Note: Descriptions are shown in the official language in which they were submitted.
CA 02402930 2002-09-19
Tareeted Ligands
Field of The Invention
The present invention relates to multispecific ligands, for example a
heterofunctional ligand comprising at
least first and second binding moieties which have cooperating functional
affinities including a
multispecific ligand, for example, a bispecific antibody, having at least a
tirst portion which binds to a
'lymphatic vessel associated' antigenlreceptor and a second portion having at
least one immune-affecting
functionality including, without limitation, functions related to antigen
presentation, immune signaling,
suppression or enhancement of immune tolerance or immune stimulation, or
binding to a target molecule,
for example a cell surface antigen, receptor etc.
Background of the Invention
Immunotherapy has gained wide acceptance as a promising measure to address
several disease states
including autoimmune disease, transplant rejection, infectious disease and
cancer. Despite rapid and
exciting progress in approaches to treatment, the disease burden attributable
to such illnesses has not
significantly abated. The complex nature of the normal and pathologic
immunologic processes associated
with such diseases, coupled with logistical problems in evaluating and
implementing methods for
immunotherapy in human subjects, continue to be some of the obstacles to
successful advances in
treatment.
Successful approaches to immunotherapy are predicated on the ability of the
immunotherapeutic molecule
to be delivered in a therapeutic, sub-toxic dose at the desired therapeutic
frequency. In the process of
selection of a suitable therapeutic molecule, it is recognized that sub-toxic
doses may be insufficient for the
desired therapeutic effect, especially where the antibody binds incidentally
to cell populations other than
the target population. In the case of an injectable preparation and especially
an intravenous mode of
delivery, in contrast to readily self administered modes of delivery, the
optimal dosing frequency for
therapeutic purposes could impose an undesirable burden on the patient and
care-giver, assuming that such
optimal frequency is to begin with deemed convenient fur clinical trials.
Numerous research efforts are underway to identify and test ligands including
antibodies, biologic effector
ligands (e.g. cyrtokines, chemokines, growth factors colony stimulating
factors) receptor agonists
orantagonists etc. which will bind to or otherwise interact with or trigger
responses in or towards target
entities, including pathogenic organisms, tissue specific cells,diseased
cells, immune cells etc. A recent
example is a renewed interest to tind molecules and methods of triggering an
interaction with CD45 (see
for example Nature (?001 ) Vol. 409 p. 349-354). Evaluating the biological
effect of interactions with such
target ligands is often obfuscated and retarded by the biodistribution of such
ligands on cells other than the
target population which results in undesired and/or conftrsing pleiotropic
effects.
The present invention facilitates scientific assessment, development, role
evaluation, therapeutic
evaluation, and delivery, particularly targeted delivery of molecules that
exert biologic functions and
particularly immune relatedfunctions. In particular, the targeting agents and
methods which are the subject
of the invention herein facilitate scientific evaluation of the biological
effects of a more targeted
biodistribution of such targeting agents, by limiting undesired or confusing
side effects. In preferred aspects
the invention contemplates compositions of matter and methods of~delivery, in
some cases using ligands
that but for the tar~~etin~~ methods herein defined would be ineffective or
have a broader effect than is
desirable; or similarly, but for the severity of the disease or the absence of
other therapeutic alternatives for
which such ligands are useful, they would otherwise be inappropriate for
therapeutic use. The present
invention accommodates evaluation of the biological role and!or effects of
such ligands for therapeutic or
other scientific purposes using such targeting strategies. In particular, the
present invention provides a
vehicle to preferentially target, on a sub-population of cells for which there
is a cell-associated marker, a
receptor or receptor ligand which is present on a more heterogeneous
population of cells.
CA 02402930 2002-09-19
Summary of The Invention
The invention contemplates a composition containing a multispecific ligand
containing at least a first
ligand binding moiety and a second ligand binding moiety. The first ligand
binding moiety specifically
binds with a pre-selected first affinity to at least a first ligand. The first
ligand has a first biodistribution.
The second ligand binding moiety specifically binds with a pre-selected
affinity to at least a second ligand.
The second ligand has a second biodistribution. The affinity of first and
second ligand binding moieties are
selected to bias the biodistribution of the multispeciflc ligand in favour of
a selected location of one or both
ofthe ligands.
The invention contemplates a composition containing a multispecific ligand.
The multispecific li~and
contains at least a first li~~and binding moiety and a second ligand binding
moiety. The first ligand binding
moiety specifically binds to a first ligand having a first biodistribution.
The second li<~and binding moiety
specifically binds to a second ligand having a second biodistribution. The
second biodistribution is
different from that of the first biodistribution, and the affinity of the
first and second ligand binding
moieties to their respective ligands are different and selected to bias the
biodistribution of the multispecific
ligand towards the first or second biodistribution.
The invention contemplates further. a composition containing a multispecitic
ligand. 'rhe multispecific
ligand contains a first ligand binding moiety and a second ligand binding
moiety. The first ligand binding
moiety specifically binds with a pre-selected first affinity to a first
ligand. The first ligand has a first
biodistribution. The second ligand binding moiety specifically binds with a
pre-selected affinity to a
second ligand. The second ligand has a second biodistribution. In this
embodiment of the multispecific
ligand, the affinity of first and second ligand binding moieties are selected
to bias the biodistribution of the
multispecific ligand.
The invention further contemplates a composition containing a multispecific
ligand. The multispecific
ligand specifically binds to a target ligand. The target ligand is specific to
a selected sub-population of a
heterogeneous cell population. This embodiment of the multispecitic ligand
contains a first ligand binding
moiety and a second ligand binding moiety. The first ligand binding moiety
specifically binds to a cell sub-
population associated ligand. The second ligand binding moiety binds to the
target ligand. In this
embodiment, the first li~~and binding moiety has an affinity for the sub-
population associated ligand higher
than the affinity of the second li~~and bindings moiety for the tartlet
ligand.
The invention further contemplates a composition containing a bispecific
ligand containing a first li~~and
and a second li<~and. The first ligand binds to a first target ligand and the
second li<~and binds to a second
target ligand. In this embodiment of the bispecific ligand. the affinity of
the first liaand is selected to
enable binding to the first target ligand independently of the ability of the
second ligand to bind to the
second target ligand. Further, the affinity of the second ligand is selected
to substantially reduce the
probability of its binding to the second target ligand without the first
ligand bindin<~ first or substantially
contemporaneously to the first target liy~and.
The invention further contemplates a composition containing a bispecific
antibody containing a first
antibody component and a second antibody component. The first antibody
component binds to a first target
ligand and the second antibody component binds to a second target li~and. In
this embodiment, the affinity
or avidity or both the affinity and avidity of the first antibody component
are selected to enable binding to
the first tartlet ligand independently of the ability of the second antibody
component to bind to the second
target ligand. The avidity or affinity or both the affinity and avidity of the
second ligand are selected to
substantially reduce the probability of its bindin~~ to the second target
ligand without the first ligand binding
first or substantially contemporaneously to the first target iigand.
The invention further contemplates a multispecitic ligand containing a first
moiety and a second moiety.
The first moiety binds to a first target ligand. The second moiety binds to a
second target li~.;and. The
affinity or avidity or both the affinity and avidity of the first moiety are
selected to enable the first moiety
to bind to the first tartlet li~.;and independently of the ability of the
second moiety to bind to the second
target ligand. The avidity or affinity or both the affinity and avidity of the
second moiety are selected to
substantially reduce the probability of its bindings to the second target
ligand without the first moiety, first
or substantially contemporaneously, binding to the first target ligand.
2
CA 02402930 2002-09-19
The invention further contemplates a multispecific li;,~and containing a first
moiety and a second moiety.
The first moiety binds to a first target ligand. The second moiety binds to a
second target liganct. 'fhe
affinity or avidity or both the affinity and avidity of the first moiety are
selected to enable the first moiety
to bind to the first target ligand independently of the ability of the second
moiety to bind to the second
target ligand. The avidity or affinity or both the affinity and avidity of the
second moiety are selected to
substantially reduce the probability of either moiety binding for a sufficient
duration or series of durations
to its respective target ligand to accomplish a therapeutic function without
the other moiety. first or
substantially contemporaneously, binding to its respective tartlet li~~and.
The invention further contemplates a composition containing a multispecific
ligand containing a first
moiety and a second moiety. The first moiety binds to a first target ligand.
The second moiety binds to a
second target ligand. The affinity or avidity or both the affinity and avidity
of the first moiety are selected
to enable the first moiety to bind to the first target liganct independently
ofthe ability of the second moiety
to bind to the second target ligand. The avidity or affinity or both the
affinity and avidity of the second
moiety are selected to enable the second moiety to bind to the second entity
in preference to the first moiety
binding to the first entity when both first and second moieties are
substantially contemporaneously bound
to the respective first and second entities.
The invention contemplates a composition containing a multispecitic ligand
containing a first moiety, a
second moiety and a third ligand bindings moiety. The first moiety binds to a
first target ligand and the
second moiety binds to a second target li~~and. In this embodiment, the
affinity or avidity or both the
affinity and avidity of the first moiety are selected to enable the first
moiety to bind to the first target ligand
in preference to the second moiety binding to the second entity when both
first and second moieties are
substantially contemporaneously bound to the respective first and second
entities, and the avidity or affinity
or both the affinity and avidity of the second moiety are selected to enable
the third target li~and to bind to
the second entity in preference to the second moiety binding to the second
entity when both the third target
ligand and the second moiety are substantially contemporaneously bound to the
second entity.
The invention further contemplates a composition containing an antibody which
specifically binds to an
epitope on a ligand. The ligand recognized by the antibody exerts a biologic
effect by binding to a target
site on a target ligand. The epitope bound by the antibody is proximal to the
binding site of the ligand for
the target ligand. so that binding of the antibody reduces but does not
prevent the affinity of the ligand for
its target lid=and.
The invention further contemplates a composition containing a muitispecific
ligand containing a first ligand
binding moiety and a second moiety. The first ligand binding moiety
specifically binds to a lymphatic
endothelial cell associated marker. The second moiety contains an independent
therapeutic function.
The invention further contemplates a composition containing an immunocytokine
containing an anti-
idiotypic antibody component and a cytokine component. The anti-idiotypic
antibody component
recognizes the paratope of an antibody which binds to a lymphatic vessel
associated ligand.
The invention further contemplates a composition containing a bispecific
antibody containin« an anti-
idiotypic antibody component and an anti-CD3 antibody or an anti-CD?8 antibody
component. The anti-
idiotypic antibody reco<~nizes the paratope ofan antibody which binds
specifically to a lymphatic vessel
associated lieand.
The invention additionally contemplates physiologically acceptable
compositions of the compositions
encompassed by the invention.
The invention likewise contemplates methods of use of the compositions
encompassed by the invention.
A composition comprisin~~ a multispecitic ligand comprising at least a first
IiV~and binding moiety which
specifically binds to a first li~~and having a first biodistribution and a
second li~and binding moiety which
specifically binds to a second li~~and having a second biodistribution
different from that of the first ligand,
and wherein the affinity of the first and second ligand bindings moieties are
different and selected to bias the
biodistribution ofthe multispecific li~~and.
3
CA 02402930 2002-09-19
Detailed Description of Preferred Embodiments
As exemplified above, the dual "aftinity" based targeting strategy of the
invention, may be understood in
one aspect, in terms of a strategic allocation of the respective affinity
properties of the multispecific ligand
to at least one "targetin~~'' function and at least one "effector: " function.
Accordingly, with respect to some
embodiments ofthe invention, the term "multifunctional" ligand is used
interchangeably.
Thus according to one preferred embodiment, at (east one of the ligand binding
moieties is a "targeting"
arm in the sense that it at least preferentially recognizes a marker that is
associated with one or more
specific target entities e~~. cell populations, and the other ligand binding
moiety is an "effector" arm which
binds with relatively less affinity or functional aftinity to a target ligand
which has a more diverse
biodistribution. In this case, the biodistribution of the multispecific ligand
is biased in favour of the
locations) of both ligands relative to the location(s'? of the target ligand
so as to limit the big distribution to
non-target entities.
Such binding or recognition is understood throughout to be specific, in
contrast to non-specific binding.
The term "effector" is used to refer to the ability to effect a biological
consequence through binding, for
example effecting a signal transduction event by activating a receptor, or
blocking the target ligand from
associating with a complementary ligand, for example blocking a receptor from
associating w kith a
complementary ligand (eg. its natural li~~and) and thereby, for example,
preventing a signal transduction, or
for example in thevcase of a decoy receptor preventing the biological
consequence (e~~. protective effect)
associated with the function of such receptor, or blocking a li'~and from
associating with a complementary
ligand eg. receptor on another entity eg. a cancer cell, infectious agent or
immune cell.
A biased biodistribution is preferably accomplished by the multispecitic
ligand contemporaneously
recognizing both ligands on the same entity e~~ cell, and may be accomplished
by such contemporanous
recognition occurring on adjacent entities or by increasing the propensity of
the multispecfic li~land to
locate in proximity to a target entity in virtue ofthe relatively high
aftinity targeting arm. The targeting arm
may itself be an effector .
In another embodiment, the biological consequence accomplished by the effector
arm is at feast minimally
retargeting, for example wherein the lesser affinity or functional affinity of
the first binding moiety is
selected to permit the multispecific ligand to preferentially bind to an
adjacent entity, for example. a
circulating entity which circulates in proximity to a lymphatic endothelial
cell to which the multifunctional
ligand is bound with lesser affinity. Again, the relatively high aftinity
first bindin<~ moiety may itself be an
effector.
In another embodiment the biological consequence accomplished by the effector
arm is minimally
cooperative targeting, for example where the biodistributions of at least one
of the first and second li~ands
extends to a diverse population of cells other than target cell population and
where binding is only possible
or consequential if both ligands are available for contemporaneous binding, in
this case due to the affinities
of the first and second ligand binding being individually insufficient for
effective targeting (eg. insufficient
for other than ephemeral binding). In the context of this embodiment of the
invention, the "cooperative
targeting" is not simply ameliorated by the effector am, it is predicated and
reliant on this arm. One or both
ligand binding moieties may exert additional eflector properties.
It will also be appreciated that any multispecitic ligand of the invention or
any component thereof may be
fused or conjugated to a separate effector as exemplified below, including
toxins, cytokines, adhesion
molecules etc.
The ligand bindings moiety is preferably an antibody or a sequence or
sequences of amino acids etc.which
are the natural ligand for the target ligand, for example where the ligand is
a cytokine or lymphokine
receptor, such as IL-? receptor, the li<,Tand binding moiety may comprise a
sequence of amino acids which
is IL.-2. 'The li;and binding moiety may also be a mutated or a newly
developed form of the natural ligand
(eg. developed through combinatorial libraries) or a natural or synthetic
chemical lieand (developed
through combinatorial chemistry).
In one aspect. the invention contemplates a composition containing a
multispecific li~~and containing at
least a first ligand binding moiety and a second ligand binding moiety, the
first ligand binding moiety
4
CA 02402930 2002-09-19
specifically binding with a pre-selected first affinity to at least a first
ligand, having a first biodistribution
and the second ligand binding moiety specifically binding with a pre-selected
affinity to at least a second
ligand with a second biodistribution and wherein the aftinity of fast and
second ligand binding moieties are
selected to bias the biological site of biologic activity of the multispecitic
ligand; and wherein the first
ligand binding moiety preferably binds with hi~~h affinity (preferably
nanamolar affinity or greater) to a
specific cell associated marker (e.g. a CD marker, a marker associated with
diseased cells, a a marker
associated cells in a particular physiological state (e.g. activated T cells,
B cells) etc. (such markers may be
associated with a particular class of cell or a subclass thereof (if
applicable) or particular subpopulation
within the subclass (if applicable). however classified, such as epithelial
cells. endothelial cells, immune
cells (lymphocytes, memory cells, effector cells) monocytes,Tcells (CD4+,
CD8+, CD45R0+).
hepatocytes, stem cells, etc.(expand) and wherein said second ii~and bindings
binding moiety binds with
relatively low, or medium affinity (preferably 0.1 micromolar or less) to a
receptor leg. chemokine, growth
factor, cytokine) involved in cell signaling or a decoy receptor, a cell
surface receptor ligand eg. the ligand
for such receptor which effects a signal or inhibits a si~~nal (e~~. CTLA4). a
li<.;and involved in cell adhesion.
a receptor or channel (ion channel) for a molecule involved in cell regulation
or homeostasis etc.
The invention contemplates that the difference in affinity will in most cases
be an essential element in
biasin~~ the location of action of the multispecitic ligand to yield an
acceptable or desired safety profile and
that the high affinity of the first ligand binding moiety for the cell
associated marker will be optimized for
this purpose insofar as the safety profile of the muftispecifie ligand
dictates maximizing its affinity
characteristics. The invention also recognizes that choosin<~ the relatively
lower affinity of the second
ligand bindings moiety may assist in this regard up to a point where its
effectiveness to bind to the second
ligand is significantly comprimised. In this regard. the invention also
contemplates that factors other than
the choice of aftinityof the first and second ligand binding moieties (and of
course the avidity effect
resulting from having two ligands on the target cell and only one on the non-
target cell) may be taken into
consideration or optimized to balance the safety and effectiveness profiles of
the multispecific ligand,
especially if such careful balance is required.
Examples of such factors, one or more of which can be employed in various
combinations, are described
hereafter:
1') the selection of cell associated marker, in terms of its cell surface
density relative to cell density of the
second ligand. The number of tirst and second ligand can readily be assessed
by radiolabelling studies or
approximated by tlow cytometric methods relative to a standard.The selection
of the cell surface marker in
this respect will depend on the function of the relatively low aftinity
binding moiety (whether it causes a
signal transduction (directly or indirectly eg. through binding to a receptor
(where less emphasis on relative
cell density may be warranted)(agonist antibodies are well known in the art
and include those described in
US 6,342,220, l1S 6,331,302, US >.635,177, lJS 6.099,841 see also Cancer Res
2001 Mar 1;61(5):1846-8
and can be made according to routine screening techniques, especially using
antibodies capable of cross-
linking receptor components (see references below) including antibodies in
which the Vtl and VL, are
capable of bindings individually to different receptor components), binding to
a decoy receptor. binding to
an inhibitory receptor etc.) or prevents a signal transduction (directly or
indirectly, e.g. binding to a
receptor, bindiny~ to receptor ligand) and in the final analysis how many
binding events per cell are required
to cause or prevent the sou~~ht-after biological effect. This can be assayed
in vitro through well known
assay methods established in the art for measuring responses to external
stimuli such as cytokines,
chemokines. growth factors, colony stimulating factors using various
immunostaining techniques including
flow cytometry (e~~r. to measure apoptosis (e.g. annexin V' binding assay)
signal transduction (e.g. using
phosphospecific antibodies that detect phosphorylarion of serine, tyrosine,
threonine), differential gene
expression etc.) depending on the type of effect that is being measured (see
for example Biosource Method
Booklets at Ettt~_ vs,_w w_hin"~uty c~isr~.r ~ofrtent
ttchC;,'.<>tnt;r~(_cy,itentirytpo~F?(..)F~:'isply ,isn.: see also
Amersham Bioscience catalogues, and those of other well known suppliers etc.)
or via animal studies. For
example, some Growth factors, lymphokines or molecules/ions required for
homeostasis are in more
delicate balance and can more easily disrupted. IL-2 depletion will cause
apoptosis of activated T cells,
which can be measured. For example. it may also be necessary or desirable for
the cell specitic marker to
approximate (preferably the the number of cell specific markers on the target
cell population is no less in
number than SO% more preferably no less than 90°%o in number relative
to the second li'~and - as stated
above, which will depend on what degree of causation or prevention ofthe
signaling/interaction will cause
the desired biologic effect) or preferably out-number (by greater than
50°,'°, preferably b_v ~'.a,reater that 100%
(greater than two fold), preferably by greater that 200°~% (greater
than 3 fold), by greater than 300°~0 (~~reater
CA 02402930 2002-09-19
than 4 fold)) the target li~~and for the relatively low aftinity bindings
moiety especially. for example, if the
goal is to block interaction of a receptor with a high affinity ligand that
exerts a biological effect in low
concentration (eg. a cytokine).
2) Furthermore, in the latter case the affinity ofthe first binding moiety
will preferably be selected to
approximate (preferably no less than one order of magnitude, more preferably
no less than 5 fold less, more
preferably no less three fold less, more preferably no less two fold less,
more preferably no less one fold
(100%) less). and will preferably equal or exceed the affinity ofthe natural
ligand.
3) Furthermore, the concentration (in virtue of the choice of administered
dose) of the multispecitic ligand
in the target cell microenvironment may also be selected to exceed that ofthe
natural ligand (MTD
permiting).
4) the choice of construct will maximize the steric blocking of the target
(IgG or F(ab')' vs diabody)_
Furthermore, in some mammalian systems (eg. mice) the hinge region is
naturally longer and this effect can
be mimicked for human antibodies through a hin~~e extension on the N-terminal
side of the hinge region
using well known neutral linkers (gly4ser) or a repeat of all or a portion of
the natural hinge sequence. 'this
extension will also permit a greater span between first and second ligands to
be bridged.
5) the choice of construct will include an Fc portion or partial Fe portion
(eg. CH2 or minibody-CH3) or
weighted Fc eg. by pegylation (site specific pegylation is well known in the
art) or 1gG subtype naturally
having additional Fc domains (e.g. an I~~E) (which Fc if it includes the CH3
is preferably mutated to
preclude its binding andior increase its half life as is known in the art see
USP 6,131, 022) so as to
maximize the shear effects on the multispecific ligand wfiich will be most
consequential in the case of
univalent binding in order to minimize the duration of such bindings (maximun
shear force is also preferred
where there is an excess in the total number of bioavailable targets of the
second ligand binding moiety
relative to the total number of bioavailable tartlets of first ligand binding
moiety(greater number of cells
and/or greater number of targets per cell and!or increased bioavailablity of
such targets eg. on normal cells
relative to cancer cells).
6) Optionally, the multispecitic ligand will include a 3''~ binding moiety
which binds to and neutralizes the
natural ligand for the receptor sought to be blocked. Such formats are well
known in the art (see for
example particularly Schoonjans R et al. A new model for intermediate
molecular wei~~ht recombinant
bispecitic and trispecitic antibodies by efficient heterodimerization of
single chain variable domains
throu~~h fusion to a Fab-chain. Biomol Eng. 2001 Jun;17(6):19_i-202.
Schoonjans R et al. Fab chains as an
efficient heterodimerization scaffold for the production of recombinant
bispecitic and trispecific antibody
derivatives.) Immunol. 2000 Dec 15;165(12):7(IS(t-7. Schoonjans R. et al.
Efficient heterodimerization of
recombinant bi- and trispecific antibodies. Bioseparation. 2000;9(p):179-8 i.
see also French RR.
Production of bispecitic and trispecitic F(ab)2 and F(ab)~ antibody
derivatives. Methods Mol Biol.
1998;80:121-34; US Patent Application No. 20020004587; Kortt AA, Dinreric and
trimeric antibodies:
high avidity scFvs for cancer targetin~~. Biomol En<~. 2001 Oct 1 ~: I 8(p):95-
108).
7) Optionally, two multispecitic ligands each binding to different cell
specific markers and each having a
second ligand binding moiety which binds to the same second ligand e~~. a
receptor, optionally to a different
polypeptide.%component of the receptor, may be employed to achieve the desired
biologic effect. One or
both may also be trispeeitic as discussed above..Accordin~~ to another
embodiment the multispecifc liaand
binds is used to protect a first target cell population in virtue of its hi<~h
affinity first ligand binding moiety
from the effects of a therapeutic entity which desirably binds to a second
tartlet cell population via the
second ligand but also undesirably binds to the first tar~,et cell population.
'therefore the second ligand
binding moiety can be used to selectively block the binding of the therapeutic
entity e.g. an interleukin,
interferon. innnunotoxin, etc. to the tirst target cell population in virtue
of the relatively low ~ aftinity second
ligand binding moiety. In this case, the multispecific ligand may also
comprise a third ligand binding
moiety which binds to the therapeutic entity, particularly where the
multispecific li~~and is first
administered tirst, optionally an anti-idiotypic binding moiety component
where the therapeutic comprises
an antibody component. Some sample targets are listed immediately below, while
others are listed later.
Greater targeting usin~~ a hi~~h aftinity tirst ligand bindings moiety which
binds to a cell associated or
specific marker may be imparted to a variety of existing antibodies with
suitably diminished affinity
6
CA 02402930 2002-09-19
including those marketed or in clinical trials or listed below which are the
subject of the patent and
scientific literature, including those listed in PharmaBusiness June 2002
No.>l
Category Functional li~"and (:ell localizing ligand for high MEode of action
Comments
for low affinity affinity arm
arm -
(locatiou of ti and)
Growth factor 11.-?. CD 4 T cells Growth Ittctor blockade for a IL-2 required
fur naive CD4 <>r CDR
blockade (soluble) specifically tbr CD4'f cell and memory CD=1'f cell
responses:.
subset: i c. sclcctiv a liinditt~ of C'Dq or CD8 would also
innnunosuppression block interaction with anti_en
_ resenting cells.
I).-1 i C D8 T cells Growth factor blockade rite a - It.-I, needed for memory
C'D8' T cell
specifically Ibr('I)8'I'ecll responses
subset: i.e. selective
inununo~tultprcssion
Chcmokine MC'P-I C'I)I lc (monocyte!macrophage) Monocyte%macrophage AI tints
ofpercutaneous coronary
' blockade ~ (soluble) chcmokines; c.=. anti- intervention (PC'I) to limit
restenosis:
inllammatorv a'~utt arthritis
i C'ell activation ' 1 hrombin CD 31 (endothelial cells) I'ruvent thrombin
binding to I Limit thrombosis and endothelial
blockade (soluble) or i thrombin receptors on ' activation . e.g at time of
PC'f
P-selectitt Ithrottthin aUivatcd ' Icukocyms or endothelial cells
endothelial cells) at endothelial surface i
Cell activation CD80l8Ec Cp83 (dendritie cells) F3lock interaction with
('1)28: At tints ofallografting to induce
blockade (dendritic antigen immuno~,uppression ~ tolerance
presenting cells) i
Inhibitory receptor Fc ganmta RII Fc epsilon I21 lnhance association of R of
acute of allergic disease.
activation (Mast cells) I (Mast cells) avtivaung and inhibitory Advantages
rner bispecific Fe fusion
rcce:ptors, reagents because of more specific cell
i tar~;eting . Sec Ihu D et aLNat ly1ed
?0U2 May:8(~lal8-21 )
Inhibitory receptor CTI.A-4 , C D8 Block C D80/86 interaction ~ Enhance
specifically CTI, mediated
blockade I v ith C l 1.,r1-~t ~ anti-tumor responses tvithout global 'I'
I ' cell activation. Anti-C"fI-A4 ahs now in
therapeutic trials to enhance tumor
_ imnntnitv
Adhesion ntolecule V("AM-I C'D31 (endothelial cells) or L:- Block VI,A-=4-
dependent f Acute Itx of MS flare-up. Anti-Vf.A-4
blockade (activated selectin (ttctivafid endothelium) ? cell and monocyle
adhesion to is in trial as 12x tar fy1S.
