonal antibody and a hybridoma cell line producing said antibody. The monoclonal antibody, a preferred embodiment of which is named 17E6, has the following properties: - reacting only with the V-chain of human V-integrins,
blocking the attachment to the integrin substrate of the V-
integrin bearing cell,
triggering reversal of established cell matrix interaction
caused by aV-integrins,
blocking tumor development, and
showing no cytotoxic activity.
Object of the invention is, therefore, a monoclonal antibody having said properties.
In addition, object of the invention is a monoclonal antibody producing hybridoma cell line having the designation 272-17E6 and deposited under accession number DSM ACC2160, as well as a monoclonal antibody having the properties given above and which is obtainable by said hybridoma cell line.
Furthermore, the invention relates to DNA sequences and amino acid sequences. The DNA sequences are coding for the antibody or parts of it. The sequences are given in Figure 17a and 17b and in the attached Sequence Protocol.

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Object of the invention is, finally, a parmaceutical composition comprising an antibody as defined above.
BACKROUND OF THE INVENTION
Integrins are a super-family of cell surface adhesion receptors which control the attachment of cells with the solid extracellular environment - both to the extracellular matrix (ECM), and to other cells. Adhesion is of fundamental importance to a cell; it provides anchorage, cues for migration, and signals for growth and differentiation. Integrins are directly involved in numerous normal and pathological conditions, and as such are primary targets for therapeutic intervention. Integrins are integral transmembrane proteins, heterodimers, whose binding specificity depends on which of some 14 -chains is combined with which of some 8 -chains. The integrins are classified in four overlapping subfamilies, containing the 1, 2, 3 or av chains, and a particular cell may express several different integrins from each subfamily. The last decade has shown that integrins are major receptors involved in cell adhesion. Reports concerning integrins are given, for example, by E. Ruoslahti ( J. Clin. Invest., 1991, 87) and R. O. Hynes (Cell, 1992, 69), and so may be a suitable target for therapeutic intervention.
Excepting erythrocytes, all human cells express one or more integrins. Their functions are regulated at many levels, but primarily, their ligand specificity depends on which a-chain associates with which chain in the heterodimer and on the activation state of the integrins (Hynes, 1992; Diamond and Springer, 1994). The cellular background in which the integrins operate (Chan and Hemler, 1993),


and the splice-variant form of the integrin which is used (Delwel et al., 1993) may also affect specificity. Given these complexities, one of the few reliable indications of integrin specificity is to directly perturb integrin function and analyse which cellular responses are affected. The history of integrin research has shown that reagents that can specifically block integrin function are decisive factors in functional analysis, from the function blocking CSAT-antibody, which first defined an integrin 1-chain (Neff et al., 1982), to the numerous vital later examples (eg. P1D6, P1B5(Wayner and Carter, 1987), P4C10 (Carter et al., 1990), AIIB2 (Hall et al., 1990), 3A3 (Turner et al., 1989) G0H3 (Sonnenberg et a!., 1987), and LM609 (Cheresh and Spiro, 1987)): the field is absolutely dependent on such reagents.
The v-series integrins are now seen to be a major subfamily, with both classical, and novel functions. As well as classically mediating cell attachment and spreading (Pytela et al., 1985; Cheresh, 1991), v integrins have also been implicated in cell locomotion (Seftor et al., 1992), in receptor internalization (Panetti and McKeown Longo, 1993a; Panetti and McKeown Longo, 1993b), as virus co-receptors (Wickham et al., 1993), in management of the extracellular protease cascades (de Boer et al., 1993), and as regulators of tumor progression (Felding-Habermann et al., 1992). The specificities of the five known av-series integrins, v 1 (Zhang et al., 1993), - 3 (Pytela et al., 1985; Cheresh et al., 1987), - 5 (Cheresh et al., 1989), - 6 (Busk et al., 1992) and - 8 (Moyie et al., 1991), have been partly defined, and they seem to exclusively recognize ligands bearing the RGD- (NH2-arginine-glycine--aspartic acid- COOH) tripeptide sequences, including those in vitronectin ( v , v 3, v 5), fibronectin ( v v 3, v 5, v ), and von Willebrand factor,

fibrino-gen, and osteopontin ( v ) (e.g. 1991; Busk et al., 1992; Zhang et al., 1993; Denhardt and Guo, 1993; Smith and Cheresh, 1990;). Dimers with related specificities may be co-expressed on the same cell (eg. v and v 5 - for vitronectin on M21 cells) (Wayner et al., 1991), but may control independent functions. However, the over-lapping ligand specificities within the v-family itself and also between v- and 1-series integrins, means that assigning a function to a defined receptor within a particular cellular environment is problematic. Function blocking antibodies have been vital in clarifying the function of v (Cheresh and Spiro, 1987; Chuntharapai et al., 1993) and v 5 (Wayner et al., 1991). However, for the other v-integrins, no antibodies which specify the complex and perturb function are known. In particular, few reagents which specify the v-chain of the complex, and perturb integrin function of the whole family are available. Lehmann et al. (1994) disclosed an v x antibody which shows no reversal of cell matrix interaction and no tumor development blocking activity.
Therefore there is a permanent interest in the function of v-series integrins in tumor development Human malignant melanoma is an increasingly prevalent aggressive skin cancer. Elevated levels of integrins 1 (Danen et al., 1993; Etoh et al., 1992), (Natali et al., 1993; Yoshinaga et al., 1993), (Hart et al., 1991), and (Hart et al., 1991) have each been implicated in melanoma progression, but the integrins most consistently implicated are those of the V-series. In particular, both the invasion from the primary tumor and distant metastases are characterized histologically by an increased expression of v integrin, 'the vitronectin receptor'. Primary non-invasive tumors and non-malignant melanotic nevi

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express little detectable v 3, a receptor rare in healthy adult tissue (Brooks et al., 1994; Buck et al., 1990; Pignatelli et al., 1992; Lessey et al., 1992; Korhonen et al., 1991; Nesbitt et al., 1993). Immunohistochemistry of staged tumors and metastases showed a progressive increase in v 3 with invasive stage (Albelda et al., 1990; Si and Hersey, 1994), screening of melanoma lines uniformly reveals a high expression of V-series integrins (Sanders et al., 1992; Gehlsen et al., 1992; Marshall et al., 1991), and in addition sprouting blood capillaries express v 3 during tumor angiogenesis (Brooks et al., 1994).
Studies in vivo also implicate v 3 in melanoma development. In the murine B16-F10 melanoma system, experimental lung metastasis could be suppressed by high levels of RGD-peptides (Hardan et al., 1993; Humphries et al., 1986), potent blockers of v-integrin function. More recently, Felding-Habermann and colleagues have shown that v-series integrins promote subcutaneous tumor growth of M21 human melanoma in immune-deficient mice. The M21 system is elegant, and consists of a suites of cells expressing different v-series integrins (Kieffer et al., 1991; Felding-Habermann et al., 1992; Cheresh and Spiro, 1987). The parent, M21, expresses v 3 and v (Wayner et al., 1991): it attaches to vitronectin and grows as a subcutaneous tumor. M21-L, a somatic variant of M21, has no detectable v (Cheresh and Spiro, 1987): it cannot bind vitronectin and develops slow growing tumors. M21-L4 is a transfectant of M21-L, stably re-expressing a full length v-chain: it binds vitronectin and grows rapidly as a subcutaneous tumor (Felding-Habermann et al..

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1992). Thus the presence of cell surface v-integrins is directly correlated with M21 subcutaneous growth.
However, M21 was subjected to extreme selection pressures during the establishment of the variant lines M21-L and M21-L4. In this invention, it was found that v-integrin function on the native M21 population can be blocked and that a surprising effect on cell behaviour and tumor development can be found. Peptidic antagonists can be synthesized easiliy, however, their use is restricted because of their poor bioavailability, short half life in vivo, and rapid clearance from the animals (Humphries et al., 1988). Syngeneic antibodies offer an interesting alternative to peptides. They have a long half life in-vivo (Tao and Morrison, 1989; Haba et al., 1985) and their binding specificities can be well demonstrated by standard techniques. Unfortunately, although there are excellent v-specific antibodies such as LM142 (Cheresh and Spiro, 1987), there are few that effectively block v-integrin functions.
The specificity and biological function of the av-fatnily members is much debated, primarily because no reagent yet exists that will knock out the function of the whole class - there exists no potent v blocking antibody. The revelation that the classical adhesion receptors of the integrin family support other less conventional biological functions has intensified the search for their molecular mechanisms of action. The search has revealed several unpredicted regions on the integrins that allosterically report their activation state, or, when ligated, can themselves activate integrin function. Inhibitors of integrin function, by contrast, generally occlude the active site, the classic example being RGD-peptides that replicate the integrin recognition site of many ligands of integrins of the v-series and of v .

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This invention describes such a novel function-blocking antibody directed against the human v-integrin chain which is designated 17E6. Many reports have discussed the irreversible nature of the interaction between v 3 and its ligand, for example, vitronectin. Here it is revealed that 17F.6 will rapidly trigger the reversal of this so-called irreversible interaction between v-series integrins and their substrates, suggesting therapeutic applications. This invention analyses the mechanism of action of 17E6 and found it an extremely poor competitor for ligand binding to v 3, while vitronectin and RGD-based active site probes compete strongly for one another at the receptor. And other antibodies compete strongly for 17E6. Thus, 17E6 acts as an allosteric inhibitor. Cross-linking experiment reveal that v 3, but not v or v , needs to be dimerized before 17E6 can block. This discovery reveals that 17E6 functions by a novel mechanism involving a manipulation of integrin-induced signal transduction pathways. The antibody according to the invention blocks tumor development and has, moreover, no cytotoxic activity.
GENERAL, MATERIALS, FIGURES, TABLES, ABBREVIATIONS:
Microorganisms, cell lines, plasmids, phagemids, promoters, resistance markers, replication origins or other fragments of vectors which are mentioned in this application are normally commercially or otherwise generally available. In some cases the above-mentioned materials are not directly purchasable. However, they are used here only as examples in order to demonstrate properties or effects of the objects according to the invention, and are not essential to fulfill the requirements of disclosure. They can be replaced, as a rule, by other suitable generally obtainable tools and biological materials.

