Ligand fishing with Biacore - Biotechnology

SCIENTIFIC REVIEW 1

Ligand fishing with Biacore®

Reprinted from BIAjournal “Special Issue” 1997

JoAnne Bruno
Biacore Inc., Piscataway,
New Jersey, USA

One of the most powerful
features of BIA technology is the
ability to detect specific binding
interactions in complex matrices.
In the receptor driven drug
discovery effort, Biacore® is
therefore an indispensable tool
for screening crude cell and tissue
homogenates as potential sources
for ligands of orphan receptors in
an approach nicknamed “ligand
fishing”.

SCIENTIFIC REVIEW 1

Figure 1 Receptor tyrosine kinases (RTKS) play a pivotal In the case of Lackmann and co-workers [2]
role in regulating cell growth and differentiation who identified and isolated a ligand for the EPH-
Screening of cell line conditioned which has stimulated considerable interest in like kinase (HEK) receptor, the RTK was identified
media for binding to immobilised identifying new members of this universal initially on the cell surface of a human pre-B-cell
HTK extracellular domain (HTKex- protein family [1]. The extensive sequence line by a monoclonal antibody (IIIA4), as
Fc). Relative response units are similarity in the tyrosine kinase domain of opposed to the homology-cloning approach
defined as Biacore response (RU) known RTKs has facilitated the homology-based cited above. The monoclonal antibody was
divided by the concentration (40- cloning of several novel receptor-like protein coupled to the sensor chip and BIA technology
80x) of the conditioned medium. tyrosine kinases which are considered to be was used from the outset of the project to guide
orphan receptors because their cognate ligands the selection of the highest soluble HEK (sHEK)
are unknown. The identification, isolation, and producing clones. The sHEK was subsequently
subsequent expression of a novel ligand for an purified and employed in ligand fishing
RTK may foster the development of new experiments.
therapeutics for diseases ranging from cancer to
muscular dystrophy. The probability of finding a new ligand
increases with the number of samples passed
Researchers from various laboratories around over the biospecific (RTK) surface. In short, the
the world have used ligand fishing with Biacore identification of a source for a ligand is a
systems to identify not only known proteins numbers game. In the search for the HEK ligand
which function as novel ligands for RTKs, but [2], Lackmann et al identified only a single
also ligands which are novel proteins. Clearly, positive (human placenta conditioned medium
BIA technology provides researchers with a (HPCM)) from 150 different sources. Sakano and
controlled platform for combining biomolecules co-workers [3] evaluated ~80 cell supernatants
whose biological interaction may be transient or to identify the ligand for hepatoma trans-
short-lived even under the best possible membrane kinase (HTK) in the conditioned
circumstances. medium of the colon cancer cell line C-1 and
KATO-III (Figure 1).
EXPERIMENTAL DESIGN – ESSENTIALS FOR
THE FISHING EXPEDITION Before embarking on their first ligand fishing
expedition, some researchers have established a
The extracellular domain of RTKs is the known “proof of principle” by evaluating known
site for ligand binding and therefore is the receptor-ligand pairs. One such example is the
logical region of the protein to use when trkB-BDNF receptor-ligand pair used at
creating a biospecific surface for ligand fishing Regeneron Pharmaceuticals Inc. [4] where
experiments. Soluble extracellular domains of researchers later fished-out ligands for Tyro3
the RTK are typically produced using (Protein S) and Axl (Gas6) [5], as well as a
recombinant methods and often the proteins are ligand for TIE2 (angiopoietin-1) [6]. The Kit-
expressed as human IgG1 Fc fusion proteins. Steel Factor model system was employed at
The Fc fusion simplifies the purification process Genetics Institute [7], where the murine ligand
and generates a dimeric receptor, which is the for Flt4 was discovered. By working with
theoretical, high-affinity ligand-binding state for biomolecules which are known to interact,
the RTK. The soluble extracellular domain is experimental conditions such as receptor surface
then covalently linked to the Sensor Chip CM5 density, injection volume, flow rate and
surface at high density (75 fmol of ligand/mm2), sensitivity may be optimised.
most often via primary amine coupling chemistry.