I i endothelial cell) VCAM-I c>n endothelial cells.
Adhesion molecule ~ IC':1M-I CDl l (endothelial cells) or E:- Block neutrophil
adhesion to i ~lcute Rx to reduce repwfusion injury
blockade )activated sclectin (activated endothelium activated ~ (myocardial
infarction, bowel
endothelial cell) . ischcmia~sur~y y)
Cell death ligand CI)9s1. l l as-ligand) C'D2.s (activated T cells) Block
activation induced cell Both ligands on same cell; CD95L
blockade ~ ( I cell ) death of I' i;ells: enhance anti- expressed on activated
. not restin5'f
tumor irnnnmitv crll_a
Protect bout IFN ;~ CDR I Block ll-'N-y toxicity towards ('ell selective block
of IFNy etlects;
another therapeutic: (soluble) endothelial cells during II -?
i.c. immunotoxins thertt v liu tuutors
Protect from 1FN-yR ~ CD31 ~ Block IFN-y toxicity towards Both ligands on same
cell. Cell
another therapeutic: (endothelial cell) (endothelial ml)) ~ endothelial cells
during II? selective block of iFNy effects;
therapy tier tumctn
Inhibitory receptor . I <iF-(~ -. CD4 or CDR Block imutunosuppressim I'C1 -
(?, ntodv of innnuuosupprcssion
blockade ~ eflvcts of tumors; enhance not clear
anti-tunu,r innnunity
I
_ -~-
Ccll type specific CD3 CD4sR() Trivalent ab so C'D3 can be I Anti-tunu>r Rx;
selectively entrance
activation ' (mcntory;'el7ector-f cells) cross-linked memory f cells: reduce
nonspecific
activation of irrclvent T cells
CA 02402930 2002-09-19
Category Functional (.'ell localizingMode of action Possible therapeutic
ligand ligand for high uses
for low affinity arm
affinity
arm
(location
of ti and)
Growth 11.-i s Growth factor I or Rx of alloerall
factor hlockadu fur rejection; 'f cull
CD8 T cell,
blockade ~ suhset specific
I specifically suppression will
ti,r C'U8 'h limit
cell
~
svlectim inluctious complications
subset: i.e. or unintended
innnuncrsup rcssion inhibiticm of ('D~l
regulators T cells
Cell activationCD80/86 C'I)83 (dcudritic At time
ofallot?ralling
cells) t3lock to induce
interaction
with C'D2R:
blockade (dendritic immunosupprussion tctlurancu_ I ar~etine
antigun to dundritic cells
resenting will enhance etlictencv/
culls)
Adhesion VCAM-1 C'D31 (endothelial
molecule cells) or I_'-
~
clock V LA-4-dependent
T Acute Rx of
MS flare-up.
Anti-VL.A-4
blockade (activated I
snleetin (activated
undotlwlium)
~ cell and utonocytu
adhesion to
~ is in trial
as Rs fitt MS.
endotheliali V(:'a\M-1 on
cull) endothelial
' culls.
Cell deathCD9sL (fas-ligand)CD?5 tactivatud
ligand T cellsl ; Block
activation induced
cell ! Enhance
anti-tumor immunity
blockade ( f cell) _ ~, death of
I cells;
Protect IFN-yR CI)31 ' Cell Block IFN-y to.vicity
from selective block tosvttrds
:,f IFNy
another (endothelial(endothelial endothelial culls
therapeutic:cell) , cell) durine 11.-? therapy
i
for tumors svfthout
impairin=other
_ useful IFN-v effects
l
InhibitoryFc ~amnrr E:c epsilon RI R x of acute
receptor RII ; Enhance association of allergic disease.
of
activation(Mast culls)(Mast cells) Advantages over
activating and hispecitie 1u fusion
inhibitory
i receptors. reagents because
of more specific
cull
i
tar~etin4_ . See
!he f) et aLNat
Iv1ed
2002 May:B(s la
18-? I )
The invention also contemplates that FAS can be selectively blocked on various
different types of cells
such as pancreas beta cells using markers such as GAD65, lA-2, IA2-B, ICA-12.
Type 1 Diabetes is
characterized by the destruction of insulin producing Beta Cells in the
pancreas. One method in which Beta
cells are destroyed is thou~~ht to be through apoptosis mediated by CD95
receptors on Beta cells. CD-9s
seems to be upregulated in Beta cells of those with Type 1 diabetes (see Ann N
Y Acad Sci 200' Apr 958
297-304; J Clin Itnmunol '?00 t Jan;? 1 ( I ): I S-8'). Similarly, usin~~
'I°g, TPO ligands as cell associared
markers CD95,TRAILR1, TRAILR2 can be blocked on thyroid cells.
Hashimoto's'fhyroiditis (liT) is
characterized by the destruction of thyroid hormone producing cells and
therefore hypothyroidism. It has
been observed that some of this cell destruction is due to apoptosis. The CD9s
receptor which is
responsible for apoptosis is up regulated in thyroids affected by HT. Blocking
the CD95 receptor by the
RLAA may reduce the amount of apoptosis. The HAA can target either Tg or TPO
which are unique to
thyroid tissue. There are also 2 other receptors suspected to be involved with
apoptosis in thyroid cells:
TRAILR1 and TRAILR2 (see Nat Rev Immunol 3002 Mar;2(.i):195-204)
Fas can also be selectively activated on distinct subsets of disease mediating
immune cells associated with
autoimmune and inflammatory disorders such as activated T cells, regulatory T
cells, CD4-~ cells, CD8+
cells etc.
Amon;,~ other embodiments hereinafter enumerated, the invention is also
directed to multifunctional li~~ands
which comprise antibodies which recognize cell type specific markers
(hereinbefore or hereinafter
exemplified), including those available commercially or published in the art,
in any combination with those
antibodies (hereinbefore or hereinafter exemplified, includin<, those
available conunercially or published in
the art) that recognize ligands on a broader population of cells including the
cell population bearing the cell
type specific marker, wherein the instrinsic affinity difference is maintained
or adjusted to one, tv~o. three,
four, five, six or seven orders of magnitude. The invention contemplates that
affinity differences (increases
or decreases in affinity) can be readily generate(1 by modifying amino acid:;
that are expected a piori to
result in changes in affinity (see for example Chowdhury P. et al. Improving
antibody aftinitv by
mimickin<~r somatic hypermutation in vitro ( 1999) Nature Biotechnol(tgy Jun:
17 p.568-572'), or by a variety
of other well-known high throughput methods that can readily be applied tt~
this task, for example by light
chain shuttling, CDR grafting, parsimonious mutagenesis, shotgun scanning
mutagenesis etc. Methods of
affinity maturation are well kwown in the art. The invention also contemplates
that two hybrisoma derived
antibodies can be digested e;~. with pepsin to create F(ab')? and can be
chemically recombined to create
bispecific antibodies. Hybridoma fusion technolo~~y can also be used to create
to tetromas for this purposes.
In this regard, the invention contemplates that hvbridoma derived I~,G
antibodies can be used for the
relatively hi~~h affinity cell targeting moiety and that hybridoma derived
I~~Ms can be used for the relatively
low affinity binding arm. ~l'he invention also contemplates that the repective
high and low affinity arms can
be conjugated. fused etcaccording to well known methods to respective
complementary ligands such as
CA 02402930 2002-09-19
fasljun strepavidin/biotin (making it possible to pre-target the high affinity
arm independently, particularly
where the aftinity of the complementary li~~ands for each other is ~~reater
than that of the second ligand
binding moiety for its target ligand.
According to one embodiment of the invention, the first liv'and binding moiety
can bind to a cell specific
marker and and said second ligand binding moiety binds to the extrracellular
portion of a li<~and involved
involved in membrane transport across the cell membrane, for example an ion
channel. vitamin recepetor
etc. to inhibit uptake or export. F'or example the first ligand binding moiety
may bind to a cancer cell
specific marker and the second ligand binsin~~ moiety can bind to p-
glycoprotein. Hi<~h affinity tumor
specific antibodies are well known and anti-PGP antibodies are well known (see
for exarnple Chen Y et al.
J Cell Biol Mar 6; l48('5):863-70: Mickisch GFI et al. C.';tncer Res. 1992 5?:
3768-3775). Some anti-PGP
antibodies function by by interfering with antibody utilization.
It will also be appreciated that a cell specific marker need not differentiate
between sub-populations of cells
that do not express the second target ligand eventhough those cells are not
the targets of action of the
multispecitic ligand.
Examples of antibodies that bind to cell specitic ligands, receptors. ete. are
abundant and well known in the
art (see for example Biosource International 3002 Research Products Catalog
e.g. pages 178-19;, Upstate
Cell SiSnalling Solutions 2002 Cataio~T and other catalogs of well known to
those skilled in the art)
Some examples are appended to the end of the disclosure and others are
mentioned or referenced
throughout the disclosure.
Definitions
The term "associated" in relation to markers that are dominantly distributed
on one or more particular
entities is used to mean exclusively expressed, primarily expressed, or over-
expressed to advantage from a
tar~~eting standpoint.
The term "receptor li~~and" means a target ligand which is a li~~and for a
receptor, for example, a receptor
on a cell or infectious absent or a receptor which circulates independently of
another entity.
The term affinity is contrasted to functional affinity which may result from
avidity.
The term epitope though technically understood to be specitic for a given
antibody. is used in a preterred
embodiments to refer to antigenic determinants that are situated proximally to
one another so that two
antibodies will be considered to bind to the same epitope if one competively
inhibits the binding of the
other throu~~h any' probative competitive inhibition experiment known to those
skilled in the art.
The invention contemplates that two antibodies with the same epitope
specificity may have substantially
the same amino acid composition ie. with possible exception of one or more
additions, deletions or
substitutions including conservative amino acid substitutions which do not
substantially affect the
specificity and an tino acid composition ofthe paratope
The teen approximately in the context of orders of magnitude variations in
affinity refers a variability that
is up to a half an order or magnitude.
Without limiting the scope of the claims it is generally understood that
biodistribution ofa multispecific
ligand in contrast to that of a ligand will be predicated on the
bioavailability of its target ligand.
The term "overlap'' and related terms connote that notwithstanding the
difference in distributions of the
first and second li~ands the first and second ligands are bioavailable for
recognition on the same entity.
This term and related terms, exemplified below, are intended to exclude a
situation where boat ligands are
preferentially expressed on substantially the same entity, for example two
different tumor associated
antigens associated differentially with a differentiated population of cells
within a tumor , most particularly
in the case where they are individually suitable tartlets for delivery of a
toxic payload. Thu sthe terms
"different" in regard to biodistributions and "heterogeneous" and "diverse" in
reference to populations of
entities are similarly understood to exclude such a common distribution, in
the appreciation that the
invention primarily represents an improved strategy for tar~~eting two
different ligands, in which one li~and
CA 02402930 2002-09-19
has a broader distribution than the other or both have distributions that may
overlap but are different from
that of the target population. It will also be appreciated that the invention
has particular application to a
situation in which at least one of the non-target populations is one on w
which one of said first and second
ligands is substantially represented (in contrast to one on which it simply
enjoys limited expression).
The term "receptor ligand" means a target ligand w which is a ligand for a
receptor, for example, a receptor
on a cell or infectious agent or a receptor which circulates independently of
another entity.
The term "antigen binding fragment" refers to a polypeptide or a plurality of
associated polypeptides
comprising one or more portions of an antibody including at least one VH or VL
or a functional fragment
thereof.
A moiety that exerts a biologic function is understood to be a "biologic
effector" in the sense that its
intended interaction with an entity in the lymphatic system or elsewhere in
the organism has a biological
consequence.
The tern neutralizing in regard to an an immune function is used broadly to
refer to any interposition,
interference or impediment which affects the function of the target entity
The terms modulating" mediating, neutralizin~~ function etc. are not intended
to be mutully exclusive and
are each used broadly, for example, without limitin~~ the generality of the
scope accorder herein or by those
skilled in the art the term modulating preferably refers to effecting a
change, and the term mediating
preferably connotes an indirect effect achieved through the instrumentality of
another entity, for example a
cell. cvtokine. chemokine etc..
The term "preferentially binds" recognizes that a given ligand binding moiety
might have some non-
defeating cross-reactivities.
The term biologic effector ligands is used to refer to any li~.:and for which
there is a complementary target
ligand on a target entity, and wfierein binding of the biologic et~ector
ligand to the tartlet ligand exerts a
biologic effect. For example the target ligand is typically a receptor and
thf: biologic effector ligand may be
any complementary ligand such as a cytokine, chemokine, hormone, colony
stimulating factor, growth
factor. receptor inhibitor, a~~onistor antagonist, which binds to the receptor
with resultin~~ biolo~~ic effect.
The term "pre-selected" in reference to the affinity of ligand binding moiety
refers to any selection or
choice of differential or cooperative affinities relative to a second li«and
bindings moiety w which is generated
as a result of a mental or physical process or both, preferably through a
process of prediction or post-facto
validation of the effects of the choice of the first and second affinities
and/or more preferably throuv~h an
empirical evaluation of different choices for at least one of the first and
second affinities, and preferably
both.
The term multi means at least two and the term li~and is used broadly to refer
to any entity or patrt thereof
which can participate in an intermolecular interaction that can result in
specific binding of suitable affinity
for the interaction in question.
The term entity includes without limitation any molecule including without
limitation. antibodies, complex
or association of molecules, drugs, drug carriers (eg.vesicles eg. liposomes,
nanoparticles,ete.) or any cell
as well as any infectious a'rent or parasite (includinw, without limitation,
spores, viruses. baceria, fungi ) as
well as any other immune or therapeutic target.
The term "low affinity' means an affinity of approximately (this term is
defined herein) 10 -'' molar to
micromolar affinity, preferably (subject to safety considerations),
approximately, !0-' molar affinity, more
preferably (subject to safety considerations). approximately micromolar
affinity, the term "medium
affinity" means approximately l0-'to nanomolar affinity. preferably
approximately l0-'' molar affinity,
more preferably approxim<rtely nanomolar affinity, and the term "high
affinity" means approximately 10-"'
affinity or greater. Thus is one embodiment the invention contemplates that
the multispecific ligand
comprises a "target-ligand" bindin'T moiety which binds with low ~ or medium
affinity to a target li~~and
present on a diverse population of cells (preferably this moiety is an
effector moiety ie_ one w ~hich~exerts a
CA 02402930 2002-09-19
biological effect attributable to its binding eg. blocking or activatir~~ a
receptor or blocking a cell
membrane channel) and a "targeting", ligand binding moiety, which binds with
medium or high affinity to
a ligand associated with a sub-population of those cells so as to bias the
biodistribution of the
multifunctional li~and in favor of said sub-population. Preferably the
multispecific ligand is adapted to be
bound contemporaneously to the same cell. In another embodiment the tirst and
second li~~ands binding
moieties each bind to ligands present on diverse overlapping populations of
entities eg. cells ( ie. neither
ligand being preferentially associated with a target cell population) and arc
adapted to be bound
contemporaneously and to both bind individually with low affinity, so as to
bias the distribution ofthe
multispecific ligand to the population of cells bearing both ligands.
As discussed elsewhere the teen approximately. in reference to "order of
magnitude" increments in
affinity, refers to up to a halt order of magnitude in affinity.
According to another embodiment, the invention is directed to an antibody
termed a "coybody". A
"coybody" is an antibody in which the on-rate contribution to affinity of the
antibody is proportionally less
than the off-rate contribution relative to a reference antibody of the same
specificity and a greater affinity
of up to several orders of magnitude, preferably a reference antibody of
approximately une to three orders
of magnitude greater affinity, preferably a reference antibody of medium
affinity or preferably hi~~h
affinity. As discussed above the reference antibody preferably comprises
cooperating light and heavy
variable regions in which at least at least one of the CDRs of at least one of
the chains, preferably at least
the CDR3, preferably that of the heavy chain. is exclusively or primarily
responsible for the binding
affinity of the coybody preferably in conjunction with the contribution of <tt
least one of the CDRs of the
other chain, such that alterations in the length and or amino acid
compositions of one or more other non-
contributin~~ CDRs can be leveraged to diminish the on-rate, for example due
to steric and/or electrostatic
hindrance. In one embodiment the on-rate is reduced by a factor of 3 to 100x.
In one embodiment the
coybody binds to a ligand which is over-expressed on a tartlet population of
entities (eb. cells) relative to a
non-target population of entities such that the biodistribution of the coybody
to the non-tar~~et population
(and target population) is diminished in a <given increment of time following
administration. This targeting
strategy is understandably adapted to situations where the resultin~~ delay in
biodistribution is preferable for
diminished toxicity attributable to reduced non-target entity binding in a
<riven unit of time especially
where the effectiveness threshold in that same amount oftime is not
significantly if at all compromised or
is preferable due to a sustained release effect (for example using a larger
antibody format that is not readily
cleared) As discussed below, advantages accrue particularly when this antibody
is coupled to a hi_~~her
affinity antibody (in the form of a multifunctional ligand) which binds to a
different ligand associated with
the target population. The invention contemplates that coybodies have multiple
independent applications,
includin~~ tempering the effects through antibody mediated neutralization of
an over-production or sensivity
to biologic effector ligands (eg. cytokines eg. ~i~NF2,r,,,~, chemokines e_~.
IL-~16 (crohns disease) etc. which
are over-produced and!or mediate or aggravate eg. a chronic medical condition
(which for example is not
an acute phase) by bindings to such li~~ands, over a prolon~~ed periods,
preferably using lar<~er antibody
formats which are not readily cleared, especially where such tempering has
side effects which are better
spread over time and.~or where effectiveness is not a limiting factor and!or
where a second therapeutic with
different non-cumulative side-effects shares the therapeutic burden and!or
where a the same antibody with
a higher on-rate is used in combination.
The term "antibody' is used broadly, unless the context dictates otherwise, to
refer without limitation, to a
whole antibody of any class or biologic origin, or chimeric combinations of
antibody reunions or domains
(eg. FRs and CDRs) of dit~ferent origins or species eg. humanized, any
combination of one or more
antibody fragments or recombinant reconstructions (scFvs) of antibodies
including dimers, diabodies,
triabodies, a myriad of known bispecific, trispecific, tetraspecific antibody
formats or monovalent, divalent,
trivalent, tetravalent or other multivalent antibody formats (see for example
review in Krian'~kum J, et al.
Bispecific and bifunetional sin~~le chain recombinant antibodies. Biomol En~~
2001 Sep;18(2):31-40 and
others herein directly or otherwise referenced) or any fragment. portion, or
reconstruction of one or more
portions of an antibody (scFv) or any truncated form a li~,and binding entity,
such antibody typically
comprising at least a VN or VL portion or both or a functional portion of same
(eg microbodies). including
single domain antibodies, F(ab'),_ Fab, Fab', Facb, Fc, etc. The term antibody
alsov includes fusions of such
an antibody so defined and other functional moieties (eg. toxins, cytokines,
chemokines. streptavidin,
adhesion molecules).
According to one aspect, the invention is directed to a multispecific ligand
with at least two different
bindings speciticities for different tartlet ligands on the same target entity
eg. a cell and which is preferably
11
CA 02402930 2002-09-19
adapted to bind contemporaneously to (ie. there are no geometric or other
constraints which preclude both
moieties from functionally interactin~~ with their respective target li<~ands
at the same tirne)the different
target ligands, said multispecific ligand comprising a first target binding
moiety which preferentially(some
cross-reactivity(s) does not preclude the utility of the invention) recognizes
a first target ligand and a
second target binding moiety which preferentially recognizes a second target
li~~and. and wherein the ability
of the second target binding moiety to bind to the second target ligand is
diminished relative the ability of
the first target binding moiety to bind to the first target ligand, the first
target bindin ~; moiety having an
ability to bind to the first target ligand which is at least sufficient for
the first target moiety to bind to the
first target ligand independently of the second target bindin~,~ moiety
binding to the second target ligand and
an off rate (with respect to the first target ligand) which at least
sufficiently exceeds the on-rate of the
second target bindin~T moiety for the second tartlet ligand to at least
provide opportunity for the second
target moiety to bind the second tar~7et li~~and when the first target binding
moiety is bound to first target
li~~and, the second target binding moiety havin<~ a relatively diminished
ability to bind and; or stay bound to
the second target ligand independently of the binding of the first target
binding moiety to the first target
ligand (such that a plurality of the multispecific ligand will bind to a
population of cells bearing both target
ligands in preference to a population of cells bearing only the second target
li~~and (ie. <rt least in part duev
to the first target bindings moiety assisting (ie. providing opportunity) the
second tar<~et binding moiety to
bind to the second tartlet ligand and preferably out of proportion to what
could be statistically attributed to
the presence of two targets ligands on the target cell e~,. the binding of the
first target bindint=, moiety
providing necessary assistance for the second target moiety to bind is
relatively increased (ie. relative to the
situation where both of are of comparable affinity).
It will be appreciated that relative number of bioavailable second target
ligands relative to the number of
the bioavailable first target ligands will influence the selection of
affinities of the first and second target
binding moieties. For example, from the standpoint of safety. the affinity of
the first target binding moiety
for the first target ligand may well be sufficient if initially
approximatingnanomolar affinity and the affinity
of the second target binding moiety for the second target ligand will be
selected to limit the number of
effective binding events on the population of cells bearing only the second
tarp>et moiety: an affinity which
is inversely proportional to the number of bioavailable second tartlet
ligan<ls on the population oi~cells
bearing only the second target ligand ie. the non-target population (relative
to the number of first target
ligands on the target population of cells). For example, this may be assessed
by determining the amount of
labelled multispecific li~~and on the target and non-target populations of
cells in vivo (or in vitro where the
number of bioavailable first and second target ligands can be roughly
estimated). This selected affinity,
from a effectiveness point of view, will then be assessed as to whether it is
sufficient for the second ligand
binding moiety to bind to the second tartlet ligand on the tartlet population
of cells, with the benefit of the
first ligand bindings moiety bound or having been bound to first tar«et
(igand. For example, where the
binding of the second target binding moiety may be assessed through an in
vitro assay (cg. an assay in
which the blockin~~ or activatin~~ of a receptor is rl~easurable eg. through
inhibition of binding of the natural
ligand for a target receptor or through some measurable parameter associated
with effective binding for
example the release of cytokines or other biologic effector li«and. The effect
of binding may be also be
assessed by comparing the effects over time relative to a hi«her affinity
second bindings moiety v~ hich is not
associated with a first ligand bindings moiety. It will be appreciated that a
more ubiquitous second target
ligand may require selecting a higher initial affinity of the first target
binding moiety for the first target
ligand eg. picomolar affinity, and selectin;~ an affinity of the second target
li'~and which may for example
be of micromolar aftinity pluslminus approximately one order of magnitude. It
will also be appreciated
that the deleterious effects of non-target cell binding will vary as will the
degree to which the first target
ligand is uniquely found on the tar<,et population of cells. In the tinal
analysis a suitable difference in
affinity between the two binding affinities may well be at least,
approximately, one, two, three. four, five,
six, seven or eight orders of magnitude. In this connection the term
approximately refers to -'- up to a half
order of magnitude (<Sx). As discussed below, the invention contemplates that
variants of a dual af~frnity
multispecific ligand may be assessed in a high throu~~hput screen or series
ofsuch screens with a view to
selecting a variant that has one or more predefined properties. alluded to
above such as a) the ability to
mediate a biologic effect on a target population relative to a negative
control; b) the ability to mediate an
improved or diminished biologic effect on a target population relative to a
positive control. 'l his ability
may also be assessed in a competition experiment of any probative type well-
known to those skilled in the
art: c) the inability or diminished ability to mediate a biologic effect on a
non-target population relative to
negative and positive controls. Such diminished ability may be also assessed
in a competition experiment
of any probative type well known to those skilled in the art:~d) the ability
to target a target population
through binding relative to controls and in a competition; e) the inability or
diminished ability to target a
non-target population relative to controls and in such competition
experiement. The invention also
12
CA 02402930 2002-09-19
contemplates that the multispecific ligand may bind to a ligand which is cell
specific in the sense that it
binds to cells to which it has been delivered by prior administration (eg an
antibody or fusion protein
thereof which only binds to the target cells or at least to cells which do not
have the ligand recognized by
the second ligand binding moiety present in any significant amount), akin to
the pre-targetting strategies
well known in the art. For example, this strategy could be used to increase:
the number of first ligands
relative to second li~~ands, where indicated.
In one embodiment, said first target binding moiety recognizes an entity-
associated ligand eg. a target cell-
associated* target ligand, for example a ligand which is exclusively
expressed, primarily expressed or over-
expressed to advantage on the target cell population and said second target
binding moiety recognizes a
non-target cell-associated target ligand which is present on target cells and
non-target cells, for example a
receptor, including a decoy receptor eg. for TRAIL. The multispecific ligand
is thereby adapted to block or
activate the receptor primarily on the target population of cells. In this
connection, the invention is also
directed to methods of evaluating or implementing the effects of this enhanced
selectivity for the receptor
on the target cell population and can be employed to diminish the adverse
consequences and evaluate the
benefits associated with using a ligand binding moiety that would otherwise
undesirably bind to receptors
on non-target cells.
The invention contemplates that a variety of different strategies that can be
used alone. or in any variety of
compatible permutations to dififerentiate between target cells and/or between
target and non-target cells.