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Monoclonal antibody 17E6 is an antibody which is produced by a hybridoma cell line having the designation 272-17E6. The cell line was deposited under accession number DSM ACC2160 at the Deutsche Sammlung fur Mikroorganismen, Braunschweig, FRG. The techniques and methods which are essential according to the invention are described in detail in the specification. Other techniques which are not described in detail correspond to known standard methods which are well known to a person skilled in the art, or are described more in detail in the cited references and patent applications and in the standard literature.
The DNA- and amino acid sequences include also slightly varied or altered sequences such as mutants and variants which can be obtained intentionally or randomly by chemical or physical processes. Generally, all such mutants and variants are included which show the described properties and functions.
The term antibody includes also, as a rule, antibody fragments such as Fab', F(ab)? or single-chain, Fvs. These fragments can be produced by usual standard techniques


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17E6 Mab
20A9 Mab
23 G5 Mab
3A3 Mab
9.2.27 Mab
ADCC antibody directed cellular cytotoxicity
AECM antibody and effector cell dependent cytostatis
AIIB2 Mab
AP3 Mab
ATCC American Type Culture collection
B16F10 cell line
CP8 Mab
CSAT Mab
cRGDfV cyclic peptide: Arg-Gly-Asp-DPhe-Val
cRGDfX cyclic peptide: Arg-Gly-Asp-DPhe-Lys
EMM31 cell line
G0H3 Mab
GRGDSPK peptide: Gly-Arg-Gly-Asp-Ser-Pro-Lys
LM142 Mab
LM609 Mab
M21 cell line
M21-L cell line
M21-L-IIb cell line
M21-L4 cell line
MAb monoclonal antibody
MTT 3-(4-5-dimethylthiazol-2-yl)-2-5-diphenyl-
tetrazolium biomide NBT-BCIP nitro Blue Tetrazolium - Bromochloroindolyl
Phosphate
NP18 Cell line
OG octyl-glucoside -(detergent)


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P1D6 Mab
PIH6 Mab
P4C10 Mab
P5H9 Mab
PMSF Phenyl-methyl-sulphonyl-fluoride
RGD Peptide: NH2- Arginine-Glycine-Aspartic acid-
COOH
SDS-PAGE sodium-dodecyl-sulphate polyacrylamide gel
electrophoresis
UCLAP-3 Cell line
WM164 Cell line
WM793 Cell line
V+B2 Cell line
Table 1.
Antibodies were compared in ELISA and cell ELISA. Reagents and
specificities are discussed fully in text. + = positive reaction, = no
reaction.
Key: - Antibody Antibodies characterized in this study (bold),
standard reagents (italicized). - Immunogen: v 3 = immobilized
human placental integrin v 3. M21= Intact M21 cells. - Purified
v 3 and llb 3 were immobilized on 96-well plates for ELISA
- the cell lines M21, M21-L and M21-L-llb, and UCLAP3 were grown
and fixed on the plates and tested for antibody reactivity in CELISA.
- Specificity: (See text) v = v chain of mammalian integrins.
200KDa = unknown 200,000 MW surface protein of melanoma cells.
v = integrin v 3. 1 = integrin 1 chain. 3 = integrin 3 chain.

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Table 2.
Levels of reactivity relative to control (secondary antibody only) were
graded as follows: 1-2 (-), 2-4 (+), 4-9 (++), >9 (+++). For example,
on M21, the relative fluorescent intensity of the control was typically
30-50 units, and that of LM142 binding was 300-500 units, giving
mean relative reactivity around 10 (400/40).
(*) M21-L was permeabilized with 70 % ethanol at -20°C.
(@)M21-L has intracellular pools of (33 chain of VNR integrin.
Table 3.
M21 and M21-L cells were harvested with trypsin/EDTA, incubated
with 17E6 antibody or control antibody, washed and injected into the
tail vein of nude mice. 7 weeks later the animals were sacraficed and
the lungs examined for surface tumor foci. Pretereatment with 17E6
lowered the numbers of foci that developed. Similar numbers of foci
developed when M21-L cells (which lack v on the cell surface) were
injected.
* = Compared to control: not antibody-dependent.
Figure 1.
Fluorescence Activated Cell Sorter (FACS) Analysis of the Alpha-V Group Antibodies and Controls to M21 and M21L Human Melanoma Cells. Cells were incubated with 10 g ml-1 primary antibodies, stained with fluorescently labelled secondary antibodies, counter stained with propidium iodide to allow gating of necrotic cells, and 10 000 cells per sample were analysed. The open peak represents intensity of the second layer antibody alone. The closed peak, the intensity of the specifying primary and secondary together. Vertical axis shows cells per channel, horizontal axis shows log. fluorescent intensity in that channel. M21 carries surface av integrin, M21-L has

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none. The pattern of staining for the alpha-V group antibodies closely matches the LM142 (av-specific) and LM609 ( v 3-specific) stainings. Especially, they react with M21 but minimally with M21-L. Antibody 9.2.27 reacts with a surface proteoglycan. 14E2 and 21H6 recognize an otherwise undefined 200KDa melanoma surface protein. Their staining patterns are discrete from those of the alpha-V group. Especially, they react similarly with both M21 and M21-L. Figure 2.
The Alpha-V Group Antibodies Immunoprecipitate Similar Proteins. Viable M21 melanoma (A) v/ere cell-surface labelled with biotin, extracted with detergent, and immunoprecipitated and resolved on non-reducing 7.5% SDS-PAGE. Standards were run in lane a and weights in KDa shown to the left. Precipitation was with the control antibodies LM142 (anti- av. lane b) and LM609 (anti- v 3: lane c), and 21H6 (lane d), 10G2 (lane e), 20A9 (lane f), 23G5 (lane g), 17E6 (lane h), 14D9 (lane i), and 14E2 (lane j). LM142 and LM609 precipitate a similar pattern of proteins to 10G2, 20A9, 23G5, and 17E6, while 21H6 and 14E2 precipitate a band at ~200KDa. 14D9 precipitates both patterns. M21 melanoma (B), M21-L av-deficient melanoma (C), and UCLAP-3 cells (D) were cell-surface labelled with biotin, and immunoprecipitated as in (A). Precipitation was with antibodies LM142 (anti- av. lane b), LM609 (anti- v 3: lane c), 17E6 (lane d), AP3 (anti- 3: lane e), and 20A9 (lane f). Molecular weight markers are run in lane a, and weights in KDa shown to the left. The position of av, , 3, bands is indicated. Figure 3.
17E6 Interferes with Initial Cell Attachment to Vitronectin. A series of melanoma lines (Mels.), and carcinoma cell lines (Cars.) were screened by FACS for reactivity with 17E6 Mab. FACS

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intensities were as described in Tab.2. The cells were then allowed to attach to vitronectin-coated substrates (0.5 g ml-1 coating) in the presence of hybridoma supematants from 17E6 (open) or 14E2 (solid). After washing, attached cells were counted as described in Materials and Methods, and the data normalized to attachment in the absence of antibodies. Note that excepting B16F10 (murine melanoma), all cells were of human origin. Malme 3 is a fibroblast line. Absolute percentage of cells attached in the absence of antibodies was for M21 (70%), WM164 (68%), A375 (75%), EMM31 (67%), WM793 (65%), MalMe3 (67%), B16F10 (70%), UCLA-P3 (76%), NP-18 (65%), SW1116 (68%), HT29 (65%). Horitontal axis: cell attachment in % of control. Figure 4.
17E6 Perturbs Adhesion Mediated by both v 3 and v integrins. Purified monoclonal antibodies (Mab) or mouse ascites were co-incubated during cell attachment to vitronectin-coated substrates (5 mg ml"1). Cell lines (A, B) M21; (C, D) UCLAP-3; (E,F) WM-793. Symbols represent the following antibodies (specificities): ( ©) = 17E6 ( av); (A)= LM609 ( v 3); (?)= 14D9.F8 ( v) ; (O)= P4C10 (B1); (O)= P5H9 ( v ); (V)= P5H9 + LM609 [dilution starting at 10 g ml-1] ( v 3+ v ). Vertival axis: cells attached (%). Horizontal axis: left side: Mab ( g/ml), right side: ascites dilution (1/x). Figure 5.
17E6 Perturbs Adhesion to Vitronectin but not to Other Matrix Components. The effect of 17E6 ( v,o ) and P4C10 ( 3, O) on cell attachment to vitronectin (A), laminin (B) or collagen type I (C) was studied. Only on VN coated surfaces did 17E6 block cell attachment. Only on collagen type I and laminin did P4C10 block cell attachment.

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Horizontal axis: P4C10 dilution(1/x) (upper axis) and Mab ( g/ml) (lower axis); vertical axis: % cells bound. Figure 6.
17E6 Reverses Established Cell-Vitronectin Contacts. M21 melanoma were allowed to attach for 24h to surfaces coated with vitronectin (VN - upper row) or fibronectin (FN - lower row). The cells attached within 30min and were well spread, and proliferating by 24h (a,e) when 17E6 was added to the culture medium (b,f). After 30mins on vitronectin (b) cells rounded , while on fibronectin (f) they remained spread. LM609 (c, g) and 14E2 (d,h) had little effect on either substrate. Antibody concentrations:- (b): 0.1 ng ml-1. (c,d,f-h): 100 ng ml-1. Figure 7.
M21 human melanoma development in BALB/c nude mice is modulated by v- integrins. A.B. Effect of 17E6 on subcutaneous M21 development 0.5 x 106 M21 (A.) or M21-L (B) were incubated with buffer (O), 17E6 (o) or 14E2 (A) antibodies and injected subcutaneously into nude mice. With time, the tumor dimensions were measured and the volume plotted. A typical experiment is shown. Error bars show the s.e.m. from 8 animals per group. Vertical axis: tumor volume (mm3), horizontal axis: days post injection. C.Effect of v M21 experimental metastasis. 0.32 x 106 (?, ¦) or 1 x 106 (O, ?) of M21 (?, ?) or M21L (¦, ?) cells were injected into the tail vein of nude mice. At the time shown, groups of animals were killed and the lungs examined for tumor nodules. All the M21 mice were killed at 42 days. For the M21-L mice, 3-6 mice were sacraficed at each point. The tumor burden after 6 weeks in the M21 mice was too high to count (>>250 per lung), so T-statistics are shown for the hypothesis that an M21-L group is from

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the same population as the control M21 group at 42 days at the 17E6 is not cytotoxic. A Effect of 17E6 on M21 cell proliferation. 5 x 104 M21 were seeded in DMEM/FCS with carrier, (o), or in the presence of 50 g ml-1, 17E6 (o) or 14 E2 (A) antibodies and the cell numbers were counted daily. Kinetics and saturation density of proliferation are unaffected by the antibodies. Vertical axis: cell number; horizontal axis: days.
B. 17E6-dependent lysis of M21 cells by microglial cells. Thymidine labelled M21 cells were mixed with BALB/c-derived microglial brain macrophages, serially diluted antibodies were added, and following incubation, thymidine release was measured. Controls: (A) M21 cells alone; (?) M21 + 17E6 (100nM); (?) M21+14.18 G2a; (A) M21 + microglia. Mean + s.d. (n=6). Experimentafs: (o) M21 + microglia + 17E6; () M21 + microglia + 14.18 G2a. 17E6 did not induce antibody-dependent cell lysis. Mean + s.d (n=3). Vertical axis: cytotoxicity (%); horizontal axis: antibody concentration (M). C. 17E6-dependent cytostasis of M21 cells by microglial cells. M21 cells were incubated with microglial cells and serially diluted antibody, and then pulsed with [H3]- thymidine to measure DNA synthesis. Thymidine incorporation ([H3]-thy ) was measured. Symbols as in B.Note the cytostatic activity of the effector cells alone (A). Microscopical observation of the assay showed that in regions of homogeneous microglial cells at 14.18 G2a > 10-10 M, no M21 cells

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survived. Vertical axis: [H3]-thyidine incorporation (cpm x 10"3);
horizontal axis: antibody concentration (M).
Figure 9.
17E6 does not affect DNA synthesis by M21 cells.
Cell lines (A) M21; (B) M21-L; (C) M21-L4 ; (D) M21-LGpllb were
cultured, and buffer (o), or the antibodies 17E6 ( o), LM609 (A) or
14E2 (A) at the concentrations shown were added. In (B) and (D), the
routine positive control, taxol (0) is shown. After 48h, the ceils were
pulsed with [H3]- thymidine and the incorporated radioactivity was
measured. The antibodies had no effect on DNA synthesis. Taxol
completely suppresses it. cf. Fig.8: 90 g ml-1 Mab = 600nM.
Vertical axis: [H3]-thyidine incorporation (cpm x 10'3); horizontal axis:
Mab concentration ( g / ml).
Figure 10.
ELISA of 17E6 and fragments on purified v 3. intact 17E6 ( ) its
F(ab')2 (O) or F(ab') (?) fragments at the indicated concentrations
were allowed to bind plates coated at 1 mg ml-1 with v 3 and bound
antibody was detected. Vertival axis: optical density (OD) at 450 nm;
horizontal axis: antibody (log10 g/ml).
Figure 11.
Cell attachment mediated by v and v but not by v 3 is
blocked by 17E6 F(ab'). Cells as indicated were allowed to attach to
5 mg ml-1 coating of vitronectin in the presence of the indicated
concentrations of 17E6, its F(ab')2 or F(ab') fragments. 100%
attachment varied from -85% (WM164) to -50% (V+B2) of total cells
added. Vertical axis: % maximum cell attachment; horitontal axis:
antibody concentration ( g/ml).