55
44
33
22
11
00
Relative response units

KG-1a
KG1a/PMA
CCRF-CEM
CCRF-CEM/PMA

SEKI
SEKI/PMA

THP-1
THP-1/PMA

5637
5637/PMA
HeLa OHIO/PMA
HeLa OHIO
K562/PMA

K562
Daudi/PMA

Daudi
HL60/PMA

HL60
Raji

Raji/PMA
C-1

C-1/PMA
C-1/TNF

T24
T24/PMA
T24/TNF

U937
U937/PMA

Jarkat
Jarkat/PMA

UT/7
UT-7/PMA
COLO205
COLO205/PMA
COLO205/TNF

MKN-74
MKN-74/PMA
MKN-74/TNF

HEL
HEL/PMA
HEL/TNF

H-1
H-1/PMA
H-1/TNF

PC-9
PC-9/PMA
PC-9/TNF
COLO320 DM
COLO320 DM/PMA
COLO320 DM/TNF

A431
A431/PMA
A431/TNF

MKN-1
MKN-1/PMA
MKN-1/TNF

KATO III
KATO II/IPMA

HeLa 117
HeLa 117/PMA

KB
KB/PMA
KB/TNF

HeLa
HeLa/PMA
HeLa/TNF

MRC-5
MRC-5/PMA
MRC-5/TNF

PC-14
PC-14/PMA
PC-14/TNF

NBT-2
NBT-2/PMA
NBT-2/TNF

MKN-28
MKN-28/PMA
MKN-28/TNF

BT20
BTSO/PMA

BT20/TNF
Hep3B

Hep3B/PMA
Cell lines

SCIENTIFIC REVIEW 1

For a screening assay conducted with Biacore HEK ligand [2], Lackmann et al observed only Figure 2
1000 or Biacore 2000, the full automation of a 5-7% reduction in the total response from ABAE conditioned medium contains
the sample handling unit allows for sample the crude HPCM. Confirmation of the results ligands for both Tyro3 and Axl:
testing with minimal operator involvement. For came from analysis of partially purified Evaluation of ABAE conditioned
Biacore 2000 in particular, the assays will be extracts (ammonium sulphate precipitation, medium (10x) for specific binding
reasonably high throughput if the cell hek-affinity extraction, size exclusion HPLC). to Tyro3-Fc or Axl-Fc immobilised
supernatant samples are injected simultaneously In all cases, the positive result is validated by surfaces (A). In order to
over all four flow cells, each containing a a demonstration of biological function, such demonstrate specificity, some
different receptor surface. In this configuration, |as the ability to stimulate tyrosine samples were incubated with 10
the different receptors (or non-relevant proteins) phosphorylation. Receptor-specific binding mg/ml soluble Axl-Fc (sAxl),
on the sensor chip will aid in determining activity from a recombinant preparation of Tyro3-Fc (sTyro3), or TrkB-Fc
specificity (see below). the ligand may also be evaluated. (sTrkB) as indicated, prior to
injection over the immobilised
The sensitivity of BIA allows for detection in the TWO LIGANDS FROM ONE SOURCE surfaces. Axl-affinity purified Gas6
ng/ml range; however, many growth factors are (B) or Tyro3-affinity purified Protein
expressed at pg/ml levels. Therefore, it is = TWO FISH ON ONE HOOK S (C) binds specifically to Axl-Fc or
common practice to use concentrated cell Tyro3-Fc immobilised surfaces,
supernatants (1-50x) in ligand fishing It is not uncommon for a ligand to bind to respectively. Soluble receptor
experiments [2-8]. In most cases the volume of other members of a family within RTKs; there- competition was conducted as
sample consumed is ~100 ml. The samples may fore, once a cell line or tissue homogenate has outlined previously.
contain additional NaCl (0.2 - 0.4 M) as well as been identified as a source for a ligand,
detergent and soluble dextran to minimise non- specific binding to other members of the ABAE conditioned medium
specific binding which may occur owing to the family may be evaluated. This test may also Binding Units (RU)
complexity of the sample matrix. verify that the ligand (or source) is
functionally relevant. B61 bound to soluble 500