The choice of strategies, may depend at least in part on the circumstances.
including the nature of the fluid
environment in question, including the rapidity and pressure of flow and the
directions) of this pressure,
the method of delivery, the medical condition for which the molecule is being
evaluated_ whether the target
is moving or stationary, or both, the location or various locations of the
target, the targeting venue or
venues that is/are most effective and the importance of the size of the
molecule for reaching the target as
well as bioavailablility, and the importance of creating immunoconjugates and
immunofusions with other
molecules (insofar as this affects the size and distribution of weight in the
molecule). The invention
contemplates Chat employing more than one than one type of construct may be
desirable and the invention
is therefore directed to the various combinations and permutation of
constructs according to the invention,
in combination with each other and other therapeutic molecules or modalities.
One of constructs
contemplated by the invention, is a multispecific antibody, for example a
bispecific antibody havin<> a
configuration which allows for binding to two antigens on the same cell. for
example a traditional four
chain immunoglobulin configuration having a hinge region (including F(ab')~
minibodies etc.), a diabody
configuration (depending on the relative positions of the target ligands) and
others herein referenced and,
known to those skilled in the ar-t. It will also be appreciated that the mode
of action of the multifunctional
ligand may be contributed to by fusing or conjugating the multifunctional
ligand to another functional
moiety, for example, as described in the literature referenced below. These
supplementary strategies are set
forth below:
Additional Strategies For Modifying Targeting Capabilities
According to one embodiment, the intrinsic affinity of the first target
binding moiety for the first target is
greater than the intrinsic affinity of the second target binding moiety for
the second target. The term ~
"intrinsic" affinity connotes a measure of the affinity of a given target
binding moiety for its target ligand
which is independent of the affinity of the at least one other target binding
moiety for its target ligand and
as used herein could theoretically be evaluated in the context of the
multispecificJ li<=and as a whole, if the
other target binding moiety had an irrelevant specificity and therefore could
not bind to its target ligand.
The invention contemplates that at least approximately onc, two, three, four,
five, six, seven or eight orders
of magnitude. differences in "intrinsic affinity" may be required to
accomplish the targeting objectives of
the invention.
According to another embodiment, the relative on-rate* of the first target
binding moiety is greater than the
relative on-rate of the second target binding moiety. 'The term relative on
rate is used to connote an
effective difference in on-rate that may be instrinsic to the individual
target binding ligand or may
attributable to its configuration or relationship vis-a-vis other parts of the
molecule.
13
CA 02402930 2002-09-19
Where the intrinsic on-rates of the first target binding moiety is greater
than the intrinsic on-rate of the
second target binding moiety, the invention contemplates that the off-rate
contribution to the affinity of the
second target bindings moiety may be proportionally greater than the off rate
contribution to the affinity of
the first target binding moiety. The invention contemplates that the binding
of the second target ligand
binding moiety to its target ligand may be more effective if its lower
affinity is attributable in part due its
reduced on-rate. The invention contemplates methods for reducing the affinity
a target binding moiety by
reducing its on rate for example by mutating or adding amino acid residues in
reunions of the VI-i or VL that
don't directly contribute to the off-rate (of a relatively high aftinity
binder for the target. for example, as
determined by modeling and structural analysis, for example, by evaluating, x-
ray crystal structure and
evaluating NMR data of the binding, or by mutagenesis, preferably by
introducing a diversity of changes in
a high-throughput manner (eg. phage display, ribsome display,microarray or
other expression library)
including substitutions, additions and deletions within various regions of the
VH or VL and determining
their effect. For example, the invention contemplates that the second target
binding moiety is generated
using a library characterized by members in which one of the red=ions of V H
or Vl.. including particularly
the CDRI and CDR?, for example the CDR1 of the VH or CDR2 ofthe VL, is
shortened andior mutated in
a manner to reduce the probability of its having any direct contribution to
the affinity of the selected
molecule (throu~~h molecular interaction). for example mutated to introduce
amino acids that are least
important for intermolecular interactions, for example by minimizing the
occurrence of amino acids that are
important for electrostatic interactions and optionally also hydrogen,
bindings, generating a binder whose
affinity will be postulated to be independent of the contribution of the
modified CDR, and then optionally
evaluating the success of this latter step through further mutagenesis (this
step is most revealing if the CDR
is shortened but not. mutated or mutated to introduce amino acids important
for intermolecular interactions)
and then using.: the library to incrementally lengthen the reunion and%or
introduce amino acids important for
intermolecular interaction at a distance (eg. electrostatic interactions and
optionally also hydrogen binding)
to introduce minimal steric hindrance or intermolecular repulsion. The
invention also contemplates that
introducing amino acids that have the greatest potential for hydrogen bonding
may introduce an aqueous
cushion into the interface region with the target ligand to diminish the on-
rate contribution to affinity. The
invention also contemplates modifying the amino acid composition of an
existing binder by introducing or
one or amino acids or mutations into a framework region at a location which is
proximal to the binding
region or a region which borders the interface of approach to the binding
rf,~~ion or any interface between
the target binding moiety and the target ligand. The invention conternplate~s
that the on-rate and off-rate can
be routinely measured using various technologies (e~~. Biacore) known to those
skilled in the art, including
various techniques of measurin~~ these rates in real-tune. for example those
that rneasurc the de;tlection
pattern of an incident form of radiation (e~~. Biosite). In one embodiment of
the method the antibodies each
have unique preferably cleavable peptide tags that are generated for example
through a random or partially
random insertion of nucleotides into the DNA encoding the antibody and that
serve to link there to their
DNA eg a phage (as per techniques known to those skilled artisans or published
in the art) and the
antibodies are evaluated independently of a phage (eg. they may even be
cleavable from the pha;~e) or other
expression system linkage which allows a more accurate measure of their true
on rates and off-rates. The
invention also contemplates that FR I could be lengthened in a relatively high
affinity second target binding
moiety to reduce its on rate. The cleanable peptide could be a unique
identifying CDR.
In another aspect the invention contemplates that the multispecitic liV~and
may comprise an Fe portion and a
hinge portion and that one or both of a) the length, amino acid composition
or* molecular weight (or
various combinations of these interrelated factors) of the Fab or Fc portion;
and b) the amino acid
composition (including len~~th) of the hinge portion (eg. any polypeptide
segment that provides means for
linking two typically heavy chains, eg. through one or more disulfide bonds,
leucine zipper fos-jun,
optionally a flexible hin~eJtypical of an IgG 1 or having one to several more
disulfide bonds eg. 1V~G3) are
selected to reduce the circurnstantial(shear rate. presence of de<~rading
enzymes) affinity of the second
ligand bindings moiety where the firsC ligand binding moiety is unbound
relative to the circumstantial
affinity of the second ligand binding moiety where the first ligand bindin;~
moiety is bound. The term
circumstantial affinity broadly contemplates that the length and molecular
weight of the Fc and the
flexibility ofthe hinge re<~ion will individually and collectively contribute
to the affinity ofthe molecule in
proportion the shear rate of the fluid environment to a degree dependinv~ on
whether the target is stationary
or moving, once the multispeciCc ligand is bound. (fbound via the second
tartlet binding moiety, any
increase in the molecular w~i~~ht especially a distribution of the molecular
weight towards the Fc or first
ligand binding moiety will serve as a lever in a moving fluid environment, to
favor disengagement from
The actual on-rate if the on-rate was to be measured independently of the on-
rate of the other binding
moiety
14
CA 02402930 2002-09-19
binding especially since the off-rate of this binding arm is relatively low to
begin with. 'This same lever
effect will impinge on the binding of the first ligand binding moiety but to a
lesser functional degree due to
its higher affinity. To an extent depending on the context in which binding
occurs, the invention also
contemplates that the high affinity ligand binding moiety will draw the
multispecitic ligand from the
circulation into a desired target tissue and that the low affinity binding
arnr will then have greater
opportunity to bind even if it does not bind simultaneously with the high
affinity binding arm. Where the
hinge region is extra flexible or has several regions of flexibility (for
example where the heavy chains are
finked through several disulfide bonds with regions of flexibiie linker
therebetween) the disenga~~ing effect
on the individual and paired binding of both the first and second ligand
binding moieties will be less
Similarly, using a truncated Fc porrtion (CH3 deleted, flab'), or minibody
format) will assist the tirst ligand
binding moiety to remain bound or foster binding of the second ligand moiety
and will assist the second
ligand binding moiety to remain bound. This construct rnay be preferred from
an effectiveness standpoint
(getting both ligand binding moieties bound), where the affinity of the second
ligand binding moiety is low
to begin with.On the other hand, decreasing the flexibility of the hinge
region by alteration to its length
and.'or amino acid composition and increasin~~ the molecular weight
distribution towards the wfree'' end of
the Fc will affect all binding, scenarios to a greater extent. The latter
strategy may be less desirable where
the Fab of the first ligand binding moiety is lengthened leg. has a longer
hinge reunion at the N-terminus of
the disulphide bond linking the heavy chains. than the: low affinity binding
arm) to increase its propensity
for individual binding. For example, in a conventional four chain or heavy
chain antibody (two heavy
chains but no light chains) the hin~~e reunion could be len«thened or
shortened on the amino terminus side
of the disulfide bond linking the heavy chains to an extent that does
interfere with the simultaneous binding
to both the first and second target binding moieties. ~fhe invention also
contemplates that the target cell
environment, naturally or through intervention, is a fluid environment (low
shear rate) or enzyme
environment which will favor a greater impact on disengagement of the second
ligand binding moiety, in
the case of an enzyme, one which will cleave off an Fc into which a cleavage
site has been introduced so
that disengagement due to the lever effect will primarily impinge on binding
of the second ligand moiety to
the non-target cell population (e~~. low shear rate or presence of MMP type
enzymes in a tar~~eted solid
tumor environment).
The invention also contemplates that second ligand bindin<~ moiety may be
selected in an environment in
which there is a selective pressure (moderate fluid flow eg~usin~~ lyive cells
or tissue, candidate ligand
binding molecules or pairs of the tar~Tet ligands on latex beads, where the
substrate to which they are bound
is on an incline or otherwise subject to fluid flow (optionally with rigid or
high r710I. weight Fe), for
simultaneous binding so that the affinity of the second ligand binding moiety
is selected on the basis of its
ability to augment the binding affinity of a first ligand binding moiety of
preselected aftinity for the first
target ligand (after or optionally before its aftinity maturation, depending
on the shear force and affinity in
question) and thereby augment the affinity of the multispecitic binding ligand
as a wfiole, while the tirst
ligand binding moiety is bound. (n this way, the strength of the binding
affinity ofthe second ligand may be
predicated on the tirst li~~and moiety bein;~ bound. 'fhe foregoin~~ strate~~y
may have accentuated or at least
equal application where the first ligand binding moiety has a longer Fab or
for example where both the first
and second li;_,~and binding_ moiety are devoid of a li~~ht chain ie. where
havin~~ the correct bindings interface
for the second target binding moiety might be more acute. The invention
contemplates that the individual
affinity of second Iigand birtdiry~ moiety selected in the above manner would
be tested to ensure that its
individual affinity was not sufficient for substantial independent targeting.
The invention also contemplates that engineering a suitable affinity antibody
for solid tumor targeting in
which the on-rate contribution to affinity is reduced (according to the
strategy suggested above) may assist
a dose of such antibody in achieving better tumor penetration. An antibods
havin~~ a reduced on rate could
be fused to a toxin such as a truncated version of PE or conju~.;ated to a
radionuclide, etc. the reduced on-
rate contribution ensuring that the antibody will be less likely to bind at
sites proximal to the point of entry
to relieve congestion in that area and better ensure its diffusion throughout
a tumor. The invention
contemplates that the strategies decribed above will better permit the
affinity to be more suitably
apportioned between the on-rate and the off rate. The invention contemplated
that a higher on-rate lower
off rate Ab could be delievered in alternating days or other cycles of
treatment. Thus the invention is
directed to an antibody conjugated or fused to a functional moiety, wherein
the on-rate contribution to the
affinity of the antibody is anywhere between 3x and two order of magnitudes
less than typical molecules
havin~~ suitable properties fbr tumor penetration through diffusion. for
example molecules having anywhere
(any increments) between I O~ and 10-"' molar attinities leg. ~x 10 '', 3x
10~") preferably increments between
10-R to 10~'° (molecules where the on rate is norrttally approx. 10-')
molar affinities. more preferably
increments betty been 5x 10-'~ and Sx I 0-'
CA 02402930 2002-09-19
It will be appreciated that the foregoing strategies could be employed for
designing a multispecific ligand
which will primarily tary~et cells which have both the tirst and second target
ligand (cg. where the ligands
together are present primarily on the target cell population) even where
neither target ligand is individually
found primarily on the target cell population, by employing a multispecitic
ligand in which neither target
ligand is of sufficient affinity in the circumstances to effectively (with
effect) bind or remain bound without
the other target ligand being available for simultaneous binding. ~1s
suggested above, it will be appreciated
that a relatively higher af~tinity ligand could initially be employed on one
of the ligand binding anus to
select a second li;~and binding arm which improves the binding properties of
the multispecific ligand under
a suitable biologically relevant shear stress and which is selected or later
modified so that it is individually
insufficient for targeting its target on non-target cells in the circumstances
in which it will be employed,
and that the high affinity ligand binding arm can subsequently be reduced to
moderate affinity with similar
lack of individual effect. In one embodiment, this construct can be employed
to evaluate the effect of
blocking two receptors on the same cell, for example chemokine receptors cg.
CCR7 and CXCR4 on a
breast cancer cell. In one embodiment. the off rate of one or optionally both
li~and binding moities is
sufficient in the circumstances to permit the mc»ety to remain bound for a
sufficient duration for the other
moiety to bind ie. it exceeds its effective or intrinsic on-rate. In one
embodiment, both arms of such
multispecitic ligand, bind to their respective ligands with low affinity. In
one embodiment, one such arm is
a "coybody...
In connection with the foregoing and ensuing strategies it will also be
appreciated that the hinge region may
be lengthened on the N-terminal side of the most N-terminus linker between the
heavy chains so as to
permit greater flexibility in the binding of different anti«ens at different
possible proximities to one
another.
'fhe invention also contemplates that the two heavy chains of an IgG (with or
without light chains an d,'or
CHIiCL domains), minibody' F(ab')~ (with or without light chains and!or
C111/CL domains), may be
linked (whether they have a full size or fully truncated Fc or elon<~ated
hinge regions) through a flexible
peptide linker (such as used for makings seFvs l.c. multiples ofgly~ser) in
order to ensure correct pairing of
the heavy chains by expressing the linked heavy chains in E. Coli, for
example, as inclusion bodies, which
are refolded in refolding solution according to well established techniques in
the art. In a construct
employing light chains, the light chains may be linked through a disulphide
bond linking according to well
known methods of makin« disulphide stabilized Fvs (dsl=vs) and the same li~~ht
chain may be employed for
both the high and low affinity arms.
With respect to each of the preceding aspects of the invention, the invention
also directed to a multispecific
ligand comprising a tirst li~~and moiety which recognizes a tirst target
ligand that is over-expressed on a
disease associated entity for example a diseased or disease-causin~~ or
mediating cell or infectious absent and
a second ligand binding moiety that recognizes a target ligand and wherein the
first tartlet ligand is
characterized in that it does not lend itself to facilitating or permitting
internalization of the second li~~and
binding moiety.
The invention also contemplates that a target li<~and can be distributed in
various concentrations for testing
purposes on cell sized latex beads, columnar packing materials or flat
substrates havin<'~ a high density
dispersion of both target ligands.
The invention is also directed to combination therapies with the foregoing
multispecitic ligands including,
without limitation, immunotoxins, drugs, therapies with other multispecitic
ligands herein described and
particularly for cancer therapies directed at interfering with the inte~~rity
of tumor cell vasculature.
Delivering Biologic Effector Ligands To A Target Entity
W ith respect to each of the precedin~~ aspects of the invention, the
invention also contemplates that the
second ligand binding moiety may be constituted in whole or in part by a
li~and which binds to a biologic
effector ligand (such as a cytokine, colony stimulatin~~ factor, chernokine,
growth factor etc. or related
extracellularly expressed regulatory molecules that control their expression
such as inhibitors, agonists,
16
CA 02402930 2002-09-19
antagonists of same, which may have corresponding biological receptors), the
ligand optionally having a
higher affinity 'for the biolo;~ic effector ligand than the affinity of that
biologic effector ligand for its
receptor, and wherein the ligand. combined with the biologic eftector ligand
(ie. bound thereto), has a
relatively diminished ability to bind and~~or stay bound to the receptor (the
second tar;: et li~~and)
independently of the binding of the first target binding moiety to the first
tar: et ligand eg. a lower affinity
of approximately one, two, three, four, five, six, seven or eight orders of
magnitude. The invention
contemplates that the foregoing construct can be used to deliver the biologic
effector ligand more
selectively to the tarjet cell population recognized by the first ligand
binding moiety. The second ligand
bindings moiety may be an antibody portion of a multispecific ligand of the
invention and the invention
contemplates that a library of second ligand binding, moieties, recognizing
multiple different epitopes on
the biologic effector ligand, can be screened for their ability to bind to the
biologic effector ligand, while it
is bound in situ to its receptor, for example, using a microarrary of such
antibodies, and the affinities of the
binders can be evaluated. The invention also contemplates that suitable
antibodies could be generated by
"panning'' (with an expression library, eg. phase display, ribosome display.
or other similar display systems
including yeast, bacterial, viral. cell based or cell-free display systems')
or otherwise screening (eg. using
antibody microarrays) against the biologic effeetor ligand while bound to its
receptor and screening for
their ability to bind to the biologic effector li;~and independently of its
receptor. Again. the affinities of the
antibody coupled to the-biologic effector ligand for the target receptor could
be evaluated. More generally,
the invention contemplates that an array of antibodies which recognize all
different epitopes on a given
biologic effector ligand could be generated and tested for their ability to
accommodate binding ofa
biologic effector ligand to a first but not a second in a related family of
receptors. This could be
accomplished by screening the array for one or more members that bind to the
biologic effcctor ligand
(BE L.) while bound to its receptor, and testing the identified members for
their ability to bind to the second
receptor, preferably by loading the biologic effector ligand onto an array of
those members pre-bound with
BEL and detecting those BEL, bound members for those which do and do not bind
to the second receptor.
Therefore the invention is also directed to an antibody which accommodates
binding of the BEL, to one
receptor but hinders the binding to at least one second receptor, preferably
by steric, charge or other inter-
molecular hindrance, attributable to the proximity of the antibody epitope on
the BEL to the BEL's receptor
binding site and optionally also the an Wino acid composition of the antibody
at that interface.
The invention contemplates that fluid flow can be simulated in a purification
or immunoaffinity column
packed with one or more known packin~~ materials to simulate flow over a
ligand coated substrate.
The invention also contemplates an apparatus and method for testing ligand
binding in a circulatin~~ fluid
environment in which the multispecitic ligands of the invention can be tested
and wherein a continous flow
of ligands, including target ligands, ligands ofthe invention and..~or ligand
bearinLg entities (eg. cells or
synthetic eg. latex spheres which can be adjusted to a cell size) to which one
or types of li'gands have been
affixedly associated accordingly to known'rnethods) can be generated. The
fluid contact interface of the
apparatus has a generally circular shape and is convex or otherwise capable of
containing the fluid and
thereby preferably permits fluid to flow around the surface continuously. For
example, this surface may be
enclosed with a bagel-shaped cylinder which is optionally open at a location
opposite; the fluid contact
surface for introducing and/or removing its contents, or it may completely
enclosed with the exception of
an access port. from which any air may optionally be displaced or evacuated.
The invention contemplates
that the apparatus (at least the fluid contact vessel) can be rotated or
oscillated (e~~. in an elliptical, oval or
similar shape welt known to those skilled in the arts of fluid mechanics and
related en'.;ineerin 'g arts) in a
variety of different planes or with rocking-like motion in multiple planes cu-
subject to peristaltic pressure
(ie. where flexible tubing is used) to generate a continuous, optionally turf'
ulence free fluid flow over the
fluid contact surface at selected rates simulating: the various shear rates of
arterial, venous, infra-lymphatic
flow (including different diameters of such vessels) or interstitial flow.
The; invention also contemplates
that the fluid contact surface may be provided with a 1 ) substrate for
linking ligands of the invention or
target ligands or ligand bearin~Ve entities to permit fluid flow trcross the
substrate in a plane suhstantially
parallel or confonoing to the axis of flow.
In another aspect the invention is directed to methods of making a
multispecific antibody in which:
a) the light chains are the same for both the Vl, domains . For example, the
light chains (assuming
the construct has two light chains) are generated for a first target binding
moiety e«, in one aspect
of the invention, the relatively high affinity binder, optionally ti-om a
light chain ~.:ermline
17
CA 02402930 2002-09-19
sequence, and this light chain is then coupled with a diversity of heavy
chains to select a pair of
chains which bind to the second target ligand, thereby constituting the second
ligand binding
moiety. which may be a relatively low affinity binder. An alternate or
concomitant strategy to
generate a lower affinity second ligand binding moiety would simply be to
substitute the light
chain of the first ligand binding moiety for that of the second ligand binding
moiety and to test the
affinity. In the case of a multispecitic which tartlet BELs to patrticular
tar~'et cells, where for
example, two high affinity binders are preferred, the heavy chain and light
chain bindings to the
BEL can be truncated correspondin~,ly at the Cf I1!CL, region so that the
VH/VL interlaces and
cysteines pairings these heavy and light chains are similarly spaced but
spaced differently from the
other VHiVL chains. By linking the heavy chains as explained above, all chains
will pair
correctly. It will be appreciated that the fore~oin~~ production strate~~ies
could be applied to the
production of heavy chain antibodies (two chains structures without associated
li~~ht chains),
wherein the heavy chains are from human or other species and that production
in this case could
be adapted to E. Coli. It will also be appreciated that deletion of a
substantial part of the CH i and
CL domains can be measured to provide a space for the BEL to sit in line with
the other Fab
which can be len<,thened in the linker or CLi 1 domain, as shown in Fi~~ure C.
fhe invention
contemplates that evaluation of a diversity of the first tarv~et binding
moiety can be accomplished
with the BEL place to best accommodate selection in the context of the entire
structure as a whole.
b) With respect to other methods to make bispecitic and bispecific fusions see
Antibody Fusion
Proteins Wiley-L.iss 1999 (infra) eg. particularly p 131 et seq., and Chapter
7 and the discussion,
Methodologies improving the correct pairing of heavy chains are well-known in
the art.
Such a construct could also be employed in conjunction with other functional
moieties fused or conjugated
thereto, for example toxins, cytokines, enzymes, prodrugs, radionuclides etc.
In one preferred embodiment, the invention is directed to a multispecific
ligand* with at least two different
binding speciticities for different target ligands* on the same tarp=et cell*
and adapted to bind
contemporaneously to the different target ligands, said multispecific ligand
comprising a first target binding
moiety which preferentially* recognizes a tirst target li~~antl and a second
tar'et binding moiety which
preferentially recognizes a second target ligand. and wherein said first
tarl;et binding moiety recognizes a
target cell-associated* tartlet ii<vand and said second target binding moiety
recognizes a non-cell-associated
target ligand w which is present on target cells and non-tary~et cells: and
wherein the ability of the second
target bindings moiety to bind to the second target is diminished relative the
ability of the first target binding
moiety to bind to the first target ligand, the first tartlet bindings moiety
havin~~ an ability to bind to the tirst
target ligand which is at least sufficient for the first tartlet moiety to
bind to the first tartlet li~~and
independently of the second target bindings moiety bindings to the second
target li~and and an oft-rate which
at least sufficiently exceeds the on-rate of the second target binding moiety
for the second tartlet ligand to
provide opportunity For the second target moiety to bind the second target
li~~and when the first target
binding moiety is bound to first target ligand, the second target binding
moiety having a relatively
diminished ability to bind or stay bound to the second target litand
independently of the binding of the first
target binding moiety to the first tartlet iigand, such that the
multifunctional ligand will bind to the target
population of cells in preference to the non-target population of cells. As
su<~~~ested above, the strategy
embodied in this preferred embodiment can also be employed in connection with
any one or any
combination of compatible strate~~ies reterred to above, to diminish in degree
the requirement of using a
low affinity second ligand binding's moiety.
In another aspect the invention is directed to heterofunctional li~~and
comprisin~~ a first moiety which binds
to a first tar~:et li~~and and a second moiety which binds to a second target
ligand, and wherein the affinity
or avidity or both the affinity and avidity of said first moiety are selected
to enable the tirst moiety to bind
to the tirst target li«and independently of the ability of said second moiety
to bind to the second target
ligand and wherein the relative avidity or affinity or both the affinity and
avidity of said second moiety are
selected or adjusted to substantially reduce the probability of the second
moiety bindings to the second
tartlet Iigand without the first moiety, first or substantially
contemporaneously. bindings to the first target
ligand. For example, in one embodiment the first moiety is divalent and the
second moiety is monovalent.