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Figure 12.
M21 attachment to vitronectin is blocked by cross-linking 17E6 F(ab'). During M21 cell attachment to vitronectin (as Fig.4,11), 17E6 F(ab') fragments or intact AP3 antibody at 0 (o), 0.1 (V), 1.0 (Q), or 10 ^g n1~1 (A) were coincubated with the indicated concentrations of cross-linking anti-mouse second layer antibody. The cross linker showed identical ELISA affinities for 17E6 F(ab') and AP3, (not shown). Vertical axis: optical density (OD) at 405 nm; horizontal axis: Goat anti mouse antibody concentration ( g/ml). Figure 13.
Ligand active site mimetics and vitronectin compete with one another for binding to v 3 in direct competition assays. Increasing concentrations of vitronectin (V), or peptides GRGDSPK (O,#) or cRGDfV (?,) were coincubated with biotinylated vitronectin (1 g mh1 == 1.5 nM) or a biotinylated cRGDfV derivative (cRGDfK-bio: 50 nM) on v 3 coated plates. Bound biotin was detected with an anti-biotin antibody. Figure assumes a mean Mr for univalent multimeric vitronectin of 0.65 MDa. Vertical axis: % ligand bound; horizontal axis: peptide/ VN (log [(M]). Figure 14.
17E6 does not compete with vitronectin for binding to v 3 in direct competition assays. Increasing concentrations of 17E6 (o), other anti- v 3 antibodies ( ) , or ligand mimetic cRGDfV (?) were coincubated with biotinylated vitronectin (1 g ml-1) on v 3 coated plates. Bound biotin was detected with an anti-biotin antibody (cf. saturation profile under the same v 3 coating conditions for 17E6: Fig.10.). Vertical axis: % VN bound; horizontal axis: [Mab] g/ml -[cRGD] M.

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Figure 15.
Ligands and active site mimetics do not compete with antibodies for binding to v 3 in pre-block competition assays. Increasing concentrations of vitronectin (VN.O), fibrinogen (FG,V), fibronectin (FN, Q), cRGDfV (66203, A), or the indicated antibodies (?) were pre-incubated for 1h on v 3-coated plates and then probed by addition (no wash) with biotinylated antibody (1 g ml-1), as shown. Bound biotin was detected. An antibody bound value of 100% indicates that the antibody binding in the presence of the concentrations of challenger shown was as in the control without competitor. The ligands do not affect antibody binding. Vertical axis: % probe anribody bound; horizontal axis (left and right side): ligand concentration (jag/ml). Figure 16.
Antibodies do compete with one another for binding to v 3 in pre-block competition assays. Increasing concentrations of the antibodies 17E6 (o), LM609 (V), AP3 (?) or 14D9.F8 ( ), at the indicated concentrations, were pre-incubated for 1h on v 3-coated plates and then probed by addition (no wash) with biotinylated antibody (1 g ml-1), as shown in each panel. Bound biotin was detected. An antibody bound value of 100% indicates that the antibody binding in the presence of the concentrations of challenger shown was as in the control without competitor. The antibodies compete with one another in cross-competition groups. Vertical axis: % probe anribody bound; horizontal axis (left and right side): antibody concentration ( g/ml).

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Figure 17 a,b.:
cDNA sequence of Mab 17E6 variable (Fv) regions.
Light chain: VL17E6Heavy chain (a) and heavy chain VH17E6 (b).
The complete nucleotide and the deduced amino acid sequence of
variable regions (including leader sequence) are shown. The leader
sequences are annotated bold; the CDR sequences are in italic. The
heavy chain has the characteristic structure of group II B and the light
chain of kappa group V, acording to Kabat classification.
FIGURE 18.
Schematic representation of the cloning process.
mRNA encoding the Fv domains of the 17E6 antibody heavy and light
chains was extracted form 17E6 hybridoma cells, transcribed into
cDNA, and PCR was used to amplify out the variable regions. These
were then cloned and sequenced.
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DETAILED DESCRIPTION
The alpha-V group monoclonal antibodies react with integrin ccv-chain Antibody screening by ELISA on purified v 3 and llb 3 revealed five clones, 17E6, 20A9, 23G5, 14D9.F8, 10G2 which reacted specifically with v 3 (Tab.1). These Mabs are termed the alpha-V group'. All were lgG1 isotype. In the same ELISA assay, anti-integrin antibodies of known specificity against the v 3 complex (LM609), the av chains (LM142), the v complex (P5H9), the allbFs3 complex (CP8), the S3 chains (AP3), and the B1 chains (P4C10), reacted as predicted from the literature (Tab.1). In ELISA on fixed cells ('CELISA'), with cells expressing avS3 and avG5 (M21), avB5 but no avB3 (UCLAP3), neither avS3 nor avS5 (M21-L), and allb 3 (M21-L-llb), the alpha-V group showed a reaction pattern consistent with their recognition of the v-integrin chain, and clearly distinct from a reaction with B3, B5, B1, or other -chains (Tab.1). The results corroborated the ELISA data with purified receptors. Mabs with specificities for B3, and Gpllb were also obtained in the screen (data not shown), and these reacted in a way clearly discrete from the alpha-v group. 17E6, 14D9.F8, 20A9 and 23G5 bound v 3 with similar apparent affinity. 50% binding was achieved at -10-20 ng ml" 1 (~50-100pM - similar to LM609). 10G2 bound similar to LM142 with about 10 times lower affinity). CP8, against llb 3 and 14E2 (see below), showed minimal binding to v 3 at concentrations up to 100nM.

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The ability of the alpha-V group to recognize native av-integrins was tested by FACS (Fig.1; Tab.2) and by immunoprecipitation from surface labelled cells (Fig 2). In FACS analysis (Fig.1), the v-expressing line (M21) reacted strongly with 17E6, 14D9.F8, 20A9,
23G5, and with the v- defining antibodies LM142 and LM609, moderately with 10G2, and also with the control Mabs 14E2 and
21H6 and Mab 9.2.27. By contrast, av-deficient variant (M21-L) reacted weakly with the alpha-V group and with LM142 and LM609, but showed similar reactivity as M21 with 14E2, 21H6 and 9.2.27. M21-L has an intracellular pool of (S3 subunits which were detected in FACS only when the cells were permeabilized (Tab.2). In FACS analysis of M21-L4 ( v-retransfected M21-L cells (Felding-Habermann et al., 1992)), the alpha-V group gave reaction patterns as on M21. The control vector transfectants, M21-L12 and the Gpllb transfectants, M21-L-llb (Kieffer et al., 1991), showed no reactions with the alpha-V group (Tab.1). UCLAP-3 adenocarcinoma reacted with the alpha-V group, with LM142 and P5H9, but not with LM609. UCLAP-3 does not express 3 (see introduction) The melanoma WM793 had the same reaction pattern as M21. In immunoprecipitation screening of M21 cells, the alpha-V group gave the same immunoprecipitation patterns as LM142 (anti- v), and LM609 (anti v 3) (Fig. 2a). A strong broad band was seen at ~92kDa and a weaker band at ~145kDa, with weak accompanying bands at
-100kDa, a pattern characteristic of surface labelled v 3 and v 5 integrins(Wayner et al., 1991). When compared to the precipitation patterns on M21-L, none of the aipha-V group precipitated (data from 17E6 and 20A9 are shown), and neither did LM142 or LM609. (Fig.2c). -specific antibodies gave similar precipitation patterns from both cell lines. In M21-L, precipitation with anti-113 antibodies

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gave a band at ~92kDa, due to intracellular 3-Iabelled in permeable (possibly necrotic) cells. UCLAP3 (Fig. 2d) gave no precipitate with LM609, but a ~95kDa/145kDa complex was precipitated, by the alpha-V group and by LM142 (Fig. 26). In summary, ELISA, CELISA, FACS analyses and immunoprecipitations of gave consistent reaction patterns and strongly suggested that Mabs of the alpha-V group react with extracellular domains on human av-integrin chains. Mab 17E6 is a potent function blocking antibody. 17E6 can modify initial cell attachment to v-ligands: av-integrins can function as receptors for vitronectin, so the alpha-V group was screened for their possible effects on ceil attachment to vitronectin substrates. After integrin analysis by FACS, cells were tested in attachment assays (Tab.2, Fig.3). In FACS, human melanoma and carcinoma cell lines reacted similarly with the alpha-V group. The reaction with 17E6 is summarized (Fig.3). The initial attachment to vitronectin of cells reacting in FACS with 17E6 was strongly blocked by that antibody, but only weakly affected by the control antibody 14E2 (Fig.3.). Other members of the alpha-V group were less potent {data not shown). The vigorous attachment of murine cell B16F10 on vitronectin was not affected by 17E6 and B16F10 did not react with 17E6 in FACS. As predicted (Cheresh and Harper, 1987), B16F10 attachment to vitronectin was sensitive to micromolar concentrations of RGD-peptides, suggesting the presence of functional surface v 3 (SLG and B.Diefenbach). Thus, 17E6 and the alpha-V group reacted with human but not mouse v. The effect of 17E6 on cell attachment was investigated. 17E6 blocked M21 attachment (~85%%) to vitronectin with an IC50 of ~0.1 g ml-1 (Fig.4a). This invention confirms previous studies (Wayner et al., 1991) by showing that M21 attachment was poorly blocked by

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antibodies to v 3 (LM609) or v (P5H9) alone, but was strongly blocked when they were added together (Fig.4a,b). Anti- integrin antibodies (P4C10) had no effect on M21 attachment to vitronectin (Fig.4b). The lines UCLAP3 and WM793 attached to vitronectin and this attachment was blocked by 17E6, and also by the complementary v -specific (P5H9/UCLAP3) or v 3-specific antibodies (LM609/WM793) (Fig. 4c-f). On v (UCLAP3) 17E6 had an IC50 of -30 ng mM. On WM793, it was -60 ng ml"1 and for LM609 the IC50 was ~ 600 ng ml-1. UCLAP3 expresses v but no v 3 (Wayner et al., 1991), while WM793 expresses high levels of a v 3 (Tab.1). The blocking specificity of 17E6 was confirmed by its lack of effect on cell attachment to other matrix substrates (Fig.5) P4C10 (anti-G1) abolished M21 attachment to laminin and collagen. Cell adhesion to these two substrates can be mediated by -series integrins (Sonnenberg et al., 1988; Takada et al., 1987). Taken together with the biochemical data, these results are consistent with the theory that 17E6 bind the av chain of various integrin complexes and disturbs their interaction with their ligands. 17E6 triggers reversal of established cell matrix interactions mediated by v-integrins : It was next investigated whether 17E6 could affect established cell-matrix interactions. When 17E6 at low concentrations (-0.1 g ml-1) was added to M21 cells, it induced extensive cell rounding after 0.5-1h at 37°C in M21 cultures even after 24h of attachment (Fig.6). The effect was fully reversible and after wash out the cells returned to their spread state within 48h. By contrast, the antibodies LM609 and 14E2 affected morphology only slightly even at high concentrations (100 g mM) . The antibodies had no morphological effects even at 100 F g ml"1 on fibronectin coated substrates (Fig.6). M21-L4 (and other scells attached via v)