After passing the samples over the receptor forms of two other receptors in the EPH/ECK
surface(s), the next step is to establish specificity family of receptors albeit with lower affinity 400
criteria for distinguishing between the true and [8], while angiopoietin-1, the ligand for TIE2,
false positives. In one of the first reports of did not bind appreciably to TIE1 [6], and the 300
ligand fishing using biacore, Bartley and co- Flt4 ligand has not been shown to bind other
workers [8] at Amgen Inc. used only a single members of the family (Flt1, PIGF, VEGF) [7]. 200
flow cell and observed numerous cell
supernatants with ECK receptor extracellular Protein S, the ligand identified for Tyro3, was 100
domain (ECK-x) binding activities above 50 found in two sources, fetal bovine serum (FBS),
resonance units (RU). At least three of these and adult bovine endothelial (ABAE) 0
supernatants, gave variable responses and were conditioned medium. Both of these sources
not pursued; however, the conditioned medium were evaluated for binding to the extracellular No Block
from two of the cell lines with the highest domain of Axl (Axl-Fc), a second member of +sAxl
reproducible binding activity was concentrated the Tyro3 family. In contrast to the high level
10-40x and passed over an affinity column of binding observed for Tyro3, FBS showed +sTyro3
containing immobilised ECK-x. The low pH little to no binding on the Axl-Fc surface. The +sTrkB
eluate of the column was identified as B61 by ABAE conditioned medium which displayed No Block
N-terminal sequence data. For confirmation of binding to Tyro3 also displayed significant
ECK-specific binding activity, recombinant B61 specific binding to Axl. That the observed +sAxl
was re-evaluated for binding to the ECK-x sensor binding activities were distinct was verified by +sTyro3
chip surface, for its ability to crosslink to full- the observation that excess soluble Tyro3 +sTrkB
length cell-associated ECK receptor, and for its would almost completely abolish the binding
ability to stimulate tyrosine phosphorylation [8]. activity on the Tyro3 surface, with only Axl Surface Tyro3 Surface
minimal reduction on the Axl surface, while A
Other researchers [2-7] publishing positive excess Axl had essentially the reverse com-
results after Bartley et al have adopted the petition profile (Figure 2). The protein Affinity purified Tyro3-ligand
practice of using a single sample injection for responsible for the Axl binding activity was
evaluating binding activities on up to four flow subsequently purified and identified as GAS6. Binding Units (RU)
cells. A different receptor or non-relevant In this case, a single source contains distinct
protein has been immobilised on each of the ligands for members of the same family. 2000
four flow cells. In addition, specificity has been (Note: Additional published studies [9,10] on
confirmed by incubating the sample with a large the ligands for the Tyro3 family suggest that 1500
molar excess of the soluble receptor outside of GAS6 and not Protein S is the ligand for
the Biacore instrument and looking for a Tyro3. This inconsistency has yet to be 1000
reduction in signal. However, the drop in resolved and may arise from differences in the
response may not be a significant percentage of isotypes evaluated (e.g. bovine Protein S 500
the total binding activity. For example, with the binding to murine Tyro3)).
0