In one embodiment the affinity of the first moiety for its target ligand is
for example up to several orders of
magnitude greater than the affinity of the second moiety for its tartlet
ligand, as discussed below. In a
preferred embodiment both moieties are capable of binding to different tar<~et
li<:,Tands on the same cell, for
example as hereinafter specified. although in the case of tumor cell
tar~~etin~, particularly with respect to
cells that are ~rrow~in~~ adjacent to another the invention contemplates that.
tho first moiety may bind to one
cell and the second moiety may bind to a neighbouring= cell. Accordingly, in
the case of receptors requiring
18
CA 02402930 2002-09-19
cross-linking for biolo'~icai activity the invention contemplates that such
same cell interactions and adjacent
cell interactions are optionally accomplished when the second moiety is
bivalent. In one embodiment, at
least one of said first and second moities comprise one or more antibody
components. In another
embodiment, said tirst moiety binds to at least one cell-surface ligand which
differentiates between cells of
the same population or sub-population, for example, at least one ligand which
diffentiates which between
populations or sub-populations of immune cells (eg. see WO 01/21641, US
6156878), for example,
activated vs. non-activated, disease-associated or non-disease-associated
(over-expressing or uniquely
expressing certain receptors or other ligands rfor example cytokine or growth
factor receptors, particular
immuno~lobulin like molecules or MHC peptide complexes] or other
differentiating markers hereinafter
exemplified or apparent to those skilled in the art), anti said second moiety,
in virtue of its binding to the
second tartlet li~and, directly or indirectly exerts a biologic effect eg. a
therapeutic effect. for example are
immune modulatin<.~ effect. In a further preferred embodiment said second
moiety has a broader target cell
population than said first moiety E~~. see W'iley H. et al. Expression of CC
Chemokine Receptc>r-7 and
Lymph Node Metastasis..., J. Natl. Cancer lnst. 91:1638-1643; Moore MA
Bioessays 2001 Aug:2 3(8):674-
6. (The invention contemplates that by targeting CCR 7 receptor selectively on
tumor cells, for example
using a relatively hi~~h affinity bindings moiety for a tumor associated
antigen and a relatively low attinity
moiety which binds to and blocks CC R7 receptor, eg, when combined in therapy
with a chemotherapeutic
agent or an immunotixun for the same tumor, metastasis can be inhibited). For
example, in one
embodiment said first moiety binds to a tumor associated antigen on a tumor
cell and said second moiety
binds to a receptor which is found on the tumor cell but also on a broader
population of cells. In another
embodiment said tirst moiety binds to an antigen associated with particular
population of leukocytes and
said second moiety binds to a receptor which is found on that population of
cells but also on a broader
population of cells (e~~. apoptosis mediating receptors Journal of Immunology
1998 1(10:3-1. Nat Med 2001
Aug; 7(8)954-960, WO 01:85782: 1CAM-R WO 00!29020: see also WO 01185768, WO
01185908: WO
01/83755, WO 01!83560, WO 01129020; Vitale et al. Prpc. Nat. Acad. Sci. 2001
May 8; 98(101:5754-5769;
CCR2 see also USP 6312689; USP 6,294,655 Anti-interleukin-1 receptor
antagonist antibodies and uses
thereof; USP 6.262,239; USP 6,268.477) . In another embodiment the second
moiety does not necessarily
bind with lower affinity to its tar~'et however it may bind to a first ligand
which in turn binds to a second
ligand on a tartlet cell (e~~. a receptor on the tartlet cell eg. a cytokine,
chemokine or ~~row~th factor receptor),
for example the receptor being on the same cell to which he first moiety
binds, and it binds in a manner in
which it partially interferes with the bindings of the tirst li~~and to the
second li~~and and thereby directs or
retargets that first li~,~and to the second li~~and in a manner which
accomplishes the intended interaction of
the the first with the second li'~and (eg a signal transduction or blockin~4
interaction ie. the second moiety
causes the e~~. cytokine to bind to its receptor without en~~enderina the
biological effects attributable to
receptor bindings eg. si<~nal transduction, which may be assessed by assayin~~
for effects of eg. signal
transduction according to well established techniques in the, art) but less
competitively relative to the first
moiety so that the fwst moiety exerts a targeting function ix. where the first
ligand bound by the second
moiety binds to a broader than desired population of cells. 'The bindings of
the second moiety rnay also be
compatible with the first ligand binding to one cell surface lip=and but not
another e~z. see WO 00164946 the
contents of which are hereby incorporated by reference. The ability to
identify ligand residues of
importance to binding or residues other these, the alteration of which
rni~,~ht interfere with binding is well
established in the art. The invention contemplates varying, by high throughput
techniques e~~. phage
display, residues of an antibody that are not involved in tirst li;~and
bindings to create variants which can be
tested for partial interference w°ith first ligand bindings to the
second liganc! e~~, receptor binding.
Examples of receptors for blocking or activation by the targetin~~ methods
described herein include tyrosine
kinase type recptors. serine kinase type receptors, heterotrimeric G-protein
coupled receptors, receptors
bound to tyrosine kinase, fNF family receptors. notch family= receptors.
guanylate cyclase types, tyrosine
phosphatase types, adhesion receptors etc. (for example receptors see those
discussed in Cancer: Principles
and Practice of Oncolo~~y 6't' Ed. De Vita et al. Eds Lippincott 2001.
includin~~ particularly Chapter ;. 7 and
18, The Autoimmune Diseases. Academic Press Third Edition, Rose;~Mackav ISBN:
0125969236,
Immunology 6'~' Edition, Mosby 2001 Roitt et al. Eds; Molecular Mimicry.
Microbes & Autoimmunity by
Madeleine W. Cunningham (Editor). Robert S. Fujinami (Editor) December 2000,
amon~~ other references
hereininbelow identified). Further mention rnay also be made of interleukin
and interferon type receptors,
HGF receptor (see for example LJSP 6,214,344), CD45, CXC family receptors
including CXCRI and
CXCR2 receptors including 1L.-8 receptor, EGFRs, receptors far molecules with
functions in apoptosis or
homeostasis. receptors such as FGF which sensitize tumor cells to
chemotherapeutic agents, etc. It is
known for example to modify receptor ligands in a way which does not
int~°rfere with a si~~naling tunction
(the residues important for siulnaling may be known or can be readly
ascertained e~. see Retar<=eting
interleukin 13 for radioimmunodetection and radioimmutrotherapy of human high-
~~rade ~~liomas. Debinski
19
CA 02402930 2002-09-19
W, Thompson JP.CIin Cancer Res 1999 Oct;S( 10 Suppl):3143s-3147s) but reduces
the affinity of the
ligand for this receptor (see also WO 0119861 ). Alternatively. the second
moiety may be an antibody
which is algonistic or antagonistic and used to block, activate, neutrali-re
etc the receptor. With respect to
EGFR family,TNF family and other receptor targeting antibodies which are
capable of causing apoptosis
directly or indirectly, see US 5,876, I 58, WO 00120576, W096, 08515, WO O I
~a4808 (P75AIRM 1 ), WO
00/29020 (ICAM-R). W'0 99r'12973, CA 2236913 etc. The invention also
contemplates that the second
moiety may also be targeted to a specific portion of a receptor which
diffc;rentiates it from other receptors
of its class and more generally contemplates that the second moiety may
contribute to the targeting ability
of the multifunctional li~~and.
In another aspect, the invention also contemplates that the first moiety binds
to a target cell and said second
moiety binds to a li<~rand, for example a natural ligand, (eg. a cytokine or
chemokine circulating at normal
levels or at higher levels attributable to a disease or treatment of a disease
with another therapeutic
molecule) and retargets that ligand (for example, the ligand may be
retar~etted from circulation) to a
targeted cell. For example the invention comtemplates that Il.-2 may be
retargeted to I,AK cells or CTI,s
via a high affinity l..cu-19 binding first moiety. For example, antibodies
including fragments thereof which
bind to cytokines or other natural li~~ands for retargeting purposes (eg.
single domain antibodies) can be
made by phage display a~~ainst the cytokine or li'~and v\hile bound in situ to
its receptor, The invention also
contemplates that the affinity for the cytokine may he adjusted to re~~ulate
the degree of tar~~eting and that
serum samples may be evaluated to assess the degree of bound cytokine and the
relative de~~ree of bound
and unbound cytokine. Amon<~ other methods, for example, the invention
contemplates that a radiolabelied
multifunctional ligand may be used assess the amount of label associated with
the multifunctional ligand
when bound to the cytokine, by capturing the 'complex' with an antibody that
r~;co;~nizes both <jnti'~enic
determinants on both the cytokine and an adjacent portion of the ligand
binding thereto ie. forming a
composite epitope), such as may be generated by phase display and assessing
the amount of label relative
to the amount of captured complex.The invention also contemplates
administration of supplemental
amounts of natural ligand to compensate for the degree in which the ligand is
retargeted insofar as such
retargeting might impact negatively on immune or other physiological
processes.
In another aspect the invention contemplates that patients treated with
antibodies to a particular biologic
effector ligand e~~. a natural ligand e'~. a cytokine, for example TNFa, may
preferably be treated with a
multifunctional ligand having a first moiety which binds to at least one cell
type and a second moiety which
binds to a natural li~~and such as a cytokine for retargeting that cytokine to
that cell type, as in a
preventative method for treatin~~ a disease, e'~. cancer. In this respect the
invention contemplates that the
antibody is capable of bindin'= to the cytokine but once bound the
cytokin'°_, the cytc>kine is incapable andlor
only weakly capable of binding to its receptor and-or that the multifunctional
ligand also comprises a
higher affinity receptor blocking moiety to minimize ret;trgeting of the
primary disease site. In one
embodiment, the fiirst moiety binds with relatively hi<~her functional
aft7nity (ie. avidity. affinity. and!or
relatively advantageous binding capacity in virtue of multiple ligand binding
arms. each binding to
different li~~ands on the target cell) to ensure bindin'~t to the retarget
cell. In another embodiment the bound
cytokine is capable of binding to the cytokine receptor at the retarget site
but incapable of binding to the
receptor at the disease site owing to differences in the receptors at the two
sites. 'fhe nvention also
contemplates using: antibodies which interfere but do not preclude binding of
the biolo~~ic efftctor to
provide a less toxic effect.
For example. patients with Crohn's disease that are treated with anti-TNFct
(see for example. Expert Opin
Pharmacother 2000 May:I (4):61 i-22 and references cited therein) may be
treated according to the
invention with a bispecitic antibody havin~~, in addition to an anti-TNFa
binding moiety. which reduces the
affinity of the bound TNF for the receptor, but also an antibody moiety which
binds to tumor antigen which
is expressed on many different tumor types or optionally a trispecitic
antibody which additionally binds to
a second multi-carcinomic antigen. preferably ane which broadens the ran;;e of
targeting against prevalent
cancers. With respect to tumor antigens mention may be made of EGFR. EPCAM,
MU('INs, TAG-72,
CEA, H 1 1 among other known multicarcinomic antigens (see also Cancer:
Principles and Practice of
Oncology 6'r' Ed. De Vita et al. Eds Lippincott 3001 Chapters 18 and 20.5). In
another embodiment, the
second moiety differentially retargets a cytokine to one receptor in
preference to another, for example, to a
TNF receptor over-expressed or. tumor cells in preference to a TNF receptor
associated with C'rohns
disease. In a related but also independent aspect, the invention contemplates
a method of screening for an
antibody which preferentially binds to a ligand when bound to a first receptor
relative to another second
receptor by screening for antibodies (eg. by pha;~e display, ribosome display,
etc.) which bind to the ligand
e~~. a cytokine, when bound in situ to the first receptcar, and selecting
among them those that bind to the
CA 02402930 2002-09-19
ligand eg. cytokine but do not bind (substractive screening) or bind with
lesser affinity to the cytokine
when bound to the second receptor, as well as to antibodies and
multifunctional ligands created by this
method (see also USP 6,046,048 and WO 99:' 12973 and references cited therein
with respect to TNF
family of receptors). Variations in the extracellular domains of such
receptors are known and can be
ascertained by methods known to those skilled in the art.
Further with respect to multifunctional ligands having a higher affinity
targeting moiety relative to the
second ie. effector moiety, the second moiety may be for example an antibody
or other ligand which
interferes with the bindings ot'the regular li~~and for this receptor. For
example, the invention comtemplates
a first ligand bindin;~ moiety which recognizes activated T-cells and a second
li~~and binding moiety which
blocks the It..-16 receptor for testin~~ the effect on Crohns disease (or
alternatively activates an IL-16
receptor on those cells eg. by usin~~ a high affinity I1.-16 bound second
moiety which becomes relatively
low affinity 1L-16 receptor ligand when bound to the antibody, anain to test
the effect on Crohn's disease
(see Gut 2001 Dec. 49(6) 795-803) For example, in one embodiment. the
invention contemplates that the
second moiety hlocks a receptor that are found on cells other than the target
cell, the blockage of w which
leads to the apoptosis of or destruction of the cell eg. CD95 (eg. see June G.
et aL,Tar~~et cell-restricted
triggering of the CD95 (,4 PO-l;Fas) death receptor with bispecific antibody
fra~~ments C.'ancer Res 2001
Mar 1:(i1(5):1846-8). With respect to blocking insulin like growth factor
receptor, insulin receptor etc. see
The IGF system in thyroid cancer: new concepts. Vella V.. Mol Pathol 3001
Jun;54(3):121-4; Insulin
receptor isoform A, a newly recognized, hi~~h-affinity insulin-like <growth
factor II receptor in fetal and
cancer cells. Mol Cell Biol 1999 May:l9(5):3278-88: Expression of the insulin-
like growth factors and
their receptors in adenocarcinoma of the colon. Freier S Cut 1999
May:44(5):704-8; Pandini G.. Insulin
and insulin-like ~~rowth factor-I (IGF-l) receptor overexpression in breast
cancers leads to insulin; lGF-f
hybrid receptor overexpression: evidence for a second mechanism of IGF-1
signalingClin Cancer Res 1999
Jul;S(7):1935-44. With respect to tar~etin~~~ beta-1 inte~~rins see e~~.
Masurnoto A, et al Role of betal
integrins in adhesion and invasion of hepatocellular carcinoma cells.
Hepatolo~~y. 1999 .lan;29( I ):68-74.
Arao S, et al. Betal integrins play an essential role in adhesion and invasion
of pancreatic carcinoma cells.
Pancreas. 2000 Mar;20(2):129-37. Xie Y, Xie H. Characterization of a novel
monoclonal antibody raised
against human hepatocellular carcinoma. Hybridoma. 1 ~)98 Oct; l 7(5):43 7-44.
Peng H, et al Production and
characterization of anti-human hepatocellular carcinoma monoclonal antibodies.
1-tux Xi Yi Ke Da Xue
Xue Bao. 1990 Sep;21(3):259-62; W'hittard JD, Akiyama SK. Activation of betal
inte~~rins induces cell-
cell adhesion. Exp Cell Res. 3001 Feb 1:263( I ):65-76 Nejjari M, et al.
alpha6beta 1 integrin expression in
hepatocarcinoma cells: re~:.ulation and role in cell adhesion and migration.
Int J Cancer. 1999 Nov
12:83(4):518-25: Yao M et al Expression of the inte'~rin alphas subunit and
its mediated cell adloesion in
hepatocellular carcinoma. .I Cancer Res Clin Oncol. 1997;1? 3(8):435-40.
The invention also contemplates a method of optimizing the cooperative
affinities of respective binding
ligands of a multifunctional li~~and described herein and the len~~th of a
linker therebetween for the above
and applications described below by pha~~e or ribosome display etc. in which
the multifunctional ligand is a
single polypeptide chain, for example, two sin~.:le chain Fvs or~sin~~le
domain antibodies linked in sequence,
or a diabody (see USP 5,837.242). by varying the DNA seduence correspondin;~
to amino acids that
represent linker andlor for example CDR regions that are postulated to impact
on affinity according, to
methods and strategies that welt known in the art for affinity maturation.
These same strategies can be
employed for engineering low°er affinity molecules. Accordingly, more
generally the invention is directed
to a phage display or similar library (e~=. a ribosome display library or a
microarray) in which the
population of variants is a multispecitic ligand. includin~~ a multispecific
li~.:and accordin4~ to the invention
herein defined.
The term ligand binding moiety includes any ligand that can be used as a
targeting arm or to bind to the
second ligand for example vytokines. chemokines. etc and optionally those that
can be modified, preferably
by high throu~~hput means to adjust its affinity and!or specificity, including
scatTolds comprising a
polypeptide, a peptide. a carbohydrate, a ribonucleic acid, a lipid and a
small molecule. or a binding moiety
consisting essentially of a combination of such types of molecules. E xample
include non-immuno~lobulin
protein scaffolds such affibodies (see LJS 5831012, US 5958736. EP 0486625)
Trinectin domains (Phylos
Inc.). TCRs, peptide mimetics. peptides located within a antibody variable
reunion scaffold (eg. see
WO 02. 44197, especially w ith respect to selectively targeting interferon
alpha to specific cell populations)
peptide fusion proteins and stabilized polypeptide loops. disulfide stabilized
loops (eg. see W'O 99,'232?2):
all known in the art for such uses. These may be linked by peptide linkers of
for example 10 to 50 amino
acids. A variety of linkersincludin~~ flexible linkers such as multiples
ofd=ty~lser are well known in the art.
21
CA 02402930 2002-09-19
In another embodiment blockage of a receptor does not necessarily lead to cell
death but may lead only to
decreased or increased release of certain cytokines etc. for example as
mediated via the IL-6 receptor. In
another embodiment the second moiety may achieve the desired therapeutic
effect by constituting the
normal ligand for that receptor or a functional substitute. The multispecific
ligand may also be fused or
conjugated to a toxic moiety or other effector. In another or further
preferred embodiment, said'rst moiety
comprises two binding ligands (cg. one or both of which may be an antibody)
which respectively bind to
two different target li~ands each of which contributes to its total bindings
capacity and neither of which are
sutfiicient to efficiently target the the cell, for example a ligand which
binds to a specit7c MHC peptide
complex and a second reduced affinity ligand which binds to a li~~and on an
APC. This approach also
obviates the need to create hi~~h affinity ligand for a particular MHC petide
complex, although this can been
accomplished. In another or further preferred embodiment the target cell is an
immune cell and the second
moiety binds to a molecule involved in cellular adhesion, a cytokine receptor,
a li~and which stimulates the
activity of said immune cell. a ligand which inhibits the activity of said
immune cell, a li'~and which causes
one or more cytokines to be released, a li~~and which prevent one or more
cytokines from bein;~ released, a
ligand which causes or facilitates apoptosis of said immune cell or a ligand
which permits internalization of
said multispecitic ligand. In another preferred embodiment the
heterofunctional ligand is fuses) or
conjugated to a therapeutic agent or a moiety that binds to a therapeutuc
agent (exemplified below) or a
li~~and which effects bindin<~ to another immune cell, for example a T cell.
In another preferred
embodiment, the multispecific ligand is a bispecific antibody. a trispecfic
antibody or a tetraspecific
antibody. In a further preferred embodiment the first moiety binds to but is
incapable of modulating the
activity of said immune cell and said second moiety modulates the activity of
said immune cell
independently of said first moiety. In another preferred embodiment the
multispecitic li'~and further
comprises a moiety that binds to at least one ligand located on the
intraluminal surface of a lymphatic
vessel, preferably a lymphatic vessel associated li~and, as hereinafter
defined. In other aspects the
invention is directed to a pharmaceutical composition comprising such a
multispecific ligand and a
pharmaceutically acceptable carrier, a method of usin~~ the heterofunctiona)
li~~and in the preparation of a
pharmaceutical composition for treating a disease, and to a method of treating
a subject by administering
same in a therapeutically effective amount.
'fhe invention is also directed to a multispecific ligand which comprises a
first ligand binding moiety which
neutralizes a ligand e'~. a natural li'~and such as a cytokine, chemokine,
colony stimulating factor or grow=th
factor and a second ligand binding moiety which binds ro a cell marker
associated with a cell through
which the natural li',and exerts a deleterious affect. Preferably the affinity
for the first ligand bindin<~
moiety for the natural ligand will be greater than that ofthe second ligand
binding moiety for the cell
associated marker. Optionally the construct will be selected so that the
bindinya interfaces are pointed in
opposite directions and the lever effect is maximized, For example a
bispecilic construct where the heavy
chains are joined directly or through an inflexible linker and are optionally
linked to their respective
(optionally common respective li~,~ht chains) throu~~h a disulphide linker via
framework residues as is well
known in the art. Optionally, the molecular wei~~ht of the first ligand
binding moiety is substantially less or
mare (by at least I 0°io, preferably at least 30°,%. more
preferably at least 2S°%) and preferably less than that
of the second ligand bindin<~ moiety as may be effected or maximized through
mutating from higher to
lower (on the first ligand binding moiety) and lower to higher mot. wt
residues (on the second li'and
binding moiety), the native residues which are not exposed ( to avoid
immuno'~enicity) and not essential for
proper folding and Function of the VHIV 1. (as may be determined from the
de~~ree of conservation of such
residues among immunoglobulins of the species, through °~o frequency
tables available throw: h the Kabat
database and well known published determinations in this regard (see for
example Vv'O 02f~0545).
Examples include neutralizing It.-2 via a marker on activated T cells,
blockin~~ IL-15 via CD8-~- ~C' cells,
blockin'~r TNFalpha on mast cells; binding thrombin via activated endothelial
cells etc.
The term heterofunctional is used broadly to refer to a linand: 1 ) comprising
at least tw ~o functional
moieties that have different functions on different capacities to perform the
same function and ?) which is
typically and preferably heterospeeitic (having two binding speeificities)_
Unless the context dictates otherwise the term avidity when used in a
comparative, quantifiable or
controllable sense is used to refer the valency of the binding entity or
moiety. The term functional affinity
is used a composite term referrin~~ to a quantitative and contollable (thou~~h
not necessarily quantifiable,
especially when its consists of both avidity and affinity components)
propensity to specific binding
attributable to one or both cafavidity and affinity effects.
22
CA 02402930 2002-09-19
In another aspect, the invention contemplates that cells, particularly immune
cells, that are expected to be
present at or proximal to a disease site (eg. at the site where an immune cell
crosses the vascular endothelial
cell wall). in virtue of the disease or a therapeutic modality which is
employed in relation to the disease or a
concurrent disease, including cells that directly mediate the disease. may be
targetted in virtue of a marker
associated with such cells, eg.markers associated with activated immune cells
or disease mediating immune
cells eg. LEU-19, a marker associated with activated or killerT-cells, etc for
example with an antibody,
which is linked to a moiety that is capable of exerting a therapeutic effeca
in relation to the disease. for
example, an immunoliposome or an antibody linked to another therapeutic
delivery system (for example
example streptavidin or biotin fused, coated or conjugated entities or other
payload carrying entities (see
for example US patents 5439686, 6007845, 5879712, 5456917, 616550?, 5079005,
_5888500, 5861 159,
6193970, 6190692, 6,077, 499, WO OOG9413, WO 01;07084, etc.). For example, an
immunoiiposome
may carry one of or a combination of cytokines, chemokines, toxins or other
therapeutic molecules suitable
for treating the disease directly or indirectly, for example by attracting or
preventing the attraction,
activating, aner~~izing or otherwise modulating the activity of immune cells
for therapeutic or related
purposes. 'thus accordin~~ to another aspect, the invention is directed to a
multifunctional ligand
characterized in that it exerts an independent hiologic function said
multifunctional ligand comprising a
ligand which binds to a non-diseased disease associated cell and: a) a
therapeutic entity; b) a ligand which
binds to a therapeutic entity; or c) a li~~and which binds to a disease
mediating entity eg. a biologic effector
molecul which is released by the disease mediating entity or the diseased
cellve~, a cytokine or other BEL
which mediates or ag~~ravates a disease process. Preferably said
multifunctional li<,and comprises at least
two of a), b) or c) and preferably all three.
The term "independent" refers to a function which is primarily exerted in
relation to an entity other than the
entity that is targeted (save for possible entity associated side effects).
The invention contemplates that
targeting a cell which localizes to a disease site will better localize the
independent effect of~the targetin<~
ligand to that locale. For example, an antibody which binds to and neutralizes
a cytokind or other BEL
associated with Crohn's disease eg. TNF alpha at the disease locale if
tar_~eted to an activated CD4't T-cell
using a marker which identifies activated T-cells.
In another aspect the invention is directed to a heterofunctional ligand
comprising a first moiety which
specifically binds to at least a first target ligand on a first entity and a
second moiety which specifically
binds to at least a second tartlet ligand on a second entity, and wherein the
affinity or avidity or both the
affinity and avidity of said first moiety are selected to enable the first
moiety to bind to the at least orte first
target ligand independently of the ability of said second moiety to bind to
the at least one second tar<~et
ligand and wherein the avidity or affinity or both the affinity and avidity of
said second moiety are selected
to enable the second moiety to bind to the seeond entity in preference to the
first moiety binding to the first
entity when both first and second moieties are substantially contemporaneously
bound to the respective
first and second entities. In one embodiment the first moiety comprises at
least one li~_and preferably at
least one antibody which binds to a first cell, for example an intraluminal
lymphatic endothelial cell and the
second moiety comprises a ligand, preferably at least one antibody which binds
to a different cell, for
example a disease associated cell (hereinafter exemplified and meaning, unless
the context implies
otherwise, diseased cells or disease causing,_.. mediating (ie, having a role
whim is known to be intermediary
or indirectly facilitating eg. antigen presenting cells) or mitigating cells
(cells, typically immune cells,
which directly or indirectly counteract the diseased or disease causing or
mediating cells). In other aspects
the invention is directed to a pharmaceutical composition comprising such a
heterofunctional liwand and a
pharmaceutically acceptable carrier, a method of using the heterofunctional
li~~and in the preparation of a
pharmaceutical composition for treating a disease, and to a method of treating
a subject by administering
same in a therapeutically effective amount.
In another aspect the invention is directed to a multispecitic ligand
comprising a first moiety which
specifically binds to at least one first target ligand on a first entity (eg.
a lymphatic endothelial cell, a
diseased cell or a cell proximal to a site of disease) and a second moiety
which specifically binds to a
second target ligand or site on a second entity, and wherein the second entity
binds to a third target ligand,
and wherein the first and third target ligands may be on the same or different
entities eg. the same or
different cells, and wherein preferably the aftrnity or avidity or both the
affinity and avidity of said first
moiety are selected to enable the first moiety to bind to the first target
ligand independently of the ability of
said second moiety to bind to the second target ligand and independently of
the ability of the second moiety
to bind to the third target ligand (the first moiety optionally comprising
more than one li~:and (which may
be the same ligand or a different ligand) one or more of which are necessary
for binding and optionally
each of which is sufficient for specific binding) to corresponding first
target libands) and preferably
23
CA 02402930 2002-09-19
wherein I ) the avidity or affinity or both the affinity and avidity of said
first moiety is/are selected to enable
it to bind to the at least first target ligand in preference to the second
moiety binding to the third target
li~and when both said first and second moieties and the second entity are
substantially contemporaneously
bound to their respective target ligands eg. to effect a tranfer or ?) wherein
the avidity or affinity or both the
affinity and avidity of said second moiety for the second entity are selected
to enable the first moiety to
bind to the first entity in preference to the second moiety binding to the
second entity and,'or 3) w herein the
avidity or affinity or both the affinity and avidity of said second moiety for
the second entity are selected to
enable the second moiety to bind to the third target ligand in preference to
the second moiety binding the
second entity when both first and second moieties are substantially
contemporaneously bound to the
respective first and second entities and the second moiety is substantially
comtemporaneousiy bound to the
third target ligand), or 4) wherein both I ) and 2) above are both operative
conditions. In one embodiment,
the tirst entity is a diseased or disease causing, mediating or mitigating
cell, for example an immune cell
(the first moiety preferably binding to a particular population or sub-
population of the tirst target entity e~~.
the immune cell, tear example activated T cells), the tirst moiety optionally
comprising two or more ligands
which may be the same or different and which bind to two or more respective
first target cell surface
ligands (though not necessarily to any particular effect (and in one
embodiment to no effect at all) utker
than to better bind to and thereby target the cell, preferably in competition
with the second entity, which in
a preferred embodiment tor~ets a broader population of cells), and the second
entity e~. a biologic etfectore
liband is an entity that binds to a third target ligand. the third target
ligand preferably being expressed on
the surface of a cell for example the same immune cell, for example a natural
cell surface li~~and, to which
binding yields a desired etTect. for example a therapeutic advantage, the
second moiety being, fbr example,
the natural ligand for the cell surface ligand or functional mimitope or
antagonist or arsonist thereof, for
example a cytokine, the third target ligand in this case bein<~ a cytokine
receptor on the immune cell. The
invention is also directed to a method of"targeted delivery' of a therapeutic
entity to a cell in need of such
therapy by administering, said heterofunctional li~~and. In this respect
numerous therapeutic entities will be
apparent to those skilled in the art, only some ofi which are mentioned herein
by referring to the therapeutic
entity itself or by referrin~~ to the third target lip=and for which such
entity is known and available or readily
made by routine skill in the art. Optionally the heterofunctional li<~and (and
similarly in the case of other
multispecitic ligands of thv invention described above which are adapted to
deliver a BEL,) is delivered
with the second entity, preferably in the same composition (preferably bound).