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were similarly affected by 17E6 on vitronectin surfaces, but not on fibronectin, collagen, or on laminin. 17E6 blocks M21 tumor development in nude mice The invention investigated the effect of the v-blocking antibody 17E6 on the subcutaneous development of M21 tumors in BALB/c nu/nu mice (Fig.7). In animal models, the development of M21 tumors in nude mice has been correlated with the cell surface expression of v-series integrins (see introduction). M21 cells were subcutaneously co-injected and endotoxin-free antibodies. 17E6 consistently (4/4 experiments) blocked the subcutaneous development of M21 tumors (Fig.7a). No tumors (0/32) have taken in the presence of 17E6, and the animals still remain tumor free- now in excess of 6 months. Control tumor take was 75-90%. Non-blocking antibodies against the av-chain itself and control antibodies against the melanoma cell surface showed variable and inconsistent effects on tumor development. In 14E2 treated controls, take of tumors was reduced depending on experiment 30-60%, but remaining tumors grew as the untreated controls and, like the controls, these animals had pulmonary micro-metastases revealed when the lungs were brought into tissue culture (not shown). By contrast, 17E6 treated animals had neither subcutaneous tumors nor metastases in lungs, liver, kidney, spleen, colon, stomach, nor in thoracic or abdominal body cavities when sacrificed at 6 months. The v 3-deficient line M21-L grew more slowly subcutaneously than M21, and was unaffected by 17E6. M21-L controls treated with 14E2 had a take reduced in comparison to untreated animals, similar to that seen in M21 cells (Fig.7b).
The growth of M21 and M21-L and the effect of the antibody 17E6 were compared in an 'experimental metastasis' tail-vein injection

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model. M21 formed many colonies in a dose dependent manner, while M21-L formed significantly fewer colonies, but did form lung nodules when injected at higher dosage (Fig. 7c). In other words, tumor growth in the lungs was also enhanced by the presence of cell surface v-integrins, and pre-incubation of M21 with 17E6 reduced (by 90%) the numbers of tumor colonies that formed. Interestingly, the level of tumor formation was similar to those acheived by M21-L cells in the same experiment. The antibody did not altering the numbers of animals in which the tumor grew (Tab.3). In summary, the presence of v at the cell surface promoted M21 tumor formation in both subcutaneous and experimental metastatic models, and the v-blocking and reversing antibody 17E6 vigorously suppressed the growth of M21. In an experimental metastasis model with M21 cells, the vigorous growth of M21 and the poor growth of M21-L closely follows the subcutaneous pattern of tumor growth and M21 growth was also suppressed by pretreatment with 17E6. This data strengthens the link between av-integrins and the development of human melanoma. Effects of 17E6 not due to cytotoxicity
After observing its potent effects on attachment, morphology, and tumor development, it was attempted to find out the mechanism of 17E6 action. 17E6 was tested for cytotoxicity (Fig.8). The kinetic of cell growth and the final saturation densities acheived were not greatly influenced by the presence of 17E6 or control antibody (Flg.8a).
Nude mice are immune deficient. Of the few cells immune competent cells which remain, macrophages are the most likely to direct a cytotoxic, antibody mediated response. To test the possibility that the antibodies were directing cellular cytotoxicity (ADCC), murine

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macrophages syngeneic to the antibodies were tested in ADCC against M21 cells. As effector cells murine brain macrophages (microglia) are especially potent mediators of ADCC (Sutter et al., 1991). The positive control, Mab 14.18 G2a caused nearly complete lysis of M21 at In order to evaluate if 17E6 might exert a cytostatic activity in the presence of effector cells, DNA synthesis of M21 microglial cocultures was measured in the presence of the antibodies (Fig. 8c). At 10-10M 14.18 G2a caused a >90% inhibition in [3H]-thymidine incorporation.
Having tested the effects on cell proliferation, ADCC and AECM, it was examined whether the levels of DNA synthesis in M21 cells were affected by the Mabs. 17E6, 14E2, and LM609 at 0.5 M had no effect on thymidine incorporation (Fig.9). DNA-synthesis in M21-L, M21-L4 and M21-L-Ilb cells were also unaffected by the antibodies. M21-L and M21-L-llb react neither with 17E6 nor LM609, but do react with 14E2 (Fig.1). Many other melanoma cell lines were also tested and their DNA synthesis was shown not to be obviously affected by the alpha-V group (not shown).
17E6 and 14E2 were lgG1 isotypes, and did not affect complement mediated lysis on M21 cells (not shown). It can be concluded that the effects of 17E6 are specific to av-integrins, and not to drastic toxic effects on other cellular systems.
17E6 action on cellular v 3 but not v or v requires receptor cross-linking: In many biological systems transmembrane signalling is initiated by dimerization of the receptors. Indeed, multimerization of v 3 alters phosphotyrosinalytion patterns in

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HUVECs (Bhattacharya et al. 1995). It was, therefore, investigated whether receptor aggregation was involved during 17E6 action. 17E6 inhibited cellular integrins v 3, v and v (Fig.4). Intact 17E6 and its F(ab')2 and F(ab') fragments had similar binding characteristics in ELISA on isolated v 3 (Fig.10), with 50% saturation being achieved at -1 ng ml-1 (~7pM intact Mab). The similar ELISA titration curves suggested monovalent interaction between 17E6 and the ELISA plates. But in cell attachment assays the fragments behaved differently. The F(ab')2 and intact 17E6 blocked cell attachment mediated by v (V+B2 cells) and v (UCLA-P3 cells) (IC50 -0.1 g ml-1 : ~700pM), and v 3 attachment (WM164 and M21 cells) (IC50 -0.5 g ml-1 : ~3nM), and the F(ab') fragment was as active as the dimeric antibodies on v and v (Fig.11). However, the F(ab') fragment was at least 1x104-fold more active on both v and v than on v 3, where it only blocked 25+10 % at the highest concentrations tested (50 g ml-1 :- IC50 " 50 g ml-1: " 400nM) - at these levels, irrelevant antibodies also produced similar degrees of blocking (not shown) suggesting this marginal effect to be non-specific.
A lack of biological activity of a F(ab') antibody fragment which is capable of binding (as 17E6 F(ab'): (Fig.10)) is a classic indicator that dimerization is necessary for antibody mediated function. To confirm that cross linking was indeed necessary for 17E6 action on v 3, 17E6 F(ab') was cross-linked during the cell attachment assay by addition of a polyclonal anti-F(ab') antibody. A classical prozone-like effect was observed where at critical concentrations of both primary and secondary antibodies, and only then, a strong inhibition of cell attachment was observed. High quantities of second layer antibody alone or of F(ab') alone did not affect cell adhesion. Furthermore, the

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effect was not the non-specific result of cross-linking v 3 integrins: cross linking of v 3 by the binding but non-inhibitory antibody AP3 had no effect on cell attachment, (Fig. 12). The concentrations of F(ab') active after addition of secondary antibody in the cross-link experiments, coincide with the IC50 of intact 17E6 on v 3-mediated attachment (cf. Fig. 11).
Together, these data indicate that cross linking of v 3 integrin is necessary to prevent WM164 and M21 attachment on the cognate ligands, while v and v do not require cross linking to be inactivated.
17E6 is a poor blocker of receptor function in isolated receptor assay:
The unusual nature of 17E6 function on v 3 was studied in detail in isolated receptor assays. The contrast between classical active site blockers and 17E6 are marked. Vitronectin binding to v 3 saturates at ~5 g mM (~10nM: assuming average vitronectin multimer size of 10 monomers) (Fig.13), and this binding is specifically competed with both classical linear ligand mimetic peptides (GRGDSPK: IC50 ~2 p.M) and cyclized peptides (cRGDfV: IC50 ~5 nM). By using a biotin labelled variant of cRGDfV, cRGDfK-biotin, it was possible to demonstrate directly that not only would the inhibitors compete for vitronectin binding but that vitronectin would compete for the inhibitor binding (Fig.13), ie. the system was symmetrical. Indeed, vitronectin, GRGDSPK and cRGDfV each competed for one another and did so both "rationally" and 'symmetrically', in that the IC50 for competition mirrored the IC50 for inhibition of vitronectin binding to the v 3 complex (Fig.13).
17E6 binding saturates v 3 at 0.01-0.1 g ml-1 (700-70 pM) (cf.Fig.10). However, the antibody is only a weak inhibitor of ligand

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binding- Even at 70nM antibody there was less than 30(+10)% inhibition of vitronectin binding. LM609 behaved in a similar way, while the avli3-binding non-inhibitory antibodies (14D9.F8, 20A9 and WAM2-4) were ineffective. The ligand mimetic cyclic peptide cRGDfV efficiently blocked receptor-ligand interaction (IC50 ~10nM) (Fig.14) but does not affect 17E6 binding. Neither intact nor monovalent 17E6 inhibited (not shown).
17E6 binds avlZ3 in the presence of saturating amounts of challenging ligand.
The weak competition of 17E6 for ligand in the isolated receptor assay and the availability of strong competitive inhibitors like cRGDfV raised the question of how 17E6 was affecting v-function. The blocking activity of 17E6 and its fragments in cellular attachment assays for v 3 integrin requires cross linking, behaviour not wholly compatible with an inhibition by competition. It was next investigated whether ligands of v 3 interfered directly with 17E6 binding. Ligands were pre-bound to the receptor, and low concentrations of directly labelled 17E6 were added (Fig.15). The binding of 17E6 was not affected (
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The ELISA data (Fig. 10) indicated that 17E6 - v 3 interactions were monovalent, which reduces the possibility that a competition artefact was observed due to very high antibody affinity combined with low ligand affinity. However, other v and 3-specific antibodies were labelled and used these to challenge pre-bound ligand (Fig. 15), including the antibodies themselves (Fig. 16). The antibodies successfully competed for one another and clearly fell into cross competition groups (Fig. 16). The 3-specific antibody AP3 competed its own binding but did not affect the binding of and was not affected by antibodies specific either for the v 3 complex (LM609) or the av-chain alone (17E6, 14D9.F8); LM609, 17E6 and 14D9.F8 all cross competed strongly. By contrast, the pre-bound ligands did not affect 17E6 binding to v 3. It must be concluded that 17E6 did not function by direct competition for the tigand binding site. Cloning and sequencing of 17E6 variable regions: A primer library designed for PCR amplification of immunoglobulin variable regions was used to clone immunoglobulin enriched cDNA libraries for the heavy and light chain regions of the hybridoma 17E6. The redundant variable region primers terminated at the start of the leader. The constant region reverse primers terminated 30bp into the constant region. The primers were designed with terminal Sal I or Xma I restriction sites for cloning. PCR products of the expected size (420-450bp (Jones and Bendig, 1991)) were obtained, cloned and sequenced (Fig. 17). The 17E6 immunoglobulin light chain variable region, VL17E6, was 381 bp in length. The heavy chain variable region, VH17E6, was 411bp in length. The heavy chain variable sequences were characteristic of Kabat group lib immunoglobulins and the light chain sequences characteristic of Kabat group V (Kabat et al., 1987). The cloning strategy is shown schematically (Fig. 18).