No Block
+sAxl

+sTyro3
+sTrkB
No Block

+sAxl
+sTyro3
+sTrkB

Axl Surface Tyro3 Surface

B

Affinity purified Axl-ligand

Binding Units (RU)

2000

1500

1000

500

0

No Block
+sAxl

+sTyro3
+sTrkB
No Block

+sAxl
+sTyro3
+sTrkB

Axl Surface Tyro3 Surface

C

SCIENTIFIC REVIEW 1

Figure 3 Biacore IS INSTRUMENTAL IN THE few micrograms of protein. These small
Preparative HIC (A) or IEX (B) DISCOVERY OF NOVEL PROTEINS quantities are a testament to the detection
chromatography of a crude HEK sensitivity of Biacore which is able to identify
ligand preparation. Protein elution Clearly, BIA is a powerful tool for the discovery ligands with starting concentrations of 1 ng/ml
was monitored at 280 nm and of novel ligands for orphan receptors (Table 1). and below (prior to concentration of
binding to a HEK immobilised For most of the examples described herein, the supernatants).
surface was determined for proteins identified as ligands for a given
samples incubated with ( ) or receptor have been sequenced previously, CHARACTERISATION OF RECEPTOR –
without ( ) 10 µg/ml soluble HEK. although their exact biological function is not LIGAND INTERACTIONS
known. With the discovery of angiopoietin-1,
References the ligand for TIE2 [6], a previously unknown Biacore is instrumental not only in the ligand
protein is found in the human neuroepithelioma discovery and purification stages, but also in the
1. Schlessinger J. and Ullrich A. (1992) cell line SHEP1-1, and the mouse myoblast cell characterisation of receptor – ligand interactions.
Neuron 9: 383-391 line c2c12ras. BIA plays a pivotal role in the
2. Lackmann M., Bucci T., Mann R.J., ligand identification process which subsequently Questions that may be addressed are:
Kravets L.A., Viney E., et al (1996) Proc. leads to the cloning, sequencing and expression
Natl. Acad. Sci. USA 93: 2523-2527 of a novel protein. • Is the affinity of the interaction consistent
3. Sakano S., Serizawa R., Inada T., with receptor – ligand interactions?
Iwama A., et al (1996) Oncogene 13: BIOACTIVITY MONITOR FOR LIGAND
813-822 PURIFICATION • How does the kinetics and affinity of the
4. Bruno J., Radziejewski C., Conn G., interaction compare for different members
Stitt T. N., Robertson A., Yancopoulos G. An added benefit of Biacore is its ability to of the same family?
(1995) 5th BIAsymposium USA function as a real-time bioactivity monitor that
5. Stitt T. N., Conn G., Gore M, Lai C., can be used to follow binding activity through- Preliminary data for the HEK ligand AL-1 pre-
Bruno J. (1995) Cell 80: 661-670 out the purification of a ligand, thereby saving dicts a Kd ~2-3 nM, which is within the range
6. Davis S., Aldrich T. H., Jones P. F., time and preserving sample activity. In the puri- for AL-1 interacting with REK7, another RTK in
Acheson A., Compton D., et al (1996) fication of the HEK ligand AL-1, Lackmann and the family [2]. HTKL, the ligand for HTK, binds
Cell 87: 1161-1169 co-workers replaced a conventional bioassay with Kd ~1 nM, which is much tighter than the
7. Fitz L. J., Morris J. C., Towler P., Long with a biacore assay for monitoring specific affinity of the interaction between LERK-2 and
A. J., Burgess P., et al (1996) 6th activity. Within a classical purification scheme HTK (Kd ~135 nM). Kinetic and affinity
BIAsymposium USA they showed a 1.8 x 106-fold purification of characterisation may also be helpful in
8. Bartley T. D., Hunt R. W., Welcher A. the HEK ligand. At each step in the purification distinguishing between cell surface and secreted
W., Boyle W. J., et al (1994) Nature 368: process HEK-specific binding activity was ligands. In general, the affinity of a cell-surface
558-560 correlated with UV absorbance in identifying ligand will be higher when ligand is clustered or
9. Mark M.R., Chen J. Hamonds G. R., the active fractions (Figure 3). This approach in a cell-surface environment.
Sadick M. and Godowsk P. J. (1996) was also used by Sakano et al for monitoring
J.Biol. Chem. 271(16): 9785-9789 the affinity purification of HTK ligand, by Stitt et THE ULTIMATE FISHING LURE
10. Nagata K., Kazumass O., Nakano T., al for the affinity purification of Protein S and
Arita H., Zong C., Hanafusa H., Mizuno Gas6, and by Fitz et al for isolation of the Biacore technology is definitely a powerful tool
K. (1996) J. Biol. Chem. 271(47): 30022- murine Flt4 ligand. In all cases, the specific for all stages of the ligand discovery process.
30027 activity is denoted as resonance units (RU) per
milligram of protein. The final yield for most From the beginning (screening cell line super-
ligands purified from cell supernatants is only a natants and tissue homogenates), through the
middle (purification, cloning and sequencing),
and to the end (kinetic characterisation of the
purified ligand) there is no better fishing pole
to bring on your ligand fishing expedition!

Receptor Ligand Source Function Reference

ECK B61 HCT-8, SK-BR-3 ? Bartley (8)
Tyro3 Protein S FBS, ABAE anticoagulant Stitt (5)
Axl Gas6 ABAE Stitt (5)
HEK AL-1 HPCM ? Lackmann (2)
HTK HTKL C-1, KATO-III ? Sakano (3)
TIE2 angiopoietin-1 SHEP, C2C12ras ? Davis (6)
Flt4 murine Flt4 BNL 1NG A.2 endothelial dev. Fitz (7)
angiogenesis?

Table 1. Summary of ligand fishing experiments

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