In the case where the second
entity is a natural li~~and circulatin~~ in the path of delivery of the
heterofunetional li~~and, some proportion
(0-100%) of the heterofunctional li~.:and may be delivered without supplied
second entity, particularly if the
treatment or the disease generates an abundance of the natural li~and. In
another embodiment the first
moiety binds to a target li~~and on a stationary cell (for example a vascular
endothelial cell or a lymphatic
endothelial cell), preferably a tissue or cell type "associated" ligand (more
abundantly expressed uniquely
expressed on target cells relative to non-target cells). and the third target
li~,and and the second moiety are
cell-suface target and ligand therefor as stated above, for example the second
moiety binds to a cytokine
and the third target li~and is a cytokine receptar. for example on an immune
cell. In one embodiment the
first moiety binds to at leant one target ligand which differentiates between
populations or sub-populations
of immune cells and the second entityin virtue of its binding to the third
target ligand, directly or indirectly
exerts a therapeutic effect, for example by modulating the activity of said
immune cell. In another or further
preferred embodiment the tirst moiety is incapable of modulating the activity
of said immune cell and said
second entity modulates the activity of said innmune cell independently of
said first moiety. In another or
further preferred embodiment the second entity binds to a molecule involved in
cellular adhesion. a
cy~tokine receptor, a ligand which stimulates the activity of said immune
cell, a li~and which inhibits the
activity of said immune cell (eg. via anergy or tolerance mechanisms), a
li~and which causes one or more
cytokines to be released, a ligand which prevent one or more cytokines from
being released, a ligand which
causes or facilitates apoptosis of said immune celll, a ligand which permits
internalization of said
heterofunctional li«and. In another preferred embodiment the heterofunctional
li~~and is fused or conjugated
to a therapeutic agent or a moiety (eg. biotin, avidin) that binds to a th
erapeutuc agent (exemplitied below)
or a ligand which effects binding to another immune cell, for example a T
cell. In another preferred
embodiment, the heterofunctional ligand is a bispecific antibody, a trispecfic
antibody or a tetraspecit7c
antibody. In another preferred embodiment the heterofunctional ligand further
comprises a moiety that
binds to at least one li~~and located on the intralurninal surface of a
lymphatic vessel, preferably a lymphatic
vessel associated ligand, as hereinafter deFned.
In other aspects the invention is directed to a pharmaceutical composition
comprising such aforementioned
heterofunctional li~~and and a pharmaceutically acceptable carrier, a method
of usin~~ the heterofunctional
ligand in the preparation of a pharmaceutical composition for treating a
disease, and to a method of treating
a subject by administerin~~ same in a therapeutically effective amount. As
suggested below, the foregoing
24
CA 02402930 2002-09-19
strategy could be used in combination with other targW ing strategies herein
mentioned or known in the art.
The invention contemplates making antibodies to second entities, for example.
while bound to their natural
receptor, by phage or ribosome display, by methods as hereinafter disclosed.
In another aspect the invention is directed to a heterofunctional ligand
comprising at least a first moiety
which specifically binds to a first tarr~et ligand on a cell and a second
moiety which specifically binds to at
least a second target ligand on the same cell, and wherein the affinity or
avidity or both the affinity and
avidity of said first moiety and the affinity or avidity or both the affinity
and avidity of the second moiety
are selected to substantially reduce the probability of tyre either moiety sin
'~ly binding to its respective
ligand for a suffiicient duration or series of durations to accomplish the
function of said heteroi-unctional
ligand unless both first and second moieties are substantially
contemporaneously bound to the cell. In a
preferred embodiment the first moiety binds to at least one target li~~and
which differentiates between
populations or sub-populations of immune cells and the second moiety in virtue
of its binding to the second
target ligand. directly or indirectly exerts a therapeutic effect, for example
by modulatin~~ the activity of
said immune cell. In another or further preferred embodiment the first moiety
is incapable of modulating
the activity of said immune cell and said second moiety modulates the activity
of said immune cell
independently of said first moiety. In another or further preferred embodiment
the second moiety binds to a
BEL, for example a molecule involved in cellular adhesion, a cytokine
receptor, a li'~and which stimulates
the activity of said immune cell, a ligand which mhrbus the activity of said
immune cell (eg. via anergy or
tolerance mechanisms). a li~_and which causes one or more cytokines to be
released, a li~~and which prevent
one or more cytokines from being released, a ligand which causes or
facilitates apoptosis of said immune
celll, a ligand which permits internalization of said hcterofunctional ligand.
In another preferred
embodiment the heterofunctional ligand is fused or conjugated to a therapeutic
a~~ent or a moiety (eg.
biotin, avidin) that binds to a therapeutuc agent (exemplified below) or a
ligand which effects binding to
another immune cell, for example a T cell. In another preferred embodiment,
the heterofunctional ligand is
a bispecific antibody, a trispecfic antibody or a tetraspecific antibody. In
another preferred embodiment the
heterofunctional li~~and further comprises a moiety that binds to at least one
li~and located on the
intraluminal surface of a lymphatic vessel, preferably a lymphatic vessel
associated ligand, as hereinafter
defined. In other aspects the invention is directed to a pharmaceutical
composition comprising such a
heterofunctional li~~and and a pharmaceutically acceptable carrier, a method
of using the heterofunctional
ligand in the preparation of a pharmaceutical composition for treating a
disease, and to a method of treating
a subject by administErin~~ same in a therapeutically effective amount.
In other aspects the invention is directed to a method of in vivo modeling or
testing using one or more
foregoing targeting strate~~ies by administering a heterofunctional ;
multifunctional li'~and as hereinbelow
disclosed as well as a method of infra-lymphatic drug delivery employinz~ such
ligand and such strategies
including adaptations thereof for such purposes, as hereinafter described. In
related aspects the invention is
directed to a test ligand in the form of such a heterofunctional '
multifunctional li~~and and compositions
thereof
In one aspect, the invention is directed to a heterofirnetional ligand,
comprising a first moiety which
specifically binds to at least one ligand located on the intraluminal surface
of a lymphatic vessel and a
second moiety which specifically binds to a disease associated cell and the
use of such heterofunctional
linand in treating or preparin~~ a pharmaceutical composition for treating
disease associated cells, includin~a
diseased cells or disease causing, mediating (ie. having a role whicn is known
to be intermediary or
indirectly facilitating e~~. antigen presenting cells) or mitigating cells
(cells, typically immune cells, which
directly or indirectly counteract the diseased or disease causing.; or
mediatin~~ cells), w ithin a lymphatic
vessel. Preferably, the ligand located on the intraluminal surface of a
lymphatic vessel is a lymphatic vessel
associated ligand.
In another aspect, the invention is directed to a pharmaceutical composition
comprising <r pharmaceutically
acceptable carrier and a heterofunctional ligand comprising a first moiety
which specifically binds to a
ligand located on the intraluminal surface of a lymphatic vessel and a second
moiety which specifically
binds to said disease associated cell and the use of such li~~and in treating
try°atin<.~ disease associated cells,
including diseased cells or disease causing or mediatin~~ cells. within a
lymphatic vessel. Preferably. the
ligand located on the intraluminal surface of a lymphatic vessel is a
lymphatic vessel associated lieand.
In another aspect, the invention is directed to a method of treating disease
associated cells, including
diseased cells or disease causing or mediating cells, within a lymphatic
vessel comprisin~~ administering to
a subject a heterofunetional ligand comprising a first moiety which
specifically binds to a li~~and located on
CA 02402930 2002-09-19
the intraluminal surface of a lymphatic vessel and a second moiety which
speciticall_v binds to said disease
associated cell.
It is to be understood that disease causing cells as used herein includes
diseased cells and pathogens,
including micro-organisms and viruses.
In another aspect. the invention is directed to a heterofunctional lir~and,
comprising a first moiety which
specifically binds to at least one ligan d located on the intraluminai surtace
of a lymphatic vessel and a
second moiety which specifically binds to a therapeutic entity for example a
cytotoxin or cytotoxin-linked-
entity or a non-tonic entity which is present in toxic amounts and to a method
of reducing the toxic effect
of such entity in a subject by administering said heterofunctional ligand to
said subject.
In another embodiment the invention is directed to a method of therapeutic
evaluation and/or targetin~~ :'
intervention in which such heterofunctional li~~and is administered
substantially contemporaneously with a
cytotoxic substance for example a cytotoxic substance useful for treatment of
cancer. The term
substantially contemporaneously is used in this connection to mean in a time
frame that permits both to
exert their respective effects, preferably one or both exvrtin« their
respective et~'ect optimally, or one
exerting its effect dominantly. It will be appreciated that this might entail
that one such entity is advanced
in its delivery over the other. Optionally, one or both of these: cooperatin;;
entities are delivered proximally
to their respective target cells, for example by cannulating one or more blood
vessels as proximally as
possible to the sites) of a tumor and/or actual or anticipated site('s) of
metastases (as discerned by using
one or more tumor and vascular imaging agents. for example, one or a
combination two or more absents
selected from a vascular opaquin~ absent, a radionuclide conjugated anti-
angiogenic antibody, and a
radionuclide conjugated anti-vascular endothelial cell marker antibody. which
cannulation may occur for
example in the course of initial surgical intervention with respect to the
primary tumor site) and-or at the
same time cannulating one or more lymphatic vessels (which may optionally be
located which the help of
a radionuclide conjugated anti-lymphatic vessel marker antihodv) leading to or
from such tumor sites or
metastases. The invention contemplates that small sections of vascular
prostheses, well known to those
skilled the art. (eg. Dacron types) may be grafted into those locations to
permit a prolonged and secure
attachment of such prosthesis to an intra-vascular cannula for secure delivery
to such vascular or lymphatic
locations for repeated and;'or prolonged administration, optionally while the
patient is mobile, optionally
using one or more portable infusion devices, including micropumps desi~~ned
for such purpose (see for
example J Neurosci Methods 1997 Mar;72( 1 ):35-8, US ~ 180 i65:lmplantable
infusion device. See also
Cancer: Principles and Practice of Oncology (infra). Numerous embodiments and
improvements in vascular
prosthesis and in such portable infusion devices and micopumps are described
in the relevant scientific and
patent literature known to thaw skilled in the art. The invention also
contemplates deliverin~~ anv
multifunctional li~~and herein disclosed in the above manner.
It is to be understood that targetin~~ strategies employing the cooperative
action of ligands with different
affinities for their targets exemplified above, may preferably have affinities
which differ, depending on the
application and their avidity, by a factor of 30°% up to a number of
orders of ma~~nitude which nr<ty one,
two, three, four. five, six and even seven or ei~~ht order of magnitude, in
order to achieve substantial
advanta~~le, as hercfter detailed in connection with one such strategy.
In another aspect the invention is directed to a heterospecitic ligand
comprising a first moiety- which
specifically binds to at leant a first disease associated ligand located on a
diseased or disease causing,
mediating or mitigating cell for example a cancer cell or an immune cell, as
well as on non- diseased or
disease causing, mediating or miti~~ating cells (non-target cells) and at
least a second moiety which
specifically binds to a second difTerent disease associated li'~and on the
carne cell and wherein each ligand
is expressed on a substantially (see detinition below) different, n-
overlappin~~, subset of non-tar~~et cells, so
that functional binding to a non-target tissue is substantially (see
definition below) precluded. In another
embodiment the functional affinities of the respective liv~ands may be
selected in accordance with the
strategies suggested above, to further facilitate targetin~~. In another
embodiment, both different li~~ands are
required for internalization. In other related embodiments. the
heterofunetional ligand comprises at least
two different pairs of binding moieties (eg. a trispecific or tetraspecific
antibody which depending on its
construction will permit 2. 3 or ~ such different pairs e~~. a tetraspecitic
sinUle domain type antibody (ie.
consistin« primarily of the heavy or li4~ht chain variable region or a
functional fragment thereon (see
discussion below regardin~~ its construction) allowing the _~rreatest
variation in such ~~eometries and
preferably simultaneous binding of more than one pair), wherein 1 ) at least
three such ligands are expressed
on a substantially (see detinition below) different, preterahly non-
overlapping, subset of non-target cells, so
26
CA 02402930 2002-09-19
as to further limit binding to non-target cells and; or 2) wherein at least
two different pairs of ligands target
a substantially different subset of cells within the same target population
eg. different cells within the same
tumor (eg. proliferating vs. non-proliferating cell -- the respective amounts
of the ditizrent types of cells
will dictate the perecenta~~e of the dose that will be targeted to one
population or another). In other aspects
the invention is directed to a pharmaceutical composition comprising such a
heterospecific li~and and a
pharmaceutically acceptable carrier, a method of usin~~ the heterospecitic
li;~and in the preparation of a
pharmaceutical composition for treating a disease, and to a method of treating
a subject by administering
same in a therapeutically effective amount. It will be appreciated that the
foregoing general strategy can be
accomplished with two or more different antibodies have differiny~ and
preferably non-overlapping normal
ie. non-tar<~eted cell distributions, preferably administered in the same
composition and preferably cross-
linked by biotin-avidin like complementary pairs to facilitate cross-linking
for internalization or targetingr
of therapeutic agents. In a preferred embodiment each such independent
antibody can'ies a different
complimentary aspect of a toxic payload e;. a different liposome (or other
payload carryinU entity for
example a micro or nano particle or sphere or albumin) which complement each
other in virtue of their
respective contents (eg. one carries the prodrug and the other the necessary
convertin~~ enzyme).
In another aspect, the invention is directed to <t multifunctional ("multi"
meanings at least two) li~and
having, at least, a first portion which binds to a lymphatic vessel associated
ligand and a second portion
comprising an immune function-exerting moiety.
'fhe tens lymphatic vessel is used to facilitate broader reference to ligands
(eg. antigens I receptors) present
on cells bordering the infra-luminal pathway throu~~h the lymphatic system
including preferably the
lymphatic vessels and optionally also parts of the lymph nodes, and refers in
the case of the lymphatic
vessels, primarily (from a functional standpoint) to the infra-luminal cell
surfaces (not necessarily to the
exclusion of non-luminal surfaces) on the infra-luminal endothelial cells (not
necessarily to the exclusion of
non-luminai lymphatic endothelial cells) of those vessels.
The term 'associated' with reference to lymphatic vessels, is used to mean
differentially expressed on the
surface of endothelial cells of those vessels for targetin' purposes. such as
to accomplish an object of the
invention, but unless otherwise expressly indicated in a particular instance,
it is used limitatively, to
reference li~~ands that are predominantly, if not exlusiveiy, found on the
aforementioned endothelial cell
surface (as well as in lymph nodes), such that the first portion of the
multifunctional li~~and is for all intents
and purposes functionally targeted to the infra-luminal surface of the
lymphatic system. for instance, it is
appreciated that the ligand in question may be targetted to a limited extent
elsewhere eg. in the case of
preferred LYVE-1 ligand discussed below. to parts of the spleen (which also
provides a venue for immune
cell interactions).
The invention is not concerned with imparting effects to or simply blocking a
receptor on the intraluminal
lymphatic endothelial cell. in this context, the multifunctional li~.;and of
the invention is intended to exclude
only, unless otherwise specifically stated in the claims, only those
embadiments disclosed in WO 98!06$_x9
or other references describing li~,ands. antagonists or antibodies which bind
to a lymphatic vessel
associated li4~and or receptor (see examples ofsuch references below), insofar
as such embodiments
comprise lymphatic vessel associated ligands as hereinabove limitatively
defined, and to this limited extent
only, the term therapeutic function exerting moiety or immune function
exertin~~ moiety preferably
excludes: 1 ) an antibody f~. recwptnr, insofar as such limitation excludes
from the scope of the
multifunctional li~~and (per se) aspects of the invention, substantially
intact naked antibodies which simply
bind to a lymphatic vessel associated ligands, as well as preferably
excluding?) cW ntoxin.s or drugs,
insofar as this excludes from the scope of the mutifunctional ligands of the
invention an antibody or
fragment thereof w which is flrsed or eonju'ated etc. exclusively to a
cytotoxic molecule (including an atom)
or drug (ie. an antibody linked to a cytotoxin or drug only, which is not per
se an or is not inte~~rated with
an ir~dep~rzdent biulo~ric or immune function exerting component) so as to
accomplish a function in relation
to cells or other entities (including other multifunctional li~ands) within
the lymphatic system other than
the cell or li;and to which the multifunctional li~and is anchored. Similarly,
the invention is not
concerned with multifunctional ligands which are adapted to be internalized
into a lymphatic endothelial
cell and the invention is specifically concerned w-ith targeting a lymphatic
vessel associated marker which
does not promote internalization andlor in which the first portion has an
af~tinity (hi~.r,h or medium) w which
limits this effect (ie. to a side effect)
In the same vein, the term immune function is broad in intent (as discussed
below, and includes particularly
any function, including binding. capable of being exerted by an ligand
preferably an antibody (e~~.
27
CA 02402930 2002-09-19
multifunctional ligands which are bispecific antibodies) however it is to be
understood that the invention
and particularly the immune function exerting moiety does not have as an
object (despite possible
incidental effects) evaluating or exertin~~ a disease responsive or immune
function vis-a-vis ligands / cells
lining the intra-luminal surface of the lymphatic system ie. insofar as such
ligands have a role in disease
(other than simple binding exclusively for anchoring purposes which is
attributable not the immune funtion
exerting moiety but to the first portion) hut rather, as evident in preferred
aspects of the invention,
preferably an independent biologic or immune function which is not predicated
on blockin4~ the lymphatic
endothelial receptor or treating cells bearing the receptor ie. exerted vis~-a-
vis targets other than the
lymphatic endothelium target, for example 1 ) in the case of stationary
diseased cells or disease causing
cells or molecules, targets at the site of the disease (which may optionally
be effected. for example, in case
of immunization or other immune cell stimulation, inhibition etc. in the
lymphatic system); and 2) in the
case of non-stationary diseased or disease causing cells or molecules, at the
site of those cells' molecules
including, preferably, within the lymphatic system, for example by binding to
or signaling those cells in the
lymphatic system.
In one embodiment, the first portion of the multifunctional ligand is an
antibody
fn another embodiment, the immune function exerting moiety binds to a target
ligand and
thereby directly or indirectly accomplishes its effect (in wtrole or part).
For example, the
target ligand may be a cytokine, for example in order to target immune cells
to the lymphatic
system to assist in, diseased, disease causin<~ or other target cell ablation
or phagocytic type
activity (eg. by the cytokine in turn binding to a ligand, for example on an
immune cell having
pha'~ocytic activity) or exertin~~ a chemotactic effect within the lymphatic
system, or to mop
up cytokines, for example, when released in toxic amounts due, for example due
to effects of
a disease or particular immunotherapy (such as anti-CD3 therapy; see for
example USP
6193969. Kummer U. et al., Immunol Lett 3001 Jan 1: 75(2):153-I 58) (with
respect to
removing disease associated antibodies from circulation see for example a
bispecific dsDNAx
monoclonal antibody construct for clearance of anti-dsDNA IgG in systemic
lupus
erythematosus. J Immunol Methods. 2001 Feb l; 248(1-2):125-138). (see also,
for example,
US 5,9b8,510 with respect to antibody-CTLA-4 fusion proteins for use in
bindings to various
tartlet li~lands).
In another embodiment, said immune function exerting moiety comprises an
antibody and
optionally both the first portion and the immune function exertin~~ moiey are
antibodies (with
respect to bispecitic antibodies, and a recent review of some of the
technologies referred to or
applicable to various aspects of the invention (see particularly, Journal of
Immunological
Methods February 2001 Vol. 2480-2) pa~.:e 1-300)
In another embodiment, said immune function exerting moiety binds to an immune
cell, a
diseased host cell or a disease causing, cell or entity (eg see US6193968).
The term disease is
used broadly to refer to any undesirable condition. The term diseased host
cell includes but is
not limited to a cancerous (in the broadest sense of that term) cell and a
virally infected cell
(these examples are given inasmuch as the invention in a preferred embodiment
involves
tar~.~etin~~ such cells for destruction) and the term disease causin~~ cell
includes but is not
limited to a virus or other infectious agent and as w ell as immune cell which
is directly or
indirectly involved in mediatin~~ or causing a undesired, deleterious or
patholo~.;ic
consequence. including but not limited to autoimmune disorders, transplant
rejectiun, and
other immune system linked diseases. The term disease causing entity is used
to refer, without
limitation, to any molecule, atom. peptide, ligand, complex, chemical,
component, epitope
etc. that is directly or indirectly involved or associated in mediatinwT or
causing a disease or
disease causing event includin~~ an antibody. Such binding to the entity may
be effected
throu~~h the instrumentality of one or mare (same or different)
multifunctional li<~ands and
through bindings to any ligand or set of li~ands, including receptors, multi-
component epitopes
etc, including_ for example, tumor "as.s~ocriuted" (ie. differentially
expressed to advantage for
targeting purposes) epitopes which may or may not or may only be partially
present on tumor
associated antigens, or commonly. for example anti~~ens ;' epitopes l ligands
' receptors etc.
w which are over-expressed in association with cancer culls: or for example,
antigens % epitopes
ligands / receptors etc. involved in immune signaling, stimulatory, co-
stimulatory.
inhibitory, adhesion or other interactions, including without limitation,
cytokine receptors,
ligands associated with immune cell adhesion (see For example L1S 5,747.()35),
li,ands to
28
CA 02402930 2002-09-19
which binding results in stimulation, activation, apoptosis, aner~~y or
costimulation, or ligands
which differentiate between different populations or subpopuiations or immune
cells.
including sub-populations of B cells and '1 cells, activated versus non-
activated lympocytes,
diseased or disease-causing cells versus non-diseased disease causing
lymphocytes and
specific immune cell clones for example those having specific Ig type and MHC-
peptide type
ligands and correlative ligands. Examples of such li'~ands include CCRS.
C'fl.A-4, LFA-1,
LFA-3. ICAMs eg. ICAM-I, ELAM-1, CD3, CD3. CD4 (eg see US 6.136,310), C'D5,
CD6,
GD18, CD?2, CD40, CD44: CD80, CD86, CDI 34 and CD154, to name only a few (see
also
US6087475: Pf4A receptor. US6135941, WO 01i 13946 Such ligand may also
selectively be
tar~~eted usin~~ any dual affinity strategy accordin~~ to the invention..
5. The invention is also directed to a multifunctional ligand and a method
which comprises using
the multifunctional ligand to assess the toxicity of directly or indirectly
targeting, for example.
primarily within the lymphatic vessel system (see discussion below), cells
having well known
markers that are associated with immune cells, for example, those exclusively
associated with
activated immune cells, in-so-tar as such targeting has a role in prolongin~~
or counteractin~~
the activated state, destroyin<~ the cell (eg. where the multifunctional
ligand is a immunotoxin)
causinv~ the cell to be destroyed (e~~. through apoptosis (eg. see WO O
119861, fas - fast, U.S
6,046,048) or assistin~~ another molecule or cell for example a T-cell or
other killing or
immune modulating cell to do the modulation or killing (markers such as CD?3.
CD25,
CD26, CD28. CD30, CD38. CD49a, CD69, CD70, are just some ofthe markers
associated
with activated immune cells) ete. (for a complete listing of marker associated
e~ ith activated
immune cells see for example Roitt t et al. Immunology, sixth edition, Mosby
20() I
referenced below and Encyclopedia of Immunology ( i 998). .Abbas et al.
Cellular and
Molecular Immunology ?000, Harcourt & Brace, the contents of w which are
incorporated by
reference herein). Antibodies for many such iigands are known or could be
readily made by
eg. phage display (see references herein including .l lntnnrnol A9ethods 1999
Dec 1 (1;?3 I ( 1-
2):65-81 ), and natural ligands for such markers or functional analogues
thereof are in some
cases known or could be made by recombinant DNA technolo~~ies referenced
herein (see also
Cellular & Molecular Inununolo~w ~1'~' Edition, Abbas Ak et al. V1~'B Saunders
and Company
X000. Antibody Fusion Proteins, Steven M Chamow ~ , Avi Ashkenazi Eds. ISBN
047 I 18358X
May 1999 V'iley: Kontermann, R.. et al.(Eds.) Antibody Engineering, Sprin~~er
2001. ISBN 3-
540-41354-5; Antibody Engineerin~~. Carl A. Borrebaeck Oxford CJniversity
Press, 199:1:
Antibody En~.eineerin<~:A Practical Approach David J. Chiswell, Hennie R.