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Tumor progression and metastasis is classically a disease where cells escape normal growth and adhesion controls and invade, migrate, attach and grow at an inappropriate site. Integrins are now known to control many cell adhesion events, and adhesion can in turn regulate mechanistically interwoven events including growth, differentiation, cell movement and the activity of protease networks, developmental events which are reiterated in the metastatic cascade (Liotta et al., 1991; Stetler Stevenson et al., 1993; Fidler, 1988). In this study antibodies are described directed against human aV-series integrins one of which, 17E6, perturbs initial cell attachment, disrupts stable v-ligand interactions and interferes with human melanoma development in an in vivo animal model (Fidler, 1986). In biochemical analyses the alpha-V group antibodies showed reaction patterns closely related to LM142- a well defined antibody to human v - but distinct from the reaction patterns of v 3-specific (LM609), v -specific (P5H9) and from other defined anti-integrin antibodies. Thus, the alpha-V group antibodies are likely to recognize the human aV-integrin chain. cDNA cloning of the 17E6 immunoglobulin transcripts revealed unique, unambiguous sequences. The heavy chain variable sequences were characteristic of Kabat group llb immunoglobulins and the light chain sequences characteristic of Kabat group V (Kabat et al., 1987). Thus, the antibody is uniquely defined.
17E6, strongly perturbed cell interactions mediated by aV. Antibodies that perturb function have been vital to our understanding of integrin function, but the aV-field has lacked a potent class-specific blocking antibody. 17E6 blocks aV-mediated cell attachment with an IC50 of -0.3-1 nM (antibody MW 150,000); by comparison, the

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peptidic blocker, GRGDSPK, has an IC50 of ~5 M. Cell lines expressing mainly v 3 (WM793) or v (UCLAP3) are blocked by 17E6, as are cells expressing mainly v . Thus, 17E6 is a general inhibitor of v-integrins.
Not only does 17E6 prevent the initial interaction of v-integrins with their ligands, but it also causes reversal of well established interactions, causing rapid retraction of cells attached on vitronectin. The interactions of v 3 with vitronectin has been described as 'non-dissociable' , proceeding in two steps, with an initial blockable interaction being followed rapidly by a stabilization reaction (Orlando and Cheresh, 1991). By contrast, 17E6 rapidly caused retraction and partial detachment of M21 and many other cells (unpublished observations) from vitronectin substrates. The rounding reversed when the antibody was removed. M21 initial attachment on vitronectin is incompletely blocked (-90%) by 17E6, but its rounding effect affects essentially all the attached cells. Previous studies could be confirmed where M21 initial attachment to vitronectin was fully blocked by mixtures of anti - v 3 and anti- v antibodies, and by RGDS-peptides, implying that both v 3 and v 5 were involved (Wayner et al., 1991). As shown in this study, 17E6 blocks several a v-receptors and can reverse stable interactions mediated by them. The difference in activity of 17E6 on attached and attaching M21 cells may lie in different function and localization of v 3 and v in each situation. On attaching ceils both are diffusely distributed, while in attached cells v 3 is found in focal contacts and v is diffusely distributed (Wayner et al., 1991; Zambruno et al., 1993). It was further investigated whether 17E6 selectively disrupts one of these contacts.

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Growth of M21 tumors in nude mice depends strongly on aV-integrins (Felding-Habermann et al., 1992; Sanders et al., 1992). As 17E6 could modulate stable aV-ligand interactions and had a long term effect its effect on tumor development was examined. It was found that 17E6 blocked the development of subcutaneous M21 tumors in nude mice, thus strongly supporting the studies of Felding-Habermann et al. In addition, it could be shown that v also promoted, and 17E6 inhibited, the development of M21 as experimental lung metastases. This invention has thus independently confirmed the important earlier study, and extended it by using syngeneic antibody-mediated 'therapy' with 17E6. These results emphasize the importance of aV-integrins in the development of the M21 tumor, and eliminate the possibility that the earlier results arose as selection artefacts of cloning.
In vitro 17E6 effects continued for > 24h following wash-out. Given a mouse volume of 20ml, the initial antibody concentration in vivo was -lOnlvl, an order of magnitude excess over the IC50 for reversing cell-ligand interactions in culture. 17E6 is syngeneic for the treated animals, BALB/C nu/nu, and the half life of murine lgG1 is 20-200h (Tao and Morrison, 1989; Haba et al., 1985). Thus, the IC50 for reversing M21-ligand interaction could be exceeded for at least 100h in this model (5 half-lives). It is interesting to compare 17E6 with RGD-peptides. In the B16F10-C57blk6 murine melanoma model coinjected peptides inhibited the development of B16-F10 pulmonary tumors (Humphries et al., 1986; Hardan et al., 1993). With the same assumptions as for 17E6, ~100mM RGD-peptide was present (Hardan et al., 1993), some two orders of magnitude over the dose required to block cell attachment to vitronectin. However, RGDS has a serum half-life of 8min (Humphries et al., 1988). As a general

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therapeutic goal, it might be preferable to generate long lived blockers for suppressing tumor development.
How might 17E6 affect tumor development? As it does not react with murine cells, the effect is on the tumor cells and obvious effects of the antibody on tumor angiogenesis can be excluded (Brooks et al., 1994). The only effects of 17E6 which can be identified are a) its binding to the extracellular domain of v-integrins, b) its ability to perturb aV-mediated cellular interactions. It can be assumed that most sources of antibody-mediated killing available to a nude mouse have been eliminated. 17E6 did not chronically or acutely affect M21 cell growth and viability, did not mediate complement fixation, did not affect DNA synthesis, and did not mediate syngeneic macrophage mediated cytotoxicity. M21 cells were sensitive to ADCC for they were effectively killed by microglial cells in the presence of Mab 14.18 G2a. The lgG1 MAb's (like 17E6) effectivelymediate murine macrophage ADCC (Herlyn et al., 1985). The number of v 3 sites expressed per cell may be below the critical threshold for ADCC to occur. Previous studies have shown that the effectiveness of ADCC correlates with the target antigen density (Rodeck et al., 1985) Macrophages in contrast to other myeloid or lymphocytic effector cells express all three classes of Fey receptors. The data argues against ADCC as the mechanism explaining the inhibition of tumor growth observed in vivo with 17E6.. The antibody was endotoxin free. It cannot be excluded that NK-cell mediated killing is activated by 17E6, but if so it is far more weakly activated by the syngeneic isotype-matched control antibodies which were used. Thus, 17E6 probably acts by impairing the function of av-integrins. Functions which 17E6 might block and where av might participate, include cell adhesion, interaction with soluble matrix components

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(Seftor et al., 1992), modulation of a protease/protease inhibitor network (Gehlsen et al., 1992; de Boer et al., 1993; Preissner, 1991), stimulating cell movement (Seftor et al., 1992; Gehlsen et al., 1992), or affecting receptor internalization (Wickham et al., 1993; Panetti and McKeown Longo, 1993a; Panetti and McKeown Longo, 1993b), but which if any is involved remains a matter for further experimentation.
The blocking effect of 17E6 on v 3 but not on v and v requires dimerization of the target. Although monovalent and divalent 17E6 antibody fragments are equally active in binding v integrin in ELISA, and in blocking cell adhesion mediated by v and v 5, the monovalent fragments are essentially inactive against v . Furthermore, when cross-linking antibodies are added to the monovalent fragments, they regain the ability to block v mediated adhesion and show a classic prozone effect in doing so. Simple cross-linking of v is not sufficient to block activity, however, because cross-linking AP3 has no effect on v -mediated adhesion: AP3 binds the chain (cf. Fig.2B:e).
A transition from mpnovalent to bivalent interaction causes a sharp increase in the affinity of an antibody for its target (Lane and Harlow, 1988}. ELISA titration of 17E6 on isolated v fails to show this transition, and therefore indicates that a monovalent interaction with both monovalent and divalent elements of 17E6 occurs in this configuration. In ligand-binding receptor assays that use the same ELISA configuration, 17E6 is a suprisingly poor blocking reagent, in comparison to peptidic ligand mimetics like EMD66203: cRGDfV. To put this into context: whereas cRGDfV gains some 2-3 orders of magnitude in activity (IC50 from -1 mM to ~ 1nM) in transfer from cellular to isolated receptor assay, 17E6 looses at least 3 orders of

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magnitude apparent activity (IC50 from -100 ng ml"1 to >100 mg ml"1), and becomes essentially inactive.
This anomaly, and the relevance of the cellular antibody cross linking data was resolved by competition experiments. These indicated that 17E6, in contrast to the peptidic blockers, did not interfere directly with the ligand binding sites. In the absence of direct evidence, it is usual to assume that a blocking reagent functions by steric occlusion of the ligand-receptor interaction site, a view fertilized in the integrin field by ligand active site-mimetics such as RGD-containing peptides (Hynes, 1992), and the ligand inactivation caused by directed mutagenesis of this site in vitronectin (Chemey et al., 1993). Our data indicates strongly that for the potent blocking antibody 17E6 this is not so. In summary: at the v integrin a) ligand mimetic peptides compete for one another and for the ligands themselves b) the ligands compete for the peptides c) neither peptides not ligands compete for 17E6 binding d) 17E6 does not compete for ligand or ligand mimetic binding e) 17E6 is fully competable by itself and by other v-binding antibodies.
17E6 is an extremely poor competitor for ligand binding to v while vitronectin and RGD-based active site probes compete strongly for one another at the receptor. And other antibodies compete strongly for 17E6. Thus, 17E6 acts as an allosteric inhibitor. Cross-linking experiment reveal that v , but not v or v 5, needs to be dimerized before 17E6 can block. This discovery reveals that 17E6 functions by a novel mechanism involving a manipulation of integrin-induced signal transduction pathways, rather than simply antagonising integrin-ligand interactions.
As v ligands are homopolymers in their active form and inactive as monomers (Preissner, KT. 1991; Stockmann, A. et al. 1993) the

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multimerization of integrins by the substrate may be essential to their function, indeed, dimerization of receptor tyrosine kinases of the growth factor receptor family by ligands are the key event in initiating signal transduction (Dougall.WC. et al., 1994). It is intuitively obvious that the stereochemistry of the substrate polymer becomes paramount to orientate the integrins in the membrane and ordain the formation of a signalling complex. Indeed the biological 'logic' of multimeric vitronectin for cell adhesion may arise from this constraint. Vitronectin multimers are from 3 to 16 units in size (Stockmann, A. et al. 1993) and can be predicted to produce ordered arrays of integrins at the cell surface and the necessary enhanced affinity by multimeric interaction. The individual lifetime of a monomeric vitronectin- v interaction is short, and the affinity low, while the rotation of the unligated integrin in the plane of the membrane is rapid, and antibody affinity and on-rate are high. Thus, it can be predicted that divalent antibodies associated with one integrin at the cell surface may capture dissociated integrins during rotation and trap them in a conformation that cannot bind ligand. This causes a progressive weakening of the molecular-array of v and vitronectin linkages and destabilizes the cell-vitronectin interaction - as if by unpeeling a molecular Velcro. This may explain the lack of function of 17E6 in receptor assays, where the receptor is cannot rotate into the necessary orientation to allow divalent binding (evidenced by ELISA data), and its efficacy in the cellular assay, explain the observed lack of necessity for active-site competition in the cellular assay, and explain the inefficiency of the F(ab') fragment in blocking v -mediated cell adhesion.
It is also implicitly obvious that, as v 5 and v 1 (and v 6, v 8 and v 1) share an overlapping ligand spectrum with v 3 it is

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conceivable that they cross regulate each others function, in a manner directly analogous to the cross regulation by hetero-dimerization of receptors in the RTK growth factor receptor family (Dougall, WC. et al., 1994). For example, v 5, having a higher affinity than v for vitronectin, could compete for v binding sites on individual multimers of this substrate - shutting down the homo-dimers of c needed to generate signal, or sustain cell adhesion, replacing them with v - v hetero-dimers. The other v-integrins may function in a similar cross regulatory fashion, but firm data to confirm these hypotheses is lacking.
In conclusion, a suite of monoclonal antibodies against the human aV-integrin chain was developed. One, 17E6, both blocks and reverses V-mediated processes in vivo. Interestingly, it also blocks aV-dependent melanoma development in nude mice. This suggests that aV-integrins may be effective candidates for tumor therapy. While this manuscript was in preparation, Lehmann et al. (Lehmann et al., 1994) described a aV-specific antibodies with some properties related to 17E6. It will be of interest to see if it can also reverse av-mediated interactions and perturb melanoma development.
THERAPEUTIC AND DIAGNOSTIC USE
The antibody according to the invention can be administered to human patients for therapy. Therefore, it is an object of the invention to provide a pharmaceutical formulation comprising as active ingredient at least one antibody or antibody fragment as defined above and in the claims, associated with one or more pharmaceutically acceptable carrier, excipient or diluent therefore. Typically the antibody of this invention will be injected intravenously or parenterally. Generally, the dosage ranges for the administration of

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the antibody (or fragments thereof) are large enough to produce the desired tumor suppressing and tumor lysing effect. The dosage will depend on age, condition, sex and extent of the disease in the patient and can vary from 0.1 mg/kg to 200 mg/kg, preferably from 0.1 mg/kg to 100 mg/kg/dose in one or more doses administered daily, for one or several days.
Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycoi, vegetable oils such as olive oils, and injectable organic esters such as ethyl oleate and other solvents known in the art which are suitable for these purposes. The antibodies of this invention can be used in a composition comprising a physiologically acceptable carrier. Examples of such suitable carriers are saline, PBS, Ringer's solution, or lactated Ringer's solution. Preservatives and other additives such as antibiotics, antioxidants, and chelating agents mayjalso be present in the pharmaceutical formulations.
The antibody (or a fragment thereof) can also be conjugated according to known methods to cytokines such as IL-2 in order to support their cytotoxicity.
The pharmaceutical formulations of the present invention are suitable for the treatment of all kinds of tumors, including melanomas,gliomas and carcinomas, as well as tumors of the circulating system and solid tumors.
For diagnostic purposes the antibody can be conjugated, for example, to a radio-opaque dye or can be radiolabelled. A preferred labelling method is the lodogen method. Preferably the antibody will be administered as or scFv fragments for diagnostic purposes. This provides superior results so that backround substraction is unnecessary.

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Table-1. Reaction Pattern of MAbs in ELISAs and CELISAs.


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Tab/e 2. Summary of Monoclonal Antibody Reactivity in Flow Cytometry.

Table 3. Inhibition of development of M21 tumor foci by 17E6 Mab in BALB/C nu/nu mice lung colonization 'experimental metastasis'assay.


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EXAMPLES
Example 1: Materials
Animals Mice for antibody production (female BALB/c; 8 weeks old) and for tumor models ("nude mice": female homozygotic athymic BALB/c nu/nu; 4-5 weeks old) were from Criffa (Barcelona, Spain). Nude mice were maintained in a sterile room in micro-isolator cages, and were given sterilized food and water ad libitum. All manipulations were performed in a laminar flow hood.
Proteins: Fibronectin (Ruoslahti et al., 1982), vitronectin (Yatohgo et al., 1988) were purified from fresh frozen human plasma, and fibrinogen (Kazal et al., 1963) from whole blood. Laminin was purified from Engelbreth-Holm-Swarm murine tumors (Paulsson et al., 1987). Where not otherwise stated, all manipulations were at 20°C, and all washings were with calcium-magnesium free PBS ("PBS": 137mM NaCI, 2.8mM KCI, 8.1 mM Na2HPO4, 1.5mM KH2PO4; pH7.4). PBS++ is PBS with added 1mM MgCl2 and 1mM CaCl2. Where not specifically stated, chemicals (Merck KGaA, Darmstadt) were of highest available purity. Cyclic peptides like cRGDfV and cRGDfK were synthesized according to known standard techniques (e.g. FEBS Let. 291, p. 50-54, 1991). Linear peptide GRGDSPK which is used as comparison compound is commercially available (e.g. by Bachem, Switzerland).
Tumor cell lines and cultures- American Type Culture Collection (ATCC) supplied SW1116, HT29 human carcinomas and A375 human melanoma and the following human cell lines were the generous gift of colleagues: M21 melanoma, its variants M21-L and M21-L4 (Cheresh and Spiro, 1987), M21-L-llb (prepared according to (Kieffer et al., 1991)), and the UCLA-P3 human lung adenocarcinoma

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(Cheresh et al., 1989) (Dr. D.A. Cheresh; Scripps), WM793 and WM164 melanoma (Dr. M. Herlyn; Wistar) (Herlyn et ai., 1990). NP18, pancreatic carcinoma (Dr.G.Cappella; Hospital Sant Pau; Barcelona). B16F10 murine melanoma originally from Dr. I.Fidler (Poste et al., 1980) (Dr.S.AIino; University of Valencia). EMM31 was established in our group from a tumor specimen defined by standard histological criteria as a primary melanoma (Jaggle et al. unpublished observations). All cells were cultured at 37°C in 7.5% CO2 92.5% air in 90% RPMI 1640, 10% fetal calf serum (FCS) plus 2mM L-giutamine, and were consistently free of mycopiasma as evaluated by a proprietary test (Mycotect Kit; Gibco).
Antibodies- Monoclonal antibody (MAb) fusions, ELISA screening, subcloning and maintenance of cultures were alt performed using standard technologies (Harlow and Lane, 1988) unless otherwise specified. Example 2:
Immunization MAbs against the v were produced by intraperitoneal (ip) injection of purified piacental v immobilized on Sepharose (80 ng v on 80 l Sepharose in 200 l PBS) or of live M21 cells (1x106 cells in 0.5ml PBS) every two weeks over twelve weeks. Four days after the last injection, PEG-induced fusion was performed using Friendly Myeloma (Ventrex) as partner. Antibodies to a 200kDa melanoma-associated surface protein were produced by immunising intact M21 cells (1x106 cells in 0.5ml PBS). Screening ELISA on receptors and on fixed M21 cells were used. For receptor ELISA, 96-well ELISA plates (Dynatech) were coated with purified v (1 g/ml in PBS, 16h;4°C), blocked (1.5% skimmed milk in PBS; 1h;4 °C) and incubated with hybridoma supernatants. Bound immunoglobulins were detected with alkaline-phosphatase conjugate

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anti-mouse Ig (Dako) using p-nitrophenyl-phosphate as substrate. For cellular ELISA, M21 or M21-L, M21-LlIb or UCLAP-3 cells on 96-well tissue culture plates were fixed (4% paraformaldehyde in PBS, 15 min. 20°C) and blocked (3% BSA, PBS; 1h;4 °C) before incubation with hybridoma supernatants and detection as in receptor ELISA. Positive hybrids were subcloned three times by limiting dilution and adapted to RPMI medium. Immunoglobulin isotype was determined using subclass-specific heavy-chain antibodies (Zymed) or light-chains antibodies (Promega).
Other murine MAbs used in the studies were the gift of our colleagues LM609 to v and LM142 to v (Cheresh and Spiro, 1987) (Dr. D.A.Cheresh ; Scripps), P4C10 to R1 integrin (Carter et al., 1990) (Telios) and P5H9 to v integrin complex (Wayner et al., 1991) (Dr.E.Wayner University of Minnesota), CP8 to ctllbfc3 complex (Dr. Ruggieri; Scripps), AP3 to the S3 chain (Furihata et al., 1987) (ATCC), 9.2.27 against a melanoma cell surface proteoglycan (Harel et al., 1990) (Dr.R.Reisfeld; Scripps) and 14.18 G2a against ganglioside GD2 (Mujoo et al., 1989). Example 3:
Antibody Purification and Scaling Up- For large scale purification, antibody supernatants were harvested from exponential phase cultures grown in roller bottles. The antibodies purified on protein A-Sepharose CL-4B (Pharmacia) were dialysed against PBS before sterile filtration (0.45 jim) and storage at -70 °C (Harlow and Lane, 1988). Purified antibodies were freed of endotoxins by passage over Kurimover-ll columns (Kurita-Vater; Tokyo). This reduced the endotoxin levels from >250 IU endotoxin mg-1 antibody to
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techniques of pepsin clevage and separation on protein-A columns (Pharmacia), followed by papain clevage and separation by gel filtration (Harlow and Lane, 1988). a.vH3 JHJdJlllbQ.3 intearin purifications
avG3 was purified from human placenta (Smith and Cheresh, 1988). Term placenta was minced and washed in ~2vols ice cold solution A (0.05% w/v digitonin, 2mM CaCl2,2mM PMSF, pH7.4), then filtered . The retained material was extracted in ~4vols ice cold buffer B (100mM octyl-S-D-glucopyranoside [OG], 1mM CaCl2( 2mM PMSF, in PBS ) and centrifuged (12000gmax, 45min; 4 °C). The supernatant was re-circulated over an LM609 antibody column (16h;4 °C). After washing with buffer C (0.1% NP-40 in PBS; -10 cv) and buffer D (0.1% NP-40, 2mM CaCl2, 10mM Na-acetate; pH 4.5 :~10cv), bound material was eluted with buffer E (buffer D adjusted to pH 3.1). The eluant was neutralized with 3M Tris (pH 8.8), dialysed against buffer C, and concentrated ~10x using Aquacide II (Calbiochem). The purified receptor was stored at -70 °C.
anbfc3 was prepared from human platelets (Pytela et al., 1986). Outdated platelet concentrates were mixed with one volume of Tyrodes buffer, pelleted (1200gmax)and the pellet extracted (1h;20 ° C) with lysis buffer (50mM OG, 1mM MgCl2, 1mM CaCfc, 1^M MnCl2, 2mM PMSF, 150mM NaCI, 50mM Tris-HCI; pH 7.4). After centrifugation (32000gmax , 30min; 4 °C) the supernatant was re-circulated (16h; 4 °C) over a GRGDSPK-conjugated CL-4B Sepharose column. The column was washed with lysis buffer (~10cv) and eluted with the GRGDSPK (3mg ml'1 in 90% lysis buffer, 10% DMSO). The peak was concentrated -5-fold , dialysed against modified lysis buffer (0.1% NP-40 substituted for OG) and stored at -70°C. The integrin preparations were - 95% pure as judged by anti-

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integrin EL1SA using a-and li-chain specific monoclonal antibodies and by SDS-PAGE. Example 4:
Surface labelling and immune-characterizations Cell surface biotinylation and extraction - Cells in exponential growth were harvested with EDTA, washed and 5x106 cells in PBS (1ml) were surface-labelled with biotin-N-hydroxysuccinimido ester (10 n g/ml; Sigma) on an end-over-end rotator (2h; 20°C), washed, and 5x106 cells per millilitre were lysed for 1h at 20°C in extraction buffer (100mM OG 2mM CaCl2, 1mM MgCl2 and 1mM PMSF in PBS). After centrifugation (12000g, 20 min) the supernatant was used for immunoprecipitation.
Immune-precipitation: Biotinylated cell extracts were immuno-precipitated with purified anti-integrin MAbs coupled to Affigel-10 beads (Biorad), or bound to protein-G Sepharose (Pharmacia). 10 mg coupled MAb was incubated with 5E6 cell equivalents of biotinylated cell extracts overnight at 4 °C. Washed beads were boiled in non-reducing SDS-sample buffer, centrifuged and resolved on 7.5% SDS-PAGE . After electrophoretic transfer to nitrocellulose (Towbin et al., 1979), bands were visualized with goat anti-biotin alkaline phosphatase conjugate (Dako) using NBT-BCIP (Biorad) as substrate.
Flow Cytometry: Cells were harvested with EDTA, washed, and 10° cells in PBS-1% BSA were incubated with MAb (10u.g ml"1I20 min, 4 °C). After washing and labelling (20 min, 4°C) with goat anti-mouse lg-FITC(Becton Dickinson), cells were incubated with propidium iodide before flow cytometry analysis (EPICS Profile II, Coulter). Living cells were selected by appropriate gates on the side and forward scatter. Fluorescein was excited at 488nm with an argon ion