Floo~~enboom,
John McCafferty Oxford University Press,l996: Antibody E.n~ineerin~~
Protocols. Sudhir
Paul ( 1995) Humana Press; Antibody Expression & En~~ineering ( 1998) Henry'
Y. Wang,
Tadayuki Imanaka, American Chemical Society). ~'he term modulation is used
broadly to
refer to any change, directly or indirectly, in an immune function or effect,
as broadly
understood. Many such forms of modulation are well know ~n vn the art (some
are excmplitied
herein), and therefore these need not be specifically recited (fir a review of
such effects see
for example Roitt I et al. Immunology, sixth Edition, Mosby '.001:
Encyclopedia of
Immunology ; ( 1998) Morgan Kaufinann Publishers, ISBN:O 123267656).
In one aspect the invention contemplates that the multifunctional ligand
exerts its function
substantially (ie. upon gaining entry into lymphatic system and when bound to
the lymphatic
endothelial cells, which is dependant on the mode of administration) within
the lymphatic
system, on cells and'or tnoiectrle.c circtrlatin~ through the !v=tttpl~crlic
.st:mertt, for example with
respect to some embodiments, for ;4reatest effect, to avoid an undesired
degree of
immunosuppression (for example, embodiments where immune cells are targeted
for ablation
and: or apoptosis). Preferably, such effect, is exerted at least in part, and
preferably
substantially to the exclusion of reunions within lymphatic system that house
at the time of
administration non-circulating cells (eg. thymus, bone marrow, and various
parts of the
secondary lymphoid tissues) or;'and with respect to some embodiments
(excluding for
example those related to immunization or mopping up toxins trr antibodies)
preferably, non-
activated cells. This specificity of tar~~eting can be accomplished in part to
the natural
distribution of the lymphatic endothelium associated marker of choice, the
mode of
administration and various tar~~etin~~ strategics herein described.
For example, the invention contemplates modes of delivery that to
wrt°in~ deb,>rce,s ensure a
greater degree of lymphatic system targetin~~, for example administration
directly within the
29
CA 02402930 2002-09-19
lymphatics, adminstration in tissues that drain to the lymphatics or parts
thereof, intravenous
delivery, as are well know ~n to those skilled in the art, preferably in each
individual case at
strategic sites of administration that are most pertinent or selective for the
disease in question,
to the extent that selectivity is desired. The invention contemplates a
variety of different size
multifunctional ligands (MRU, sin~~le domain, scFv, Fab, minibodies, F(ab)~.
F(ab')~.
substantially w hole antibodies etc. and known or obvious multimers thereof
referenced herein
and in the referenced literature) that are most suitable (e~=. for small
enou~~h or, for example,
having lon~~est half life in circulation) for par-ticlar modes of
administration to the extent that
this is a limitation (eg. size, where drainage into the lymphatic system is
sou~~ht to be
increased or optimized).
In a preferred embodiment the invention contemplates that the immune function
exerting
moiety of the multifunctional ligand comprises (eg. by way of recombinant
fusion,
conjugation etc.), or binds to (such antibodies are known or may be made by
phage, ribosome
or other such 'display' methods), so as to present the functional part of an
adhesion molecule
(molecule involved in cellular adhesion). for example an endothelial adhesion
molecule such
as a selectins, ICAMs (eg. ICAM-1. ICAM-?) V-CAM, M.AdCAM-I or functional
analo~~ues
or portions thereof (see for example USP6143?98, 5512660, 5861 I51, 54895;3,
5.5;8,725,
6037454, 5565550. C'ir~culatio~a 2001 Feb 27; 103(8):1 128-1 134, and specific
examples,!referenees recited below) in order to control cell traffic including
facilitating cell
anchorin~~ within the lymphatic system, including for example to facilitate
interaction with
another "arm" (functional moiety) of the multifunctional listand or a second
etc.multifunetional ligand or an immune cell (or a cell-sized latex sphere as
described herein -
for this pupose the adhesion molecule may be on the surface of another,
preferably
multifunctional-ligand-anchored latex sphere or on a similarly anchored cell)
as well as
combination therapies, for example, with therapeutic entities that enhance or
inhibit leucocyte
adhesion, or multifunctional ligands or antibodies that bind to one of their
correspondin~~
ligands on immune cells (eg. intergrins) or other ligands eg. CD44, to
facilitate control and/or
some selectivity of cell entry into the lymphatic system, for example. for
reactivity with the
multifunctional ligands of the invention. The invention also contemplates that
such adhesion
molecules may be the subject of targetin~~ with dual affinity ligands of the
invention and that
such ligands may include a moiety which binds to a lymphatic endothelial cell.
he invention also contemplates that one or more multifunctional li'~ands in
which the
immune function exertin~~ moiety comprises an antibody type molecule tar<~eted
to a
particular cell surface li~,~and may be able to mimic effect of such adhesion
molecules, as
discussed below (any such discussion of an antibody mimickin~~ this function
is unless
otherwise stated not intended to limit the broader concept of utilizin~~ any
class of molecule
that would facilitate anchoring or controlling, a<~. slowing the passage of
cells throu~~h the
lymphatic vessels). It is to be understood that there may be limitations in
the number of cells
that can be targeted for ablation in the lymphatic system by ~slowin~~ the
passable of cells,
particularly for the purpose herein specified of allowing thorn the requisite
period of residence
within the lymphatic system for immune cell targeting or interaction or
prolonged interaction
with multifunctional li~ands ofthe invention for binding purposes while bound
to the
lymphatic system endothelium, for example, certain end stage
lyrnphomas'leukemias. In this
particular context it is to be understood that: 1 ) the invention may ham
greatest application
when the multifunctional ligand is administered so as to primarily tar~.;et
cells within the
circulatory system, or as an adjunct therapy, or for remission or near
remission conditions, or
when combined with hyaluronic acid therapy. For example, the invention
contemplates that
an effective amount of hyaluronic acid is pre-administered to tissues
drainin~~ to the lymphatic
system so as to initially occupy bindings sites on l_YVf-1 primarily in the
smallest lymphatic
vessels and thereby minimize excessive binding within the narrowest vessels.
10. In a preferred embodiment said first portion binds to LYVF- I or
podoplantin described
below ~.
1 I . In a preferred embodiment, said tirst portion is fused, conjugated or
otherwise linked directly
or indirectly to an immunizing moiety, for example an antigen, epitope,
mimotope or peptide
etc. presenting/incorporating entityiscaffold that generates by itself or with
the help of one or
CA 02402930 2002-09-19
more cytokines, costimulatory molecules and,'or adjuvants etc. an immune
response to a
desired antigenic determinant (this term is used broadly to correspond at
least in scope to the
overlapping groupings: antigen, epitope, mimotope or peptade), for example an
anti-idiotypic
antibody. an antibody component which is capable of binding to a T cell
activatin~~ entity for
example a cell (cg. an APC see Int Inrman-ml ?000 Jan; 12( I ):>7-66 or other
cell having cg.
immune modulating activity cg. see USP 6,00=1,81 1 ) which is for example
genetically
en~~ineered to express relevant ligands for activating (or with respect to
functions not
necessarily related to immunizing, aner~.:izing, tolerizin~~ or otherwise
Inodulatin~ the activity
ot), an immune cell for example a B cell or T-cell, for example an MHC-peptide
and B7 co-
stimulatory molecules for activation of '1'-eel Is ( see for example Proc ,W
nl .4c«ct Sci (S A
?001 Jan 2; 98(1):241-246 see also ~fham EL et al. J of Immunological Methods
~'ol. ?49(1-
2)(2001 ) p I I 1-I l9 with respect to latex spheres that can be used for this
purpose). or for
example a CTLA-4 scaffold, a peptide fused to an Fc domain (see WO 01!18?03) a
HSP-
peptide cornplex/conjugate, an MEiC protein or peptide complex etc. Antibody-
MHC''
complex fusions and antibody-B? costimulatory 'fusion molecules are known and
the
invention contemplates that fusion molecules with anti-lymphatic marker
antibodies could be
made and used to~~ether for immunization purposes. For example the invention
contemplates
that two Fabs (or linked Fvs, with linker extensions of suitable len '.;ths
comparable (l.c. in a
rankle of up to two fold shorter%longer when compared to extensions used in
the art see Park et
al. A divalent recombinant immunotoxin formed by a disulfide bond between the
extension
peptide chains. Mol Cells. 2001 Dec .'> 1:12(3): 398-402) which reco~>nize a
lymphatic
endolethelial marker can be disulttde linked and fused respectively to an MEtC
petides and a
B7 molecule) It is also contemplated that the absence of costimulatory
molecules for
presentation in a co-stimulatory fashion with an MI~C peptide complex will
cause a tolerizing
effect. Accordingly the invention is also directed to a multifunctional
li~~and comprising an
immune function exerting moiety which comprises an MHC'.. preferably complexed
or
otherwise linked to a peptide. Peptide linking may for example be effected
independently,
naturally or for example through CausIn~T release of peptides from an MH(.'
peptide or HSP
peptide complex by injecting a weak acidic solution into tumor cg. ,just prior
to excision.
Suitable such solutions which may for example be combined with a cytokine '
e~~.ll,-12 and-or
adjuvant are known in the art.
12. In a preferred embodiment said immune function exerting moiety comprises
an anti-idiotypic
antibody, for example an antibody' that a) mimics, for example, a cell surface
expressed tumor
associated epitope, a virus or other infectious agent associated surface
epitope. a toxin, an
immune stimulatory, costimulatory, inhibitory, or otherwise interactive
ligand; or b) serves
to bind to the idiotype (lc, paratope) bearing antibody to which it binds as
an anti-idiotype, for
example an autoimmune antibody. etc. or an antibody bearing a toxic moiety for
removing
such antibody from passage into the circulation.
13. In a preferred embodiment, the invention contemplates that the first
multifunctional ligand is
used for development, therapeutic evaluation or combination therapy in
conjunction with a
second different multifimetional ligand of the invention, to achieve a
cooperative effect (for
example. in the same composition or substantially contemporaneously
administered (lc. to
reach the same or an interrelated destination in a cooperative time frame) or
in necessary or
desired sequence,'interval, etc.). An example of such cooperative effect is an
interaction (not
necessarily simultaneously) with two different immune cell surface ligands
(for example via
an antibody binding interaction), or to deliver different payloads cg. toxins.
to a diseased cell
see (USP 6,077,499). 'fhe invention also contemplates a method of effecting
substantially
coordinated interactions of ditfering temporal and spatial complexities,
ran~~in~~ from a
somewhat proximal and contemporaneous delivery (cg. in the same composition)
of a first
multifunctional ligand having, for example, a cancer cell binding second
portion, and a
second multit'unctional ligand havin~~, for example, a cytokine bindim~ Ab,
e~;. to reduce any
toxic effects associated with toxic levels of cytokine release. a cytokine
component ( far
example to harness the effect of such component as a means to attract one or
more immune
cells to kill a diseased cell or to harness the inhibitory effect of such
component (e~~. using one
or more cytokines employed by cancer cells to evade innnune eel( targeting)
cg. on undesired
immune cell elimination or immune cell elimination ofthe multifunctional
ligand, or a 1-cell
bindings component (cg. anti-CD3) t.o harness the effects of such component on
cancer cell
killing optionally with a concomitant object of assessing> possible
counterproductive immune
31
CA 02402930 2002-09-19
cell elimination (cg. as would be enabled by using a radiolabelled
multifunctional ligand and
determinin~~ the disposistion of the label over time) of the multifunctional
ligand.
l4. Also contemplated are methods to implement more spatially and.'or
temporally sensitive
interactions. For example, when administered in empirically determined
suitable proportions
and in empirically determined sufficient total amounts for, at least, partial
and;'or local
lymphatic-vessel-associated-ligand saturation or partial saturation to achieve
proximal
bindin<~ of a tirst to second multifunctional ligand (having reheard to the
route of
administration e~~. local saturation can be more readily accomplished by
administration into
the lumen of the lymphatic vessel). Two different such multifunctional
li~~ands may be used,
for example, to deliver two different immune function exerting moieties in
substantial
proximity to one another for contemporaneous interaction with two different
li'~ands on an
immune cell (ie. when it approaches the luminal wall of a I~~mphatic vessel).
For example, this
approach may be used to implement one or more effects including increased
avidity to the cell
for proton<~ed cell anchoring, which may positively impact on desired (in some
embodiments)
transfer of the multifunctional ligand from the lymphatic vessel wall to the
target cell cg. for
achieving an inhibitory etfect via ligand binding (e~T. assessed via duration
of multifunctional
ligand binding e~. quantitative or radioima<,e approximated label
elimination)(N.B. this effect
may be assessed with multiple copies of the same multifunctional li~Tand),
delivery of a
cooperative payload cg. different entities which contribute to the same or a
different
mechanism of cell killing, counterparts in a two component interaction (biotin-
avidin), which
preferably yields evidence (preferably quantifiable evidence) of the
interaction, tier example
an enzyme-substrate interaction to quantitatively assay the amount of an
enzyme converted
substrate (cg. usin« a conjugated prodru« and pro-drug conversion akin to
ADEPT and
assessing the extent of prodru~ conversion e~ by labeled anti-drtr~~ specific
antibody). For
example, the invention contemplates the use of a respectively linked catalytic
antibody
component (see for example US5658753:Catalytic antibody components) and
labeledy
substrate or RNAase and labeled RNA etc.for this purpose. Another example,
discussed in
more detail below is the use of one multifunctional ligand for targetin~~
(selectivity) purposes
and another for implementing directly or indirectly a desired therapeutic
effect, both ligands
optionally b~in~~T required to to ~~ive rise to a substantial probability of
binding (the invention
also contemplates that this strate~w could be used with a sin=le
multifunctional li~and having
two infra-luminally directed bindings moietios).
15. The invention contemplates that such interactive entities may be
conjugated fused or
otherwise linked to a respective first and second multifunctional ligand for
achievin~~ a
cooperative interaction between adjacently bound such ligands.
16. The invention contemplates that adjacently interacting multifunctional
ligands yielding
detectable evidence of the interaction, could be use in a method to assess a
f;. a) luminal ligand
saturation for dosing" b) multiple simultaneous binding interactions, and c)
perhaps most
spatially sensitive, development ofa process to achieve cross-linked binding
with multiple cg.
immune cell li~~ands cg, a eostimulatory innnune effect (ie. the effect of
different
simultaneous interactions cg. on stimulation, inhibition etc.of cg. an immune
cell for example
combining a first multifunctional ligand capable of selectively binding to.
conjugated to or
fused to a B7component (see .l Jmnrar~aothc 2001 Jan-Feb; 24( 1 ):27-36; J
Jntn~trnol 2001 Feb
1 >; 166(4):2505-2513; Chalitta PM et al. J. Immunol. 160:3419-3426) and a
second
multifunctional li~~and capable of selectively bindin;~ to, conju~~ated to or
fused to an MHC
molecule delivered initially with Yor without peptide. For example, the
invention
contemplates using.; various amounts.~proportions of multifunctional ligands
having antibody
components fused or conjugated to or capable of binding selectively to, for
example an MHC
class I or II peptide complex and recombinant B7-I-Fc and'or B7-2-Fc
respectively (see Eur J
Immunol 2001 ,Ian; 31( I ):32-38; E;aar J JnrnrurmJ 2001 Feb; 31 (2):440-449)
(for tumor
reactive peptides see forexample,/Irnmnnother2001 Jan-Feb; ~4(1):I-9). In this
latter
connection (cross-linking type interaction). am d:-odor permanence of binding
or ease of
attachin;~ other cooperative entities (for example biotin coated or
conju~~ated radionuclides,
liposornes or other payload carryings entities (cg. see for example US'patwts
5439686,
6007845. 587971?, 5456917, 6165502, 5079005. 5888500. 5861 159. 6193070.
6190692, WO
00!69413, WO O 1:'07084) the invention contemplates biotinylatin~~ the two
multifunctional
ligands and linkin~~ the two biotinylated cooperative mulfunctional ligands
with avidin,
32
CA 02402930 2002-09-19
streptavidin (or other modified forms thereof e~l. de~~lycosysylated avidin or
using other
complementary linkin~~ components- see a<~. US Patent (USP) 6,077, 499).
17. The invention also contemplates enhancin~~ the cross-linking ofthe
multifunctional ligands of
the invention through complementary components such as biotin and avidin.
18. Preferably. with respect to, for example. increasing selectivity of
targeting certain cells (e~~.
to induce immune tolerance), the invention also contemplates that a first
multifunctional
ligand is used to bind to a marker specific to a particular kind of cell (eg.
activated immune
cells) and a second multifunctional ligand (which may not be specilic for
activated immune
cells) is used to modulate the activity of the immune cell (for example
inactivate it or reduce
its disease causin<~ capability directly or indirectly by binding to it ). For
example, where the
marker is used to determine the selectivity of the tar~~eting but cannot be
used for modulating
its activity, it is contemplated that the functional affinity of one or both
the first portion and
second portions of one or both of the cooperatin<, multifunctional ligands can
be selected to at
least partially control the selective modrrlatin~, effect of the pain, for
example both interactions
would be required for the second multifunctional ligand to have an optimal
opportunity to
bind. For example, the functional affinity for the tar~,~et cell is relatively
weak for the purpose
of attachin<~ to the eg. immune cell for a sufficient duration (cg. so as to
yield the effect of
becoming attached to the immune cell in preference to the lymphatic vessel),
compared with
that of the first multifunctional li~~and (ie the one that accomplishes the
selective recognition
through binding) to rectzzce the likelihood that the second moiety will bind
in the absence of
binding of the first moiety (notably a similar type of coordinated interaction
ie. two binding
interactions, is naturally used for fell adhesion). (NB. this type of
coordination has application
ie. both specificities are optimally required for bindings. to a single
multifunctional ligand,
having a divalent immune function exerting moiety eg a triabody or tetrabody
or for cross-
linking and other types of coordinated interactions). In a preferred
embodiment, if transfer of
binding of the first multifunctional ligand to the in nnune cell is not
desired its functional
affinity of the first portion to the lymph vessel can be greater than that of
its second portion.
while the reverse could be true for the second multifunctional li~~and. It
will also be
appreciated that antibodies which cross-link for example an integrin and a
marker of immune
cell activation could be used to limit the number of activated innnune cells
that migrate
through the lymphatic system. For example bispecitic d Abs, diabodies, etc. in
which the
functional affinity of each specitic bindin<v portion individually does not
sron~~ly favour
bindingJ. could be used to selectively tarv~et specific sub-populations of
immune cells or even
specifically activated immune cells (for example antibodies that recoy~nize
particular antigen l
peptide specitic T cell or B cells).
l9. Accordingly. more generally speaking.:. the invention is directed a
bispecitic li~~and, preferably
a bispecitic antibody, having a tirst portion which binds to a ligand which
differentiates
between members of the same immune cell population (eL~ a particular type of T
cell) and a
second portion which binds to a second ligand on the same cell, which binding
exerts directly
or indirectly a desired et~fect_ wherein the functional affinity of said first
and second portions
are selected so as to substantially increase amount of immune cells in which
both such
portions are bound to their respective ligands relative to those which a
single such portion is
bound to a single li~~and and preferably wherein the amount of immune cells to
which the
bispecific ligand is not bound is substantially greater than the number of
immune cells that are
not bound when compared to using a bispecifie ligand having the same
specificity and for
example a 10' to 10~' (preferably I 0' to 10'', preferably 10' to P 0'',
preferably 10' to 10')
increase in aflinity of one or both portions. This invention also contemplates
that binding to
the li~~and which differentiates between members of the same population (a
particular type of
T cell) does not have a negative consequence other than to cause the molecule
to be
ineffectual unless both of its portions are bound and that its binding is
itself sufficient for
binding and'or stron~~er relative to the second portion by two fold to ~
orders of magnitude
,preferably I to 3 orders ofma~~nitude. The term substantially greater imports
medical
significance and rnay preferably be 1 >°ro - 10000°'0 ~,reater.
The foregoin~~ examples are not
meant to be limitative.
20. In a preferred embodiment, the invention more broadly speaking
contemplates a two lioand
interaction (using one or more multifuncaional ligands) wherein for example
both are required
or increase the likelihood of interaction and w herein the interaction of at
least one contributes
33
CA 02402930 2002-09-19
to specificity, though not necessarily to modulation, thus permitting a
broader selection of
modulators including those that but for the selectivity enhancing effect of
the cooperating
ligand and the lymphatic system venue, would be toxic in the desired
therapeutic dose.
Examples of markers that could assist in selectivity include those are unique
to, for example,
activated B cells or f cells or those having particular speciticites in virtue
of unique I~~ type
receptors. C:xamples of li<~ands on, for example immune cells, through which
modulation'inhibition.'stimulation etc. (including, for example apoptosis),
for example by
antibody binduy~ or supply of a natural interactive ligand, are well known.
Some examples are
provided herein. Combinations and permutations of markers and li~ands for
selectivity and
exerting an immune effect such as modulation; inhibition,~stimulation referred
to herein or in
the literature incorporated herein by reference or well known in the art are
contemplated to be
within the scope of the invention.
21. It will be appreciated that a combination of factors, such as dose, using
additional molecules
that increase or decrease migration or adhesion optionally in a tissue
targeted manner, route of
administration leg within tissue that best drain to lymphatic vesssels or a
portion thereof. use
of cytokines, etc. and irnrnune rnodulatin~~ drug's, as well combination
therapies with known
entities, can be employed in various combinations for strategies of harnessing
the unique
properrties of the multifunctional ligand of the invention, to achieve a
selectivity enhancing
and or modulatorylinhibitory,'stimulatory etc or otherwise cooperating effects
with respect to
the desired target population of cells. Unless their function are self-
evidently contlictin~~ the
invention contemplates all permutations ofthe multifunctional ligands
disclosed herein or in
the literature incorporated by reference hererin as well as those evident to
persons skilled in
the art whose mention is omited.
32. In a preferred embodiment, the immune function exerting moiety binds with
greater
functional affinity to its target ligand than said first portion binds to its
target ligand. For
example said immune function exerting moiety may bind with ~~reater avidity
(preferably at
least 2 times ~~reater (divalent vs. monovalent) and lesser or greater
affinity (e~~. within a range
of 1 ~ 10-' to I x 10' fold) or with the same avidity and greater affinity
leg, up to 1 x 10'' fold). In
applicable aspects, the invention contemplates that this increased functional
affinity can be
employed to effect transfer of a lymphatic vessel bound multifunctional ligand
leg. a
bispecific antibody) to a cell passin~~ through lymphatic system. The
invention also
contemplates a method comprising radiolabelling the mutifunctional ligand to
assess, for
example. the degree to which immune cells at a disease site have passed
throu;h the
lymphatic system. Certain aspects of the invention, discussed herein, relate
to a
multifunctional ligand based system of tar4~etin~~ a particular immune cell
li<~and for
stimulation, inhibition etc. predominantly within select portions of the
lymphatic system that
contain mi<~rating cells (although some ~~eneral tars~etin~~ can controllably
occur before the
multifunctional ligand binds to the lymphatic system or when the
multifunctional li~and
releases from the lymphatic system without havin~~ found its target within the
lymphatic
system) will have at least a partially selective effect on tar~~etin~~ disease
causincrimediating
immune cell> leg. activated with a specificity that effuses the disease) as
opposed to non-
disease causin'~lmediating cells, in the case where such ligand is also
expressed on such other
immune cells eg. of the same type eg. T cells. This pennies tar~~etin'~ of
immune cells
primarily within the portions of the lymphatic system that contain migrating
cells particularly
disease causing;'mediating cells while minimizing immune system dysfunction.
This effect
can be even more selectively accomplished, for example, by delivering the
multifunctional
ligand directly into the lymphatic system and within a time frame which is
shorter that the
normal duration of binding of the multifunctional lit.;and deterrninin~, the
degree to which the
multifunctional li~~and is bound to such diseased related cells at the disease
site and similarly
the degree to which it is bound to the cells unrelated to the same disease
e~~. via radiolabel. As
discussed more fully below, the invention also contemplates a multifunctional
ligand based
system of assessing the effects of certain types of immune stimulation e~~.
how~ stimulatin~~
enhanced migration or adhesion, will differentially affect disease activated
cell migration
through the lymphatic system to enhance such disease cell targeting within the
lymphatic
system. For example, for tumor cell targetin<,~ and stimulation ofdisease-
activated immune
cells the invention contemplates evaluatin~~ cytokine leg. 'fNFoe) linked anti-
angiogenic
marker antibodies, optionally, preferably in combination with anti-tumor
vaccination
strate~~ies, to direct disease activated immune cells to tumor site and the
lymphatic system for
34
CA 02402930 2002-09-19
further immune stimulation. Based on a "bait and trap" type approach, ligands
such as OX40L
and CD44 may also be assessed for this purpose.
23. In this connection and more generally the invention also contemplates
using a bi-specific
antibody, for example having a lymphatic endothelial binding first portion and
for example a
cytokine binding second portion, wherein the cytokine binding portion has a
lower
functional affinity for the cytokine (for example I x 10 -6 to 0.9 fold)
compared with that of
its natural inceptor on an immune cell. It is contemplated that a
multifunctional ligand of the
invention could be used optionally in conjunction with a multifunctional
ligand which
displays a functional adhesion molecule (a selectin, ICAM, etc.) to assess the
optimal
parameters for transfer of the cytokine. for example, as is known to occur by
monitoring the
effects of cvtokine release attributable to such cytokine transfer. It will be
appreciated that
this information or approach could be used to optimize the bindings parameters
for other
li~aands as w ell (eye, anti CD3) and could be employed not only in lymphatic
system but to
locally deliver inhibitory or stimulatory cytokines or other ligands to
certain tissue targets. for
example new- blood vessels formin~~ within tumors or other Tissue specific
markers.
24. The foregoing strategies could be used as part of a primary, adjunct or
low ~ disease burden
therapy.
25. In a preferred embodiment, the second portion comprises a ligand which is
capable of binding
to an immune cell for example B cells. T cells etc. preferabl~Y in one
embodiment to assist in
cell killings or immune modulation of a target cell (re NK cells see for
example I.IS
5770387)(see also US6071517:Bispecific heteroantibodies with dual effector
functions;
Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells.
.llmnurnnl .h-9crhuds
2001 Feb I; 248(1-2):l 13-123; Bispecific antibody-targeted phagocytosis of
HER-2meu
expressing tumor cells by myeloid cells activated in vivo..l Immunol Methods.
2001 Feb 1;
348(1-3):167-182 as well asJ lmrnur~ol a-fethucls 2001 Feb 1:248(1-?1:103-1 I
1 ).