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laser and the emitted fluorescence (525nm) was recorded. In some
experiments, M21-L cells were stained following a brief fixation and
permeabilization step (70% ethanol; 5min x -20°C).
Complement dependent cvtotoxicitv (CDC): M21 cells (10,000) were
plated in a 96-weII plate with 50 ^l of complete medium and 20 yA of
antibodies. Rabbit serum (50 \±\, 1:5; Ben ring) was added as source
of complement and plate was incubated (37°C; 60 min). Lysis was
quantified by MTT technique (Mosmann, 1983). Percent of lysis was
calculated using wells with 1% Tween-20 as 100% lysed controls,
and without antibody as 0% lysed control.
Example 5:+
Cell Growth, Viability and Activation-
Proliferation assays: In order to test chronic antibody effects, 109
cells were incubated in presence of MAbs (70 g mM PBS; 1h; 20°
C) on an end-over-end rotator, washed, then resuspended and further
cultured in RPMI- medium. Growth and viability were estimated by
Trypan blue dye exclusion. Activation was measured by the MTT
assay as described above, and cell numbers were counted dailey
using a cell counter (Coulter electronics).
Adherent melanoma cells were detached (0.05% Trypsin/0.02%
EDTA). Washed cells were seeded into 96-weII flat-bottom microtest
plates (1 x 104 cells per well) and cultured without (control) or in the
presence of serially diluted antibodies in RPMi medium with 10%
FCS. After 48h, cells were pulse labelled for 18h (18.5 kBeq [3H]-
thymidine per well ) and harvested. Incorporated radioactivity was
measured and expressed as counts per minute.
Example 6:
Antibody-directed cellular cvtotoxicitv assays (ADCC): Preparation of
BALB/C microglial cells and conditions for ADCC and antibody-

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effector cell mediated cylostasis (AECM) were essentially as desribed (Sutter et al., 1991).. M21 cells were pulsed for 18h with [3H] -thymidine (18kBq per 4x103cells per well) and incubated on 96-well plates with BALB/C microglia (5x104cells per well) in 200^1 DMEM/FCS (10%) in the presence of antibodies. After 48 hrs [3H] label retained in the nuclear compartment of target cells was measureds. MAb dependent cytotoxicity was calculated using the formula : [(experimental ccpm- spontanous ccpm) / (maximum ccpm-spontanous ccpm)] x100 Example 7:
Antibody and effector cell dependent cytostasis (AECM) : As for ADCC, but instead of pulsed cells, freshly passaged unlabeled M21 cells were used. After 24 hrs the effector-target cell cocultures were pulsed with [3H]-thymidine (18kBq per well) for an additional 24hrs and the incorporated nuclear [3H label measured. Cell Attachment Assays were as previously described (Goodman et al., 1991), using hexosaminidase activity (Landegren, 1984) to detect attached cells. Dilutions and cell suspensions were in attachment buffer (RPMI, 1% BSA, 25mM HEPES; pH7.4). Matrix proteins were coated onto 96- or 48-well plates, the wells were blocked with BSA and serially diluted MAbs were added to the wells followed by cells (2.5x104 - 5x104). After 1h at 37°C, non-adherent cells were washed away and attached cells counted against a standard curve run in parallel. Inhibition of attachment was calculated by using wells with no MAbs as reference. Typically, over 70% of added cells had attached to vitronectin at 1h. Cell attachment to wells coated with BSA alone was routinely less than 5% of specifically attached cells. Surface crosslink assay. As in the cell attachment assay, cells were allowed to attach to matrix-coated plates in the presence of serially

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diluted 17E6 or its F(ab')2 or F:(ab') fragments, and increasing amount of goat-anti-mouse-F(ab') as cross-linking reagent. Washing and detection of attached cells was as for the normal cell attachment assay.
Attachment reversal assay: Cells were plated in attachment buffer on
wells coated and blocked as for cell attachment assays, and
incubated at 37°C. After spreading (60-75min), serially diluted MAbs
were added and the cells returned to the incubator. Alternatively, the
cells were allowed to attach for 24h before addition of antibodies.
After 2-3h in the presence of antibody, the supematants were
cautiously removed and replaced 5 times by fresh, pre-warmed
attachment buffer - resulting in a final dilution factor of > 1x105.
In vivo tumor development: Tumor cells in exponential growth were
harvested with EDTA, washed and examined for viability by trypan
blue dye exclusion. Viability was between 96-99%. For primary tumor
growth, cells (0.5x106 cells in 0.2 ml PBS++) were injected
subcutaneously into the flanks of nude mice. Tumor growth was
followed by measuring tumor diameters with callipers and the tumor
volume was calculated using an approximated formula for a prolate
ellipsoid volume = [(a*b2)/2]
where a is the longest axis of the tumor and b the shortest. A minimum of eight animals was used per group. For experimental lung metastasis, cells were harvested (0.05% Trypsin/0.02% EDTA) and were injected into the tail vein of nude mice (0.5x106 cells in 0.2 ml PBS++). 7 weeks later the animals were sacrificed, the lungs removed and fixed in a Bouins* solution, and the tumor foci on the surface of the lungs counted. For antibody treatment, harvested and washed cells were incubated with purified endotoxin free MAbs (70 ng per 106 cells 0.5ml PBS++) for 30 min at

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20°C in an end-over-end rotator before dilution to 0.5x106 cells in 0.2ml PBS and injection. Cells viability was assessed by Trypan blue dye exclusion before and after completing the injection schedule, where no significant difference was found (viability pre-injection = viability post injection +_5%). The tumor inhibition data was assessed using the 2-tailed Student T-test. Example 8:
Molecular Biological techniques
Unless otherwise stated, all molecular biological techniques were as previously described in detail (Sambrook et al., 1989). RNA and cDNA preparation:. Total RNA was isolated from 17E6 cells by guanidinium thiocyanate extraction and the RNA was isolated by caesium chloride density gradient ultracentrifugation (Chirgwin et al., 1979). First-stranded cDNA was synthesized using a commercial kit (Pharmacia). The synthesis was done in a volume of 15 \x\ with 5 ^g of RNA, for 1h at 37°C. First strand cDNA was used directly for PCR amplification.
PCR amplification: The mouse light chain variable and heavy chain variable regions were amplified using a redundant and semi-degenerate sets of PCR primers designed to hybridize to murine Ig leader sequences (Jones and Bendig, 1991). A mixture of 61 oligonucleotides served as 5' primers or the heavy chain variable domain, and a mixture of 405 oligonucleotides served as 5' primers for the kappa chain variable domain. 3' primers were designed to anneal in the constant region: specific lgG1 and kappa mouse primers were used.
For amplification, with a thermostable DNA polymerase, 25 reaction mixture containing: 1 t of the cDNA-RNA hybrid, 250 nM of the appropriate 5* and 3' primers mixture, 200J.LM of each dNTP, 1mM

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MgCl2 (for the light chain), 2mM MgCl2 (for the heavy chain), and 1 U of Taq polymerase (Cetus), was overlaid with mineral oil and subjected to a hot start at 60oC to the 55°C annealing temperature. After 30 cycles (1"x55°C: 1.5'x 72°C: 45s x 92°C), one tenth of the PCR reaction was run on a 1% agarose TAE gel electrophoresis and ethidium bromide-stained to visualize the resulting PCR products. Molecular cloning and sequencing: 1jal of each PCR-product was ligated into TA vector (Invitrogen, San Diego). The two DNA ligation reactions, for heavy and light chain variable regions, were transformed by heat-shock into competent E.coli strain TG1 cells to create two DNA libraries, one light variable region of 17E6 Mab and the other one the heavy variable region of 17E6 Mab. Colonies were selected on LB plates with lOu^g/ml carbenicilin and picked for further analysis. Positive colonies were detected by PCR screening or using plasmid digestion (Gussow and Clackson, 1989). Double-stranded plasmid DNA was prepared (Wizard preps, Promega Corp.) for sequencing from transformants. Thermal cycle sequencing using the dideoxynucleotide chain termination method (Sanger et al., 1977) was carried out using Taq polymerase. Up and down-stream sequencing primers complementary for the TA vector were used. Intearin lioand binding and competition assays : Biotinylation of ligands Ligands in PBS were diluted with 5-fold concentrated ligation buffer (end concentrations: 1 mg mM protean, 100mM NaCI. 100mM NaHCO3 pH8.2) in an end volume of 1mL Freshly prepared N-hydroxysuccinimido biotin solution (100 ^l; 1 mg mM in DMSO) was added with mixing and the reaction continued for 2h at 20 °C with end-over -end rotation. After exhaustive dialysis at (4°C: PBS, 0.05% NaN3), the protein concentration was assessed and the biotinylated ligand was stored at 4°C and used within 21 days. The synthesis of

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the biotinylated peptide cRGDfK (cyclic-Arg-Gly-Asp-DPhe-Lys = N-biotin-aminohexanoic acid) has been described in the prior art or can be achieved according to standard techniques. Direct Competition Assays. Were based with some modifications on earlier methods (Smith et al. 1990). avR3 was diluted to 1 ^g ml"1 in buffer A (150mM NaCI, 1mM MgCfe, 1mM CaCl2, 10 ^M MnCl2, 20mM Tris-HCI; pH 7.4) and 100 ml was applied overnight at 4°C to 96-well microtitre plates. The plate was washed once with buffer B (3% BSA (w/v), 100mM NaCI, 1mM MgC^, 1mM CaCl2, 10 uM MnCl2, 50mM Tris-HCI; pH 7.4) and incubated 2h at 20°C in the same buffer.
After rinsing with buffer C (buffer B with BSA at 0.1% (w/v)) was added containing biotinylated ligand (1 ^g mM end concentration) and serially diluted test peptides or proteins. After incubation (3h, 30° C), unbound ligand was washed from the plate by 3 rinses with buffer C, and incubation continued for 1h with anti-biotin-alkaline phosphatase conjugated antibody (1:10000 in buffer C) (Sigma) followed by PBS wash and detection of bound antibody with NBT-BCIP (Biorad) as substrate. ECM molecules and antibodies were routinely biotinylated and used in such an assay configuration. Assays were routinely performed in triplicate or quadruplicate, and repeated at least 3 times to derive an IC50 from an unweighted sigmoid curve fit. Results were normalised against external control peptides to allow intra-assay comparison. Linear RGD peptide GRGDSPK and cyclic peptide cRGDfV were routinely included in titrations as external standards, as were control wells where binding of biotinylated-ligand to blocked but integrin-free wells, and of anti-biotin antibody to ligand-free integrin were measured: the signal from the control wells was
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Pre-b!ock-competition assay Plates coated with receptor and blocked as for the Direct Competition Assay were pre-incubated with two-fold concentrated antibodies, matrix molecules or competitive peptides (50 ^1 in buffer C: 1h; 30°C) before addition of biotinylated probing ligand or antibody (50 \i\ in buffer C) and continued incubation (3h; 30° C), followed by washing and detection of bound biotin as described for the direct competition assay.

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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Merck Patent GmbH
(B) STREET: Farnkfurter Str. 250
(C) CITY: Darmstadt
(E) COUNTRY: Germany
(F) POSTAL CODE (ZIP): 64271
(H) TELEFAX: 49-6151-727191
(ii) TITLE OF INVENTION: Anti-alpha-V Integrin Monoclonal Antibody
(iii) NUMBER OF SEQUENCES: 17
(iv> COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 381 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal

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(vi) ORIGINAL SOURCE:
{A) ORGANISM: mouse (B) STRAIN: BALB/c
(vii) IMMEDIATE SOURCE:
(B) CLONE: 72-17E6
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..129
(D) OTHER INFORMATION:/function= "Leader- and FR-1 sequence (leader: 1 to 60; FR-1: 61 - 129)"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:130..162
(D) OTHER INFORMATION:/function= "CDR-1 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:163..207
(D) OTHER INFORMATION:/function= "FR-2 sequence" (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:208..228
(D) OTHER INFORMATION:/function= "CDR-2 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:229..324
(D) OTHER INFORMATION:/function= "FR-3 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:325..351
(D) OTHER INFORMATION:/function= "CDR-3 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:352..381
(D) OTHER INFORMATION:/function= "FR-4 sequence"

-59-
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 1:
ATG GTG TCC TCA GCT CAG TTC CTT GGT CTC CTG TTG CTC TGT TTT CAA 48
Met Val Ser Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin
15 10 15
GTT ACC AGA TGT GAT ATC CAG ATG ACA CAG ACT AC A TCC TCC CTG TCT 96
Val Thr Arg Cys Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser
20 25 30
GCC TCT CTG GGA GAC AGA GTC ATC ATC AGT TGC AGG GCA AGT CAG GAC 144
Ala Ser Leu Gly Asp Arg Val lle Ile Ser Cys Arg Ala Ser Gin Asp
35 40 1 S
ATT AGC AAT TAT TTA AGC TGG TAT CAA CAG AAG CCA GAT GGA ACT GTT 192
lie Ser Asn Tyr Leu Ser Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val
10 1 5 10
AAA CTC CTG ATC TTC TAC ACA TCA AAA TTA CAC TCA GGA GTC CCA TCA 240
Lys Leu Leu He Phe Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser
15 1 5 1
AGA TTC AGT GGC AGT GGG TCT GGA ACA GAT TAT TCT CTC ACC ATT AGT 288
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser
5 10 15 20
AAC CTG GAC CAA GAA GAT ATT GCC ACT TAC TTT TGC CAA CAG GGT AAT 336
Asn Leu Asp Gln Glu Asp He Ala Thr Tyr Phe Cys Gln Gln Gly Asn
25 30 1
ACG TTT CCG TAC ACG TTC GGA GGG GGG ACA AAG GTG GAA ATG AGA 381
Thr Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Met Arg
5 15 10
(2) INFORMATION FOR SEQ ID NO:: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: 1inear

-60-
(ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Val Ser Ser Ala Gin Phe Leu Gly Leu Leu Leu Leu Cys Phe Gin
10 15
Val Thr Arg Cys Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Asp Arg Val Ile Ile Ser Cys
35 40
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Arg Ala Ser Gin Asp Ile Ser Asn Tyr Leu Ser
15 10
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu lie Phe
15 10 15
(2) INFORMATION FOR SEQ ID NO: 5:

-61-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Tyr Thr Ser Lys Leu His Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
15 10 15
Leu Thr He Ser Asn Leu Asp Gin Glu Asp He Ala Thr Tyr Phe Cys
20 25 30
(2) INFORMATION FOR SEQ ID NO:: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
Gin Gin Gly Asn Thr Phe Pro Tyr Thr
1 5

-62-
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Phe Gly Gly Gly Thr Lys Val Glu Met Arg
15 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: mouse
(B) STRAIN: BALB/c
(vii) IMMEDIATE SOURCE:
(ix) FEATURE;
(A) NAME/KEY: CDS
(B) LOCATION:1..57
(D) OTHER INFORMATION:/function= "leader sequence'

-63-
(A) NAME/KEY: CDS
(B) LOCATION:58..147
(D) OTHER INFORMATION:/function= "FR-1 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:148..162
(D) OTHER INFORMATION:/function= "CDR-1 sequence1
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:163..204
(D) OTHER INFORMATION:/function= "FR-2 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:205..255
(D) OTHER INFORMATION:/function= "CDR-2 sequence'
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:256. .351
(D) OTHER INFORMATION:/function= "FR-3 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:352..378
(D) OTHER INFORMATION:/function= "CDR-3 sequence"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:379..411
(D) OTHER INFORMATION:/function= "FR-4 sequence"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATG GGA TGG AGC TGG GTC TTT ATC TTC CTG TTT TCA GTA ACT GCA GGT 48
Met Gly Trp Ser Trp Val Phe Ile Phe Leu Phe Ser Val Thr Ala Gly
15 20 25

-64-
GTC CAC TCC CAG GTC CAG CTT GAG CAG TCT GGG GCT GAA CTG GCA GAG 96
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Ala Glu
15 10
CCT GGG GCC TCA GTG AAG ATG TCC TGC AAG GCT TCT GGC TAG ACC TTT 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
IS 20 25
AGT AGT TTC TGG ATG CAC TGG GTA AAA CAG AGG CCT GGA CAG GGT CTG 192
Ser Ser Phe Trp Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu
30 1 5 1 5 10
GAA TGG ATT GGA TAC ATT AAT CCT AGA TCT GGT TAT ACT GAG TGT AAT 240 Glu Trp lie Gly Tyr lie Asn Pro Arg Ser Gly Tyr Thr Glu Cys Asn
15 10
GAG ATA TTC AGG GAC AAG GCC ACA ATG ACT GCA GAC ACC TCC TCC AGC 288
Glu lie Phe Arg Asp Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Ser
15 1 5 10
ACA GCC TAC ATG CAA CTG AGT GGT CTG ACA TCT GAG GAC TCT GCA GTC 336
Thr Ala Tyr Met Gin Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val
15 20 25
TAT TAC TGT GCA AGT TTT CTG GGA CGA GGG GCT ATG GAC TAC TGG GGT 384
Tyr Tyr Cys Ala Ser Phe Leu Gly Arg Gly Ala Met Asp Tyr Trp Gly
30 1 5 1
CAA GGA ACC TCA GTC ACC GTC TCC TCA 411
Gin Gly Thr Ser Val Thr Val Ser Ser
5 10
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10

-65-
Met Gly Trp Ser Trp Val Phe lie Phe Leu Phe Ser Val Thr Ala Gly
15 10 15
Val His Ser
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Ala Glu Pro Gly Ala
15 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser
20 25 30
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
Ser Phe Trp Met His
1 5
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS: (A} LENGTH: 14 amino ac ids (B) TYPE: amino acid (D) TOPOLOGY: 1inear

-66-
(ii) MOLECULE TYPE: protein
(xi> SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie Gly
15 10
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Tyr lie Asn Pro Arg Ser Gly Tyr Thr Glu Cys Asn Glu He Phe Arg
15 10 15
Asp
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gin
15 10 15
Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ser
20 25 30
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids

-67-
(B) TYPE: amino acid (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Phe Leu Gly Arg Gly Ala Met Asp Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

-68-WE CLAIM:
1. Monoclonal antibody having the following properties:
- reacting only with the v-chain of human v-integrlns,
- blocking the attachment to the integrin substrate of the
aV-integrin bearing cell,
- triggering reversal of established cell matrix interaction
caused by v-integrins,
-blocking tumor development, and
- showing no cytotoxic activity.
2. Monoclonal antibody as claimed in Claim 1, wherein the integrin
substrate is vitronectin, fibrinogen, or fibronectin.
3. Monoclonal antibody as claimed in Claim 1 or 2, wherein the tumor, the
cell growth of which is blocked, is melanoma.
4. Monoclonal antibody as claimed in Claim 1 to 3 comprising the amino
acid sequence of the FRs and CDRs of the fight chain given in Figure
17a and the heavy chain given In Figure 17b or mutants and variants
thereof.
5. Monoclonal antibody fragment having the properties of the monoclonal
antibody as claimed in Claims 1 -4.
6. Hybridoma cell line having the designation 272-17E6 and deposited
under accession number DSM ACC2160 capable of producing a
monoclonal antibody as claimed in Claims 1 -4.

-69-
7. Monoclonal antibody obtainable by the hybridoma cell fine DSM
ACC2160.
8. Amino acid sequence comprising the sequence given in Figure 17a.
9. Amino acid sequence comprising the sequence given in Figure 17b.
10. Amino acid sequence as claimed in Claim 8 starting from position 21.
11. Amino acid sequence as claimed In Claim 9 starting from position 20.
12. DNA sequence comprising the DNA sequence of Figure 17a or
mutants and variants thereof coding for the FRs and CDRs of the light
chain of the variable region of a monoclonal antibody having the
properties as claimed in Claim 1.
13. DNA sequence comprising a DNA sequence of Figure 17b or mutants
and variants thereof coding for the FRs and CDRs of the heavy chain
of the variable region of a monoclonal antibody having the properties
as claimed in Claim 1.
14. DNA sequence as claimed in Claim 12, comprising the leader
sequence given Figure 17a.
15. DNA sequence as claimed in Ciaim 13, comprising the leader
sequence given in Figure 17b.

-70-
16. Pharmaceutical composition comprising a monoclonal antibody as
claimed in one of the Claims 1 - 5 qr 7, and a pharmaceutical
acceptable carrier.
17. Method for the manufacture of a monoclonal antibody as claimed in
one of the Claims 1-4, by immunizing a mouse with purified v
integrin, selecting clones binding to the purified concerning v
receptor by ELISA and producing according to standard techniques a
specific cell line which produces said antibody.
18. Method as claimed Claim 16, wherein the integrin is vitronectin.
19. Method for the manufacture of a monoclonal antibody of as claimed in
Claim 7, by immunizing a mouse with purified vitronectin, selecting
clones binding to purified vitronectin receptor by ELISA and producing
according to standard techniques the cell Vine as claimed in Claim 6
which produces said antibody.
The invention relates to a novel monoclonal antibody, a hybridoma cell line producing said antibody, DNA sequences coding for said antibody, and amino acid sequences. The monoclonal antibody, a preferred embodiment of which is named 17E6, has the following properties:
- reacting only with the V-chain of human V-integrins,
- blocking the attachment to the integrin substrate of the V-
integrin bearing cell,
triggering reversal of established cell matrix interaction caused by V-integrins, blocking tumor development, and showing no cytotoxic activity.

Documents:

01646-cal-1995 abstract.pdf

01646-cal-1995 claims.pdf

01646-cal-1995 correspondence.pdf

01646-cal-1995 description(complete).pdf

01646-cal-1995 drawings.pdf

01646-cal-1995 form-1.pdf

01646-cal-1995 form-18.pdf

01646-cal-1995 form-2.pdf

01646-cal-1995 form-3.pdf

01646-cal-1995 form-5.pdf

01646-cal-1995 letters patent.pdf

01646-cal-1995 p.a.pdf

01646-cal-1995 priority document others.pdf

01646-cal-1995 priority document.pdf

1646-CAL-1995-FORM-27-1.pdf

1646-CAL-1995-FORM-27.pdf


Patent Number 205485
Indian Patent Application Number 1646/CAL/1995
PG Journal Number 14/2007
Publication Date 06-Apr-2007
Grant Date 05-Apr-2007
Date of Filing 15-Dec-1995
Name of Patentee MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER
Applicant Address 64271, DARMSTADT,GERMANY, A GERMAN COMPANY.
Inventors:
# Inventor's Name Inventor's Address
1 FRANCESE MITJANS C/O.MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT,GERMANY.NO.1TO 4 ARE CITIZENS OF SPAIN, NO. 5. IS BRITAIN NATIONAL AND NO. 6. IS U.S. CITIZEN.
2 JAUME PIULATS, C/O. MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT
3 ELISABERST ROSELL C/O. MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT
4 JAUME ADAN C/O. MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT
5 SIMON GOODMAN C/O. MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT
6 DIANE SANDER C/O. MERCK PATENT GESELLSCHAFT MIT BESCHRANKTER HAFTUNG, 64271, DARMSTADT
PCT International Classification Number C07K 16/28
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 94120165.9 1994-12-20 EUROPEAN UNION