36. W ith respect to avidity. affinity and other elements of design includin~~
size, blood clearance,
additional functionality etc.the multifunctional li~~and may be. for example,
a bispecitic
antibody having a monovalent tirsc portion and a monovalem second portion, a
bispecific
antibody havin<~ a divalent first portion and a divalent second portion, a
trivalent trispecitic
antibody havin~~ a monovalent first portion and a second portion comprisirt~~
a monovalent
immune function exerting moiety which binds, for example, to a target li~~and
on a tartlet
diseased. disease causin~~ or immune cell, and for example, a monovalent
portion which binds
to an immune cell which assists in killing or modulation tbr example anti-CD3
or anti-CD28
antibody component, a tetravalent trispecific antibody having. a monovalent
first portion and a
second portion comprising a divalent immune function exertin~~ rnoietv which
binds, for
example, to a target li~and on a target diseased, disease causing or immune
cell, and for
example, a monovalent anti-C D3 or- anti-CD28 antibody component (it is
contemplated that
this orientation might advantageously position the anti-CD3 component for
interaction with a
T-cell almost exclusively when the first portion is not bound To the luminal
wall of a
lymphatic vessel). a trivalent bispecific antibody havin~~ a monovalent first
portion and a
second portion comprisiny~ a divalent innnune function exertin~~ moiety, for
example, one
which binds, for example, to a tar~~et ligand on a tartlet diseased, disease
causin~~ or innnune
cell. fhe antibody' subunit may be for example, a Fab, a substantially intact
antibody, a single:
domain antibody (see also Hutton SE. Dis Markers 2000;16( 1,2):37 Single
domain 11Ur11ar1
immuno~~lobulin fold-based biomolecules; Antigen specificity and high affinity
binding
provided by one single loop of a camel sin~~le-domain antibody. J Biol Chem.
?001 Jut
13;276(28):26285-90. Optimal Desi~~n features of Carnelized Human Single-
domain
Antibody Libraries. J Biol Chem. ?001 Jul 6;276(27):24774-?4780; Recognition
of antigens
by sin~~le-domain antibody fra~~ments: the superfluous luxury of paired
domains.'Urends
Biochem Sci. 2001 Apr;26(4):230->; l..lama heavy-chain V regions consist of at
least four
distinct subfamilies revealing novel sequence features. Mot Immunol. 2000
Au<~:37(10):579-
90) a minibodv an scFv or a minimal recognition unit (MRU cg see
US6174691:Mirtimum
recognition unit of a PEM mucin tandem repeat specific monoclonal antibody).
27. In a preferred embodiment, the multifunctional liy~and binds to an immune
cell which is
associated with an autoimmune reaction, for example a CCRS-expressing cell.
(see also
CA 02402930 2002-09-19
Apoptosis <genes and autoimmunity. Curr Opin Immunol. '000 Dec; 12(6):719-24.
for
application herein)
28. In a preferred embodiment, the second portion comprises a cytokine
component.
?9. In a preferred embadiment, the second portion comprises a cytotoxic
component.
30. In a preferred embodiment, the second portion of the multifunctional
li~~and comprises an
internalizing antibody and a cytotoxic component.
31. In a preferred embodiment, the second pardon consists of an antibody which
binds to T cells,
for example, an anti-CD, antibody or an anti-CD28 antibody.
32. In a preferred embodiment, the second portion consists of a cytokine
component.
33. In a preferred embodiment, the second portion comprises an antibody which
binds to a target
diseased. disease causing or immune cell and further comprises one or more
cornpanents
selected from the group consistin~~ of a cytokine componem, a cytotoxic
component and an
anti-CD.i:'C'D28 component.
34. In another aspect the invention is directed to a composition comprisin~~ a
multifunctional
ligand and a pharmaceutically acceptable excipient.
;5. In another aspect the invention is directed to a composition comprising a
plurality of different
multifunctional li~~ands.
36. In another aspect the invention is directed to methods and compositions
for develaping and
evaluating the therapeutic value of stimulators, mediators, inhibitors etc. of
immune cell
signaling (~~~. stimuVatory, inhibitoy, costimulatory), adhesian,
rnigration,ete. including the
effects of li~aand~'receptor blockin<~ and supply of specific cooperative
ligands, using the
multifunctional ligands of the invention.
s7. In a preferred aspect, the multifunctional ligands of the invention may be
used to assess the
effects of such compositions on the sub-populaton of cells ttrat migrates into
lymphatic
vessels. In particular, the invention is directed to assessing the expectation
that some disease
causing, mediating or otherwise disease active immune cells have an enhanced
ability%opportunity (and, or can be enhanced in their abilitylopportunity to
make their way into
the lymphatic system) so that targeting of relevant li~~ands on that sub-
population of cells
within the lymphatic system will cause at least a parrtial selective
tarwetin~~ effect, preferably
with positive effect onythe dosing capability and choice of lif;ands ie. in
terms of limiting
more universal and: or deleterious consequences. fhe invention is also
directed to a method of
reduein~~ the toxic side effects of a pharmaceutical composition comprisin~~ a
multifunctional
li~~and in which the immune function exerting moiety is tar~~eted to a ligand
that is not found
exclusively on disease causing, mediatin~,~ or otherwise disease active
inunune cells, by
administerin~~ said composition in a manner in which it enter more directly
into the lumen of a
lymphatic vessel. (ft contemplated that immunization within the lymphatic
system can also be
enhanced in virtue of such selective tar~~eting.) In particular, the invention
is directed to a
multifunctianal li~and. a pharmaceutically acceptable composition therof and
methad of using
same for assessing enhanced migration or enhancing migration ofdisease-active
immune
cells, said multifunctional ligand comprising an immune function effecting
moiety which has
an immune effect on an immune cell surface li~~and ie. effects including
signaling (e~.:.
stimulatory, inhibitory, costimulatory. antagonistic, agonistic), includin~~
tlr adhesion and
mi~~ration eftects,etc. This may be accomplised practically, tc~r example
throu~~h
ligandreceptor blockin~~ e~~, via antibody. or by antibody
fusions;~con.jugates etc. that supply
the natural li'aand or a functional fi-a~~ment c>r chemical%biolo~;ical
mimotope thereof: In a
preferred embodiment the invention is directed to a multifunctional ligand in
which the
immune function exerting moiety is an antibody that binds to a ligand
selected. for example
from the ~~roup consisting of CTLA-4. ll.-? receptor, GCRS. CD44, CD 134. CD3,
C'D?8,
CD2.
36
CA 02402930 2002-09-19
38. In another aspect the invention is directed to a composition con uprising
a plurality ofdifferent
multifunctianal ligands which exert a potentially cooperative immune effect
with respect to an
immune cell. for example bindin;.: to two or more different li~~ands on an
immune cell,
wfierein said ligands are selected, for example from the group consisting of
CTLA-4, ll.-2
receptor, CC'R>, CD44, C'D134. from any of the ligands herein mentioned or
referenced or
preferably ('D3, CD28, CD3.
39. The invention is also directed to a method of inhibiting metastasis
durin~~ the course of
surgical removal of a tumor comprising administering to a patient prior to
sur~~ical treatment
of the tumor site, a pharmacetical composition comprising a multifunctional
ligand in which
the immune function effecting moiety binds to a tumor associated epitope on a
cancer cell.
40. In another aspect the invention is directed to an immunocytokine having an
anti-idiotypic
antibody component which reco~~nizes the paratope of an antibody which binds
to a lymphatic
vessel associated ligand and a cyiokine component fused therewith or
conjugated thereto. For
example the cytokine component comprises IL-? or a functional fragment thereof
and.~or II.-
13 or a functional fragment thereof. In addition to their individual use in
fusion proteins for
tumor cell killing, combinations of 11-3 and lL-13 have been used successfully
for this
purpose. It is contemplated that such cytokine fusion could be used to tartlet
T-cells or
phagocytic cells to a multifunctional ligand that has bound to its disease
causing or diseased
cell target. preferably having left the lymphatic vessel endothelium in
preference for binding
its target. In this connection it is contemplated that the functional affinity
of the anti-idiotypic
Ab for the first portion would he less than that of the first portion to the
lymphatic
endothelium. so as to minimize competition between the two. It is also
contemplated that the
delivery of the immunocytokine occur substantially contemporaneously but
separately and
after that of the multifunctional li~~and. optionally by a different route of
administration.
41. Similarly the invention contemplates for the same purpose, a bispecific
antibody having an
anti-idiotypic antibody component which recognizes the paratope of an antibody
which binds
specitically to a lymphatic vessel associated ligand (preferably with lower
affinity than that of
the Ab forJits target) and for example an immune cell bindin;~ portion eg. an
anti-C D3
antibody or an anti-CD28 antibody component.
42. Thus the invention is directed to a method of targeting a diseased or
disease causing cell for
destruction by the immune system comprising administering separately but
substantially
contemporaneously to a subject frostings the diseased or disease causing cell.
preferably in
sequence with an interposed interval and'or by different routes of
administration, first a
multifunctional ligand in which the immune function effecting moiety binds to
a diseased or
disease causing cell surface associated epitope. and an innnunoeytokine or
bispecitrc antibody
as decribed in the immediately preceding two paragraphs.
43. In a preferred embodiment the invention contemplates modification of the
mufti-functional
ligand to substitute one or more amino acids which reduce without functional
impact on the
molecule the number of immunogenic MHC II class peptide sequences within the
molecule.
This can be accomplished through procedures available to those skilled in the
art, for example
through the I:)elmmunisation services of Biovation Limited (see also US 5821
1?~ and related
Xoma patents).
44. Inasmuch as the invention is predicated on intraluminal lymphatic system
targeting such
lymph associaton may be alternatively implemented, in suitable circumstances
by the method
of delivering the multifunctional ligand, for example into the lumen of a
lymphatic system
vessel or ('where the multifunctional li_~and is not of an unsuitable size
(see for example
Ikorni, F. et al. 1_ymphology 32 ( 1999) 90-122, within a portion of body that
drains to the
lymphatic system (ie a portion ofthe lymphatic system), for eventual
mi~~ration to the
lymphatic system. Particularly. with respect to embodiments of the invention
in which the
immune function exerting moiety is targeted with greater functional affinity
to a therapeutic
target (ie, not the lymphatic system target), such lymphatic system oriented
modes of deliven~
coupled with preferred tarrgeting to the therapeutic target may combine,
absent saturated
37
CA 02402930 2002-09-19
binding to the therapeutic target, to better accomplish functional lymphatic
tar<~eting.
Accordingly, in a broader aspect the invention is directed a lymphatic system
tar~~eted
multifunctional ligand in which the second portion is as described herein and
in which the
specificity of the first portion exclusively for a lymphatic system is
inessential. In this
connection, the invention contemplates tar~~etin<~ markers on lymphatic
vessels that are also
present. for example on blood vessel endothelial cells leg. VI~CiF2). (with
respect to lymph
specific markers see also Birner P. et al. Clin Cancer Res ?001 Jan; 7(1):93-7
"Selective
immunohistochemical staining of blood and lymphatic vessels reveals
independent prognostic
influence of hlood and lymphatic vessel invasion in early-stage cervical
cancer'' and
published references to the markers therein mentioned.)
4~. In the case of purely sustained release aspects of the invention where the
tirst portion is
temporarily anchoring a second portion for eventual release Luck into the
circulation, the use
of term immune function affecting moiety with reference to the role of the
second portion
does not adequately accommodate the breadth ofthe invention since any form
ofdisease
palliating active moiety or entity which exerts its effect elsewhere than at
the lymphatic
endothelial cell may gain advanta<~e from this form of delayed delivery (depot
effect) or
anchorin~~.
46. Furthermore, in another preferred aspect, the second portion is capable of
binding directly or
indirectly (e'.=. binding to an entity which in turn binds to a target entity)
to a target entity, fbr
example a therapeutic entity (for example to mop up excess such entity that
does not
imtnediatelv reach its target leg. an entity that is toxic elsewhere in the
body), a toxic entity
including an entity which is not per se toxic but the presence of which is
undesirable at a
particular tune or in particular amount or concentration leg. a cytokine, for
example when
released as a result of anti-CD3 therapy), to rediy~ec~t an an entity to a
tartlet, for example a
therapeutic entity, for example through the instrumentality of an antibody
portion that is
directed to that target leg. a multifunctional ligand in which the second
portion comprises an
anti-tumor antibody portion that is conjugated to streptavidin. to retarget
biotin conjugated
radionuclide back to the tumor (see Martin J. et al. ( 1997) Cancer Chemother,
Pharmacol.
40:189-301 ). to temporarily anchor liposomes or other carriers of entities
leg. drugs) having
an direct or indirect beneficial effect elsewhere.
In a preferred embodiment, the invention provides a multifunctional li~and
having, at least, a first portion
which binds to a lymphatic vessel associated antigen 'receptor (and thereby
exerts, not necessarily to the
exclusion of other effects) at least an anchoring_ function, and a second
portion having at least one
independent immune function. The term "immune function" is broad in intent
including but noc limited to
direct or indirect and primary or corollary effects related to simple
tar~~eting, tolerance, immunization,
stimulation, inhibition, modulation or various other immune related effects
(other than simply forming pan
of the entity which blocks the lymphatic endothelial associated ligand). The
term independent is used to
exclude only an effect specifically targeted tow bards the ligand (bloc-kingi
or cell bearing the ligand to
which the first portion of the multifunctional li;~and is bound, which is not
contemplated as an object of the
invention. The invention contemplates rather that the immune function is
exerted. for example, vis-a-vis
immune cells or molecules or a~~ainst cancer or infected cells to affect an
immune function that relates to
assessment, diagnosis, therapeutic modelins:. or treatment of various disease
states such as autoimmune
disease, transplant rejection, cancer and infectious disease. In a preferred
embodiment, the invention
contemplates that the independent immune function is exerted throu<~h a
physical ligand-ligand interaction.
In a preferred embodiment the multifunctional ligand has an ability to bind in
the manner of an antibody in
virtue of at least one of the tirst or second portions, and preferably at
least the first portion. The lymphatic
system directed first portion may in some embodiments (LYVE-1 ) be hyaluronic
acid or analogues thereof
that have the appropriate bindings capacity. In a further preferred embodiment
the second portion binds to a
target ligand on a cell or molecule leg. a cytokine or autoimmune antibody)
which passes through the
lymphatic system. In a more preferred embodiment the multifunctional ligand is
a bispecitc antibody. The
term antibody is used to refer to any antigen binding ti-a'~ment of an
antibody that substantially has the
binding capability of an antibody including anti-idiotypic antibodies, and
therefore the term hispecific
antibody is used (unless the context implies a more specific usage) in a
functional sense to refer to at least
two different speciticities (including trispecifc antibodies etc.) and
includes well known entities wfiich are
38
CA 02402930 2002-09-19
diabodies, triabodies, tetrabodies, minibodies, scFv dimers, etc., and
entities in which one or both binding
moieties are seFv or single domain type antibody fragments or dimers etc of
such fragments (with respect
to single domain antibodies see for example Carrel single-domain antibodies as
modular building units in J
Biol Chem. 2000 Oct 2>, R Mulligan-Kehoe U.S. patents ).
The term "anchoring function" is used hroadly to refer to physical attachment
for a period which renders
the second portion of the mufti-functional ligand capable of exerting its
immune function. For example
where the function ofthe second portion is to interact with a cell passing
through the lymphatic vessels, for
at least a period which permits sufficient interaction for the desired effect.
The term ligand is used very broadly herein to refer to any moiety, preferably
in some cases, a specitically
interacting moiety includin~~ binding moieties (eg antibodies, receptors etc.)
and bound moieties (e~~
antigens. epitopes etc) and;'ine;luding otherwise interacting moieties (e'~,
chemotactic interactions or
interactions that require multiple points of interface eg. cross-linkin~~ or
mufti-component epitopes). In
other words, the term li~~and is used broadly to refer to any entity or part
thereof which can be subject to an
intermolecular interaction that can result in bindin<.;. The term moiety is
used broadly and non-limitatively
to refer primarily to a functional part of an entity, and may refer to even
the whole of the entity depending
on the context in light ofthe broadest concept of the invention.
Optionally, dependin~~ on the mode of delivery and the relative functional
aftinity of the respective first and
second portions, the mufti-functional ligands of the present invention. may
exert their immune function
primarily in lymphatic system and also significantly bet>re and optionally
after entry into the lymphatic
system. In a preferred embodiment the multifunctional ligand is capable of
simulatin~~ a depot effect by
bindin<~ for a prolonged period to the intra-luminal lymphatic endothelium for
later release over time back
into the circulation. 'fhe choice (avidity effect resulting ti-om multiple
binding "arms") and affinity of the
binding molecule as well as various, preferably controllable factors impacting
on any "undulating"
movements of the lymphaticvessels (eg. water consumption)or competitive
bindings is contemplated to
impact the binding time.
With respect to the depot and delivery aspects of the invention discussed
herein, it is contemplated the
second portion of the mufti-functional ligand of the invention may have at
least primary medicinal effects
that are not immune function related as broadly understood.
It is to be understood that a use of a slash (.% ) means the broader of "or''
or "and.~or" unless to the context
dictates otherwise.
Some innnune interactions require. prefer or are capable of being enhanced via
coordinated ligand
interactions, for example for optimal immune stimulation, for example,
specific costimulatory li~~and
interactions eg. CD80/CD86 interactions with CD28, or for example.
interactions aimed at tolerizing or
otherwise inhibiting or reducin~~ immune effects or preventing such inhibition
(for etample usin<~ anti-
CTLA-4.!CD152 see related U.S. patents, for example 6,051.327, 5.844,095) (see
also Hodge JW et al.
Ernst Schering Res Found Workshop 2000 (30): 23-53 and Immunological Reviews
Vol 172 Dee 1999.
Entire Issue).
The invention contemplates modeling, evaluating and-or effectin~~ these
interactions for therapeutic
intervention within the lymphatic system through the substantially
contemporaneous use of different
multifunctional ligands of the invention. Furthermore, control of the relative
proportion of each of the
different li~~ands permits different spatial interspersion of these li~~=ands
on the intraluminal suface of the
lymphatic system (primarily) so as to provide controlled variability of
spatial configurations appropriate for
optimizing the coordinate interaction with multiple li~~ands on another
entity, tUr example immune cells or
cancer cells. This strategy also permits controls on avidity that extend
beyond the choice of valency for a
given single multifunctional ligand for controlling the nature and duration of
the coordinate interactions
including the duration of temporary anchoring, for example to allow cancer
cells to be killed by immune
cells, as well delivery of, for example. cytokines (through cytokine antibody
fusions), superantigens etc. to
the site of interaction. Such coordinate interactions may be substantially
contemporaneous or sequential, for
example the effect of a tirst interaction with a first multifunctional li~~and
slowing the progression of a cell
or infectious agent throu~~'h the lymphatic system for eventual rection with
another first multifunctional
ligand (ie ofthe same type) or reaction with a second type of multifunctional
li~~and. The invention also
contemplates as a strategy. alone or in combination witft other strategies: I
) delivery of a multifunctional
ligand of the invention to a particular site of action f<>r the purpose of
e~x~rting, for example a local effect,
39
CA 02402930 2002-09-19
with the result of causing the multifunctional ligand (whether or not it has
exerted its effect, provided that
or to the extent that it remains functional in at least one aspect) to
subsequently be targeted to the lymphatic
system for exerting a second effect (be it the same or a different disease
counteracting effect) including
simply elimination, or return back to the circulation lie. where the ligand is
selected leg. based on size,
immunogenicity etc.) to be preferably minimally eliminated (at least not
maximally eliminated) by the body
in the course of its circulation, havin5 regard to competin~~ desi~~n
considerations) for example, in the case
of multifunctional ligand which is an anti-tumor li<7and that has some
residual binding to normal tissues, to
set up, in effect, a site of competitive binding that advantageously impacts (
ie_reduces) undesired binding
more than desired target binding; 2) delivery of a multifunctional ligand of
the invention or an entity that
binds to a multifunctional ligand of the invention to a particular site of
action eg. local disease mediating
immune cells, for the purpose of simple binding with the expectation that a
delayed immune or other
effect will be exerted within the lymphatic system. Accordingly, the invention
is also directed to a
composition comprising at least one and optionally a plurality of different
multi-functional ligands of the
invention. The invention is also directed to such a composition when combined
with a pharmaceutically
acceptable carrier for example those that may be suitable for one or more of
the various well known and
heretofore used routes of administration including intravenous. intradernnal
etc which (for present purposes)
are preferably not incompatible with delivering a multifunctional ligand ofthe
invention to the lymphatic
system. The invention is also directed to therapeutic compositions comprising
a multifunctional ligand of
the invention and to methods of treatment using such compositions. 'the
invention is also directed to
method of : 1 ) evaluating the therapeutic effect of a particular therapeutic:
entity against a particular target
with reduced effect on undesired targets; 2) facilitating elimination a
therapeutic entity; -- by
administering the therapeutic entity as part of or in circumstances which
permit interaction with, a
multifunctional ligand of the invention.
The invention also contemplates cannulating particular portions of the
lymphatic system to localize the
delivery of a multifunctional ligand (see United States Patent 4,911,690 ) ,
for example 1) to accommodate
or further accommodate the treatment of conditions in which the immune
affectin<, molecule has an
undesirable systemic or localized side-effect if delivered otherwise; 2) for
the localized delivery, as
required, of larger molecules, complexes leg. for temporarily anchoring MHC-
peptide complexes) or
otherwise associated (at bast temporarily) entities lie. associated other than
through complex formation)
etc. andior 3) for the localized delivery of additional compositional elements
e';. adjuvants. cytokines (see
Immunological Reviews 2000 Vol 177 p. 5-246: Nature Innnunology Feb 2001 Vol 2
No. 3 page 89), or
for affecting only subsets of populations of cells or molecules that pass
through the lymphatic system or a
desired portion of the lymphatic system or are found with ~~reater
concentration within the lymphatic
system. The invention also contemplates methods of selective, enhanced or
localized, targeting! delivery by
administering multifunctional ligands ofthe invention as well as methods
(includin<~ methods directly or
indirectly employing the multifunctional ligands of the invention) of
er~hancin~>:' inc~trciy= entry of cells or
molecules, particularly immune cells lie. cells having an immune system
function as broadly understood)
or subsets thereof, to the lymphatic system or a portion of the lymphatic
system, for example for the
purpose of direct or indirect interaction with the multifunctional ligands of
the invention (in order to be
acted on directly or indirectly, by multifunctional li<~ands of the invention)
or for recruitin<~ cells that will
for example kill or modulate the activity of other cells, for example kill
cancer cells or infected cells that
will have, are havin' or have had direct or indirect interaction with the
multifunctional li~ands of the
invention, as discussed further below ~. for example in the case of cancer. by
tar~~etin~~ immunocytokines to
the disease affected tissue e~~. using cytokines eg. 'fNFa fused to antibody
that binds specitic;ally to tumor
cell markers or markers for an~~io~~enesis. Similarly tissue targetted as
opposed to disease targeted
immunocytokines could be used selectively recruit immune cells within that
tissue for example a diseased
tissue to enter the lymphatic system for such purposes including for example
interaction with a
multifunctional ligand of the invention.
It is also contemplated that a single multifunctional li~~and can have
multiple requisite interactive
functionalities for example to stimulate, attract, anergize (or otherwise
inactivate) sub-populations of B-
cells of T cells via the use, for example, of trivalent or tetravalent
antibodies and antibody
conjugates.-fusions thereof having multiple li_and interactive capabilities
(see also for example technologies
being developed for selection of successful binders by phage or ribosome
display (see for example WO
01/00866; Acfv Protein C'herrr 2000; 5>:367-403). A particular application of
this technology for
application to this invention are antibodies which retarget T-cells to tumor
cells (see for example Manzke
O. et al. Int. J. Cancer 83. 700-708 (' 1999); Br~ ,l C'urrcer 2000 Jan:
82(3):472-9; J C'oratrol Relc~u.se 2000
Feb 14: 64( l-3):229-39 as well as related relerences, cited therein or citing
these publications,
CA 02402930 2002-09-19
The present invention accommodates such technology through multispecific
antibodies or alternatively
obviates the need for combining a T-cell receptor type molecule with the
primary immune function
effecting moiety leg, a cancer cell binding moiety) by using a separate
multifunctional ligand which
combines, for example, a first portion and a second portion comprising a T-
cell interacting moiety leg. anti-
CD3). This is accomplished by administering in the same composition or
substantially contemporaneously
an amount of the second multifunctional ligand that provides, as may
empirically predicted by assessing the
dispersion ofthe marker on the endothelial cell, a strong probability leg.
.001-100°'°, optionally I-100%.
optionally 5-100°%. optionally 10-100°~0, etc) that the T cell
will be targeted in the vicinity of a <,iven
lymphatic endothelial cell that happens be proximal to the cell sought be
targeted eg the cancer cell. It is
self=evident that a 50.!50 proportion of the first and second multifunctional
li~and will yield a stronC~ chance
that a second multifunctional ligand will be immediately adjacent on a
particular given side (assuming for
the sake of argument that there are sides when in reality the dispersion of
the lymphatic endothelial marker
is governing). It is also contemplated that adjacent multifunctional ligands
may be linked for example
through linkage effective pairs of li<~ands (avidin-biotin), the second
portions having an antibody
component which binds to a common ligand (e'~ on a iiposome (see US 6197333
and refs. therein cited) or
other pharmaceutically acceptable micro/nano partiele/sphere of preferably
selectable size for optimal
spacer or endothelial cell protective purposes) and that such entities could
optionally also be employed to
house and deliver a payload to a given target vicinity.
In one aspect the multi-functional li~~ands of the present invention provide
for a method and preferably a
means for evaluating and-or inducing immune tolerance (with respect to B cells
see strategies discussed in
Immunolo~~ical Reviews ?000 Vol. 176 pp. 5-247).
It is believed that immune tolerance is enhanced or prolonged through
prolonged /strategic exposure to
tolerance inducing and!or enhancing molecules for example prolonged antigen
exposure (see Wand Y et al.
Eur. J. Immunol. 2000; 30( 18):2226-2234; Encyclopedia of Immunology : ( 1998)
Morgan Kaufirann
Publishers, ISBN:0122267656: Hoyne GF et al. lmmurzol~y 2000 Jul; 100(3):281-
8: Lerner CG et al. J
Immunol. 2000 Apr. I 5: 164(81: 3996-4002: Grossman I. et al. .Scrrir lnununo!
2000 Jun; l 2(3):197-
203; discussion 357-344 Textbook of the Autoimmune Diseases by L..ahita R. et
al. ISBN: 0781715059
Lippincott Vv'illiams K, Vfilkins; Multi-Systemic Auto-immune Diseases : An
Integrated Approach
Dermatological & Internal Aspects ISBN: 0444818960 Elsevier Science :
Arthritis and Allied Conditions
- A Textbook of Rheumatolo~~y, Thirteenth and Fourteenth Editions, William J.
Koopman. MD 14'~':1SBN:
0-7817-2240-3, November 2000; Principles of Dru~~ Development in
Transplantation cY Autoinununity
Landes Bioscience, ISBN:0412100614; Cancer c~, Autoimmunity by Ciershwin M. et
aI.,ISBN:
0444503315 Elsevier Science : J.9utoi~nmun 200(1 Jun: 14(4):278-82; The multi-
functional ligands of the
present invention, depending on their mode of administration (direct
application by cannulatin~~ a lymphatic
vessel or conventionally e~~. intradermally or intravenously), can be
advantageously employed to provide
prolonged.%strate~~ic exposure to tolerance enhancin~~ molecules (for example
by employing a multivalent
eg. bi-specific Ab fragment or diabody which hs a first portion which binds to
a lymph associated antigen
and second portion which optionally comprises anti-idiotypic Ab portion
mimicking the desired A~~ or the
antigen itself or a suitable portion thereof fused or conjugated to the first
portion) on the intra-luminal
surface of the lymphatic vessels, optionally. in addition to its conventional
effects, when administered
intradermally or intravenously, etc.. It is anticipated that the multi-
functional li~~ands of the present
invention would be useful to assess and/or effect toler<rnce induction (see
Bassadona GP et al. Proc Natl
.4enct Sci U S.a 1998 Mar 31; 95(7):3821-6; USP 6,106,834; USP 6,099,838;
US6010902: Antibody
heteroconjugates and bispecific antibodies for use in re~yulation of
lymphocyte activity: as well as
additional examples cited below with reference to examples of suitable anti-
idiotvpic antibodies).
It is also contemplated that a multispecitic contruct as described in
WO99/37791 could be used with
respect to various aspects aspects of the invention..
Additional Applications of Various Aspects of the Invention
It is contemplated that the present invention could be used to strategically
mediate, CD45 (or
variants/other PTPs) related ''cell signalin~'~. for example through signaling
molecules leg. inhibitors)
using multifunctional li~~ands ofthe invention including but not limited to
bispecific antibodies, antibody
fusions/conjugates e~~. where the immune affectin~~ antibody portion or other
moiety is conjugated, fused
etc. to an antibody or fi~a~~ment that binds to an entity associatied marker
e~~. LYVE-1 ( 1999) Journal of
Cell Biology Vol 144 No 4 p. 789-801 ) (see for example ISP 5,914,1 I I
Sievers EL., Cancer Chemother
Pharmacol 2000 46 Suppl s18 ?2 W09946268, Neel BG Curr Opin Inmunol 1997 Jan
9(3) 405-420:
Front Biosci 1998 Nov I 3:D-1060-96, Slifl:a MK et al. J. Mol. Med 2000 78(2)
74-80 Goodnow CC Ciba
41
CA 02402930 2002-09-19
Found Symp 1997 204: 190-202; Mustelin T'. et al. Front Biosci. 1998 Nov l; 3:
D 1060-96; Gaya A.
Leuk Lymphoma, 1999 Oct 35 (3-4): 237-43; Sievers EL, Curr Opin Oncol. 2000
Jan 12( I ): 30->;
Thomas ML, et al. Imnrul. Today 1999 Sep 20(9): 406-41 l; Appelbaum FR, Semin.
Hematol. 1999 Oct;
36 (4 suppl. 6): 2-8; Ulyanova T; Immul. Res 1997 Feb: 16(1 ): 101-13; re PP32
for example USP
5,846,822 and Brody JR, et al. J Biol Chem. 1999 Jul l 1i; 374(29):20053-~
regarding the functional moiety
of PP32 which is necessary for interaction with CD45, and for example IISP
6,981,351 with respect to
methods of~identifying such molecules).
In preferred embodiments the invention is directed to multifunctional ligands
that comprise immune
function exertiny~ moieties havin~~ funetionalities of molecules currently in
clinical trials or proposed for
clinical trials (see for example G lennie MJ et al. Aug 2000, Immunology Today
408 Vc>I 21 (8); sce also
Journal of Immunological Methods 237 (2000) 13 I-145; Mol Immunol 2000 Jun;
37(9) 515-526; Annu
Rev Med 2001; 52:125-145: Annu Rev Med 2001 52:63-78: Q J Nucl Med 2000 Sep;
44(3) 268-83)
including those that have an anti-CD2 functionality (see USP 5,795,572) anti-
CD4 functionality (see for
example USP 6.136,310 Herzyk D, J Infect Immun 2000 Feb; 69(2): 1(132-1043)
anti-CD3 functionality
(for example WO 00/41474; WO 9813936 3; USP 6, I I 3,901; Transplantation 2000
Dec 27 70 ( 12')
1707-12); Anti-CD44 functionality see for example Weiss L, et al., Proc Nat
Acad Sci IJSA 2000; ,Ian 4
97( I ) 285-290; Sugiyama K. Immunol Invest ( 1999) Mar-May 28(2-3) I 85-200;
Brocke S. et al. Proc
Nat Acad Sci USA 1999 Jun 8 96( 12) 6896; Mickeez K et al. Nat MecJ I '1995
Jun; 1(6); 558-63;
Ahrens T et al., J Invest Dermatol. 2001 Janl 16(1) 93-101); with respect to
control of migration ofT-cell
lymphocytes see Nohara C, et al. J Immunol. 2001 Feb I ; 166(3) 2108-21 15),
anti-CD20 functionality
(see Crit Rev Oncol Hematol 2001 Jan 37(1):13-25) etc. anti-CD22 functionality
see for example Newton
DL, et al. Blood 2001 Jan I 5: 97 (?): 538-5_i 5. (JSP 5. I 84,892; Anti-
CD4U!CTLA-4 see for example J
Immttnol 2000 Oct 1; 165(7):3612-9. Microsur~~ery 2000: 2c (8); 448-452; USP
5874082: USP
6056959; USP 5,801,227; USP 6004552; USP 5677165; USP 6087329; IJSP 5961974:
USP
6051228; White CA,et al. Annu Rev Med. 2001; 52: 63-78 (see also reviews and
specific applications
referred to in Ditzel et al., Immunol Res. 2000; 21(2-3):185-93; USP
6,010,902, IJSP 5876950; USP
5876718; CJSf 5.601,819, USf 5981251, USP 5885579 and 5885796: C'crncc>r
Immtttzol Imnutttothc>r
2000 Jun; 49(3):173-80: Oman K, J Neuroimmunol 3001 Feb I, I 13(1) 129-141;
Bellido M, Fur J.
Haematol 2001 Feb. 66(2) 100-106: Broeren et al. J Immunol (2000) DeclS
165(12) (i908-14:
Alexandroff AB et al Mol Immunol 2000 June 37(9) 515-526; Werkerle T ,I
lmmunol. 2001 i,eb I S
166(4) 231 1-2316; Howard LM J Immunol 2001 Feb; 1 16(3) 1547-53 anti-CD154:
.I I'hurntac'okinet
Biophurnt 1999 Au_~; 37(4):397-420,./('liza Ut~cn12000 Apr; 18(8):1622-36,
Leukemia 2000 Mar;
14(3):474-5, Clin C'czncv>r Re.s 2000 Feb; 6(2'):372-80, Lezrkemiu 2000 .Ian:
14( 1'):129-35, ,I ,Nucl rLlc:ct 1999
Nov; 40(1 I ):1935-46, f3loocl 1999 Nov 15; 94(10):3340-8, Blood 1999 Aug l5;
94(4):1237-47. C'cznc;er Res
1999 May 1; 59(9):3096-101, 1'crccira~ 1999 Apr 9; 17( 15-16):1837-45, l3loocl
1998 Dec I; 92( 1 1 ):4066-71,
J Rltezrrzzutol 1998 Nov; 25( 1 1 ):2065-76, C'!in Phnrnurcol Ther 1998 Sep:
64(3):339-46, ;lltrh .Scler 1996
Jul: 1(6):339-42. C.'ctnc'er lttatnuzanl hratrttenotltcrr 1997 Jul: 44(5):265-
72. 7rctn.splcrnt Pnoc 1996 Dec;
28(6):3210-1, Arthritis Rheum. 1996.1u1; 39(7):1 102-8, Itttttmnodu~~t' 1996
May; 88(1):1 3-9 and USf
5,876,7 I 8).
The invention contemplates assessment and therapeutic benefit of lymphatic
localization in the case of
antibodies and multispecitic ligands which are toxic to non-target cell
populations which express the
targeted ligand to a limited extent or in the case of toxic cross-reactivity
of the second potrtion e~~. antibody
for its desired target with an undesired target ('see eg. l,cancer 1999 Nov
13: 354(9191 ):1691-5). It is
contemplated that the toxic effect of a given effector rnoietv of a
multifunctional ligand of the invention
could be alternated usin~~ an additional binding arm for a lymphatic marker.
Antibody Structure and Function
Antibody' structure and function has bee extensively described in the
literatue. For example see Antibody
Engineering 2"'~ ed. Carl A.K. Borrebaeck, Oxford University Press 1995 p 3-
44.
Production of Bispecific Antibodies
A variety of different constructs have been developed tbr the production of
bispecific antibodies including
conventional four chain antibodies (including truncated version thereof such
minibodies (see IJSP
42
CA 02402930 2002-09-19
5,837,821), F('ab')~_ (see ,Antibody Fusion Proteins, Steven M Chamow , Avi
Ashkenazi Eds. ISBN
0471 18358X May 1999 Wiley p.136-144; or using CH3-truncated heavy chains),
diabodies (see USP
5,837,242 Multivalent and multispecific binding proteins, their manufacture
and use) constructs in which of
one or two diabody molecules are heterodimerized by creatin~~ a fusion protein
with the CL and CH 1
immunoglobulin constant domains (see WO 02:02781 ).
In recent years, a variety of chemical and recombinant methods have been
developed for the production of
bispecific and/or multivalent antibody fragments. For review, see: Kriangkum
J, et, al. Bispecific and
bifunctional simile chain recombinant antibodies. Biornol Eng 2001
Sep;18(2):31-4(1, Holli~~er P. and
W inter, G., C'urr. Opin. f3iotecvhnol. 4. 446-499 ( 1993 ); Carter, P. et
n1., J. Hemulotherapv 4, 463-470
(1995); Pluckthan, A. and Pack, P._ Inaurrrraotec~hraolos~o 3, 83-105 (1997).
Bispecifieity and/or bivalency has
been accomplished by fusing two scfv molecules via tlexible linkers, leucine
zipper motifs. C"C,,--
heterodimerization, and by association of scFv molecules to form bivalent
monospecitic diabodies and
related structures. Multivalency has been achieved by the addition of
multimerization sequences at the
carboxy or amino terminus of the scFv or Fab fragments, by using for example,
p53, streptavidin and helix-
turn-helix motifs. For example, by dimerization via the helix-turn-helix motif
of an scfv fusion protein of
the form (scFv I )-hinge-helix-turn-helix-(scFv2), a tetravelent bispecitic is
produced having two scFv
bindings sites for each of two target antigens.
Production of IgG type bispecitic antibodies, which resemble IgG antibodies in
that they posses a more or
less complete IgG constant domain structure. has been achieved by chemical
cross-linkin;~ of two different
IgG molecules or by co-expression of two antibodies from the same cell. Both
methods result in production
of significant amounts of undesired and non-functional species due to
mispairin~~ among the component
heavy and light chains. Methods have been employed to reduce or eliminate
mispairing.
One strategy developed to overcome unwanted pairing's between two different
sets of IgG heavy abd light
chains co-expressed in transfected cells in modification of the C,,3 domains
of two heavy chains to reduce
homodimerization between like antibody heavy chains. Merchant, A. M., et crl..
( 1998) h'cu. l3iotcchnolo~v
16, 677-681. In that method, light chain rnispairin~~ was eliminated b;'
requiring the use of identical light
chains for each binding site of those bispecific antibodies.
To produce bispecific antibodies, Kostelny et al (J. Immunology 148:1547 (
1992)) fused Fab fra~~rments of
antibodies to the leucine zipper portions of fos and jun proteins in the
absence of a sin<~le chain construct
for the anti~~en combinin~~ region. These methods are well described in the
literature and summarized with
references in Antibody Fusion Proteins, Steven M Chamow . Avi Ashkenazi Fds.
ISBN 0471 18358X May
1999 Wiley; Kontermann, R., et al.(Fds.) particularly at pages 139-145. Pack
and Pluckthun, fused a single
chain antibody to amphipathic helices froth a four helix bundle or from
leucine zipper proteins,
Bispecific antibodies that are in a conventional I~~G-like and Fab-like format
have been developed by Ihu
as tetravalent or bivalent molecules, respectively with each of the chains
serving to anchor a binding moiety
(see WO 01/90192 and Figure 1 therein'), preferably consistin~~ of a scF'v. In
the bispecific I~~G-like
construct, each side of the molecule comprises a Cli l domain and a Cl, domain
and each CH and CL
domain is linked through its N-terminus to a scFv of different specificity.
The invention herein
contemplates that this construct can readily be adapted to have each each halt
of the molecule associated
with a polypeptide e~~. a sefv of the same specificity so that each half of
the molecule is monospecific (or
to have each half of the molecule associated with different pairings of scFvs)
so that each half of the
molecule is effectively monospecilic. The invention herein contemplates that a
bivalent relatively low
aftinity second ligand binding moiety is used to activate receptors that
require cross-linkin~u for activity.
The invention also contemplates that numerous permutations in which the
functional affinity of the first
ligand binding moiety v'hether monospecitic or bispecitic can be accentuated
relative the functional affinity
of
the second ligand bindin<~ moiety including ernployin'~ a first ligand high
affinity seFvs for a sin~~le
antinstances in which the second li~;and binding moiety is effectively
monovalent (has one. or one useful
binding moiety). The invention also contemplates that this construct can have
a truncated Fc portion and
various known methods in the ar-t for improving the pairing efficiency of the
heavy chains. The invention
also contemplates that the C'. H I and CL domains of the second li~~and
birtdin~~ moiety can be truncated as in
camelid antibodies for efticient delivery eg. of biolo~~ic effector ligands.
43
CA 02402930 2002-09-19
Methods of Generating Antibodies That Bind To Selected Target Ligands
A variety of technologies for generating antibodies with desired specificity
have been extensively
developed and become well known to and routinely practiced by those skilled in
the art including phage
display (see review in Basic Methods in Antibody Production R Characteriztion
G.C. Howard et al. eds.
CRC Press 2001 p. 105) and other display systems (ribosome display, display on
the surface of various
cells), immunizin~~ mice, including particularly mice having human Ig genes,
and antibody microarray
technologies. These methods have also been extended to making antibodies with
dual specificites such as
diabodies (USP 5,837,2-F2 Multivalent and multispecitic bindin~,~ proteins,
their manufacture and use)
and are the subject of extensive scientific and patent literature. For
example, see US patents of Winter et al.
6,29 I ,650; 6,291,161; 6,39 I , I 58; 6,017,732; 6,225,447: 6. I 72.197:
6,140,47 I . 6.0 I 0.884
5,969,108, 5,871,907. 5,858,657; 5°733,743, 5,723,287and those of Dyax,
Morphosys, and Cambridge
Antibody Technology .
Affinity Maturation
Methods of codon based mutagenesis have been extensively developed t'or
engineering the antibody
binding site. For example, the use of such methods in a filarnentous pha;~e
display system is described in
Antibody Engineering 2"~ ed. Carl A.K. Borrebaeck, Oxford University Press
1995 p I 17-128 see also
pp.53-84 with respect to techniques of phage display of antibodies (see also
!~-rnrl~~r ru.rrm-R l~iilnrl .5J.
~fds.~ ~'f>t!li~\otibacfv I n~inc~rin;~ ISBN: 3-540-4J1354-5. --_ _ __ -
Methods of Generating Single Domain Ligands
The ability of a single variable fragment of an antibody to bind with
specificity and suitable selected
affinities in the nanomolar- range has been extensively demonstrated using
camelid and human VH
fragments. Methods of generating VHs with the desired specificity have been
extensively described (see
USP 6,248,516 Single domain ligands, receptors comprising said ligands methods
for their production, and
use of said ligands and receptors). (see also literature referenced herein on
this subject).
Methods of Making Antibodies In E. Coli
The expression of recombinant antibodies, including diabodies in E. Coli has
become routine. General
precepts, and methods are discussed in Antibody Engineerin~~ 2"'~ ed. Carl
A.K. Borrebaeck, Oxford
University Press 1995 p229-266 see also Antibody Therapeutics WJ Flarris et
al. eds. CRC Press 1997 p.
221: see also review in tzis~l~~hr~cslc~";e, 1'r~lrrn~c 5.~,
I~cc°c~r~ri~inant I'r°ot~in~< ~~-l~scraaitcanarl antiFsss~ticH,
zsrrd 'F~trer.rl~~aata~ i~erris ~. '~'1,.;,t~t~;:i~3. 1=. '~ ; .~~~=,r._w
wtU,c~zz_L,~.~~ 1.'° ft',. . _ ?"-'~ ~' "-' tf~~t~sa:~ Issg
Arstit~od~ I'rocitrsli<3rs:la~~r~tiarl ies~hrriq~rc~('~c~°, l
I=)°'~<.~1~~3~.t:E.l;? c3,i)'y"4','il~clrri;~ !t>z3"a:rr_I
Antibody Therapeutics
Production. Clinical Trials, and Strategic Issues, By Rathin C. Das, Ph.D.,
M.B.A. & K. John Morrow. Jr.,
Ph.D., D&MD Publications October 2001 Chapter 3.
Eukaryotic & Other Expression & Production Systems
Approaches for the eukaryotic expression of antibodies and antibody fusion
proteins and the preparation of
vectors for use in such methods are well known and extensively described in
the literature. General
precepts, and methods are discussed is Antibody F_ngineerin~; 2°'r ed.
Carl A.K. Borrebaeck, Oxford
University Press 1995 p267-293 (see also Antibody Therapeutics WJ Harris et
al. eds. CRC Press 1997 p.
183-220; see also review in F3is=tec°Irr~r~le3gi, 't t>lurt'c~ 5;4,
(~c~c}rnl~iraarrt i'r°c~t~ins. ylixnc~rlto~:~f
:la~lit~oclit~;, <~rr~t fl-l~~~r.~t~errlis° (~er3cs :y. '~-It, . ...~
.~. ( - ~c;. t3ie~tffit:t °~c:oc;.nhms, I'~t°:~: _ ~?~-~~ z ( ~-
_s.
1-~iie°,..J,~~E~ra:u 1;3a~cj,~~, ~~ntal>ckcl~
#'~c~<iustic~c~:las~s~ti.ill'c.°cl~r~i~lrse~!':s~Y.l..)G? ~5"~ti'v:t)-
~?I-
~)7~t~i>-~ ~tU~:;v 1~.~~~ l~u> % a;r~l Antibody Therapeutics Production,
Clinical Trials. and Strategic Issues,
44
CA 02402930 2002-09-19
By Rathin C. Das. Ph.D.. M.B.A. & K. John Morrow, Jr.. Ph.D., D&MD
Publications October 2001
Chapter 3.
With respect to a review of immunotoxins set also Antibody Therapeutics WJ
Harris et al. eds. CRC Press
1997 p 33
With respect to Methods for producing recombinant vectors see also ~.S>62,25s
Methods for producing
recombinant vectors
Formulation, purification and analytic methods involving antibodies are well
knoen to those skilled in the
art and have been extensively reviewed. With respect to formulation,
purification and analytic methods see
for example, reviews in Antibody Therapeutics Production, Clinical 'Trials,
and Strate<1ic Issues, By
Rathin C. Das. Ph.D., M.B.A. & K. John Morrow, Jr.. Ph.D., D&MD Publications
October 2001,
Chapter -1.
W ith respect to methods of generating antibodies against self antibodies see
USP x.885.793 Production of
anti-self antibodies from antibody segment repertoires and displayed on phage
Antibody Conjugates
Methods of chemical manipulation of antibodies for attachment of ligands
(eg.biotin), radionuclides etc. are
well known in the art and have been extensively reviewed (for example see
review in Basic Methods in
Antibody Production & Characteriztion G.C. Howard et al. eds. CRC Press 2001,
p. I 99; with respect to
therapeutic principles see for example, Antibody Therapeutics WJ Harris et al.
eds. CRC Press 1997 p >3-
88).
The applications of bispecific antibodies, including methods of making and
using them have been
extensively reviewed (ee for example van Spriel AB, van Ojik HH, van De Winkel
J(i. Immunotherapeutic
perspective for bispecific antibodies. Immunol Today. 2000 Aug; 21(8):391-7;
Weiner LM. Bispecific
antibodies in cancer therapy. Cancer J Sci Am. 2000 May; 6 Suppl 3:5265-7l.
Barbet .l, et al. Pretar~,~eting
with the affinity enhancement system for radioimmunotherapy. Cancer l3iother
Radiopharm. 1999 Jun;
14(3):153-66. de Wolf I=A, Brett GM. Li~~and-binding proteins: their potential
for application in systems for
controlled delivery and uptake of ligands. Pharmacol Rev. 2000 Jun; ~2~(?):207-
36.: Wang F-t, Liu Y, Wei
L, Guo Y. Bi-specific antibodies in cancer therapyAdv Exp Med Biol. 2000;
46:369-80; Staerz UD, Lee
DS, Qi Y. Induction of specific immune tolerance with hybrid antibodies. In
nnunol Today. 2000 Apr;
2((4):172-6: 1999 Dec; 43(4):336-43. Elsasser D, Stadick H, van de Winkel JG.
Valerius T. GM-CSF as
adjuvant for immunotherapy with bispecific antibodies. Eur J Cancer. 1999 Aug;
35 Suppl 3:S2>-8.
Molema G. Kroesen BJ, Helfrich W. Meijer DK, de Lei,j LF. The use of
bispecific antibodies in tumor cell
and tumor vasculature
directed immunotherapy. J Control Release. 3000 Feb 14_ 64(1-3):229-.,9. Bodey
B, Bodey B, Siegel SE.
Kaiser HE. Genetically engineered monoclonal antibodies for direct anti-
neoplastic treatment and cancer
cell specific delivery of chemotherapeutic a;~ents. Curr Pharm Des. 2000 Feb;
6(3):261-76. Kudo T, Suzuki
M, Katayose Y, Shinoda M, Sakurai N, Kodama H. Ichiyama M. Takemura S, Yoshida
H, Saeki 11, Sai,jyo
S, Takahashi J. Toimina«a T. Matsuno S. Specific targeting immunotherapy of
cancer with bispeciiic
antibodies. Tohoku J Exp Med. (999 Aug; 188(4):376-88. Koelemij R, et al.
Bispecitic antibodies in
cancer therapy, from the laboratory to the clinic. J Immunother. 1999 Nov;
22(6):514-34. Se«al DM,
Weiner GJ, Weiner LM Bispecific antibodies in cancer therapy Curr Opin
Immunol. 1999 Oct; I 1 (~):558-
62. Hudson PJ. Recombinant antibody constructs in cancer therapy. Curr Opin
Immunol. 1999 Oct;
1 1(5):548-~7. Barth RF et al, Boron neutron capture therapy of brain tumors:
an emergin~~ therapeutic
modality. Neurosurgerv. 1999 Mar: 44(3):433-50; Fleckenstein G, Osmers R.
Puchta J. Monoclonal
antibodies in solid tumours: approaches to therapy with emphasis on
gynaecological cancer. Med Oncol.
1998 Dec; 15(4):212-21. Guyre CA, Fanger MW. Macrophage-targeted killings and
vaccines. Res
Immunol. 1998 Sep-Oct: 149(7-8):655-60 Cao Y, Suresh MR. Bispecific antibodies
as novel
bioconjugates. Bioconjug Chem. 1998 Nov-Dec: 9(6):635-44. Farah RA, et al, The
development of
monoclonal antibodies for the therapy of cancer. Crit Rev Eukaryot Gene Expr.
1998; 8(3-4):321-56.:
VOItII M. Multidrug resistance and its reversal.Anticancer Res. 1998 Jul-Au~;
18(4C):290~-17. Rouard IH,
et al, Fc receptors as tartlets for immunotherapy.lnt Rev tmmunol. 1997; 16(1-
2):147-8~. Fan Z et al.
CA 02402930 2002-09-19
Therapeutic application of anti-growth factor receptor antibodies; Curr Opin
Oncol. 1998 Jane 10(1):67-
73. de Gast GC, et al,Clinical perspectives of bispecitic antibodies in
cancer. Cancer Immunol
lmmunother. 1997 Nov-Dec; 45(3-4):121-3. Carter P, Merchant AM. Engineering
antibodies for imaging
and therapy.Curr Opin Biotechnol. 1997 Aug; 8(4):449-54. Pluckthun A. et al,
New protein engineerin<>
approaches to multivalent and bispecific antibody fragments. Immunotechnology.
1997 .lun; 3(2):83-105.
Rihova B. Targeting of drugs to cell surface receptors. Crit Rev Biotechnol.
1997; 17(2):149-69. Molema G
et al,Tumor vascular endothelium: barrier or target in tumor directed drug
delivery and immunotherapy.
Pharm Res. 1997 Jan; 14( 1):2-10. Bodey B, et al, Human cancer detection and
immunotherapy with
conjugated and non-conjugated monoclonal antibodies. Anticancer Res. 1996 Mar-
Apr; 16(2):661-74
Hartmann F et al, Treatment of Hodgkin's disease with bispecitic antibodies.
Ann Oncol. 1996: 7 Suppl
4:143-6. Wels W, et al, Intervention in receptor tyrosine kinase-mediated
pathways: recombinant antibody
fusion proteins targeted to ErbB2. Curr Top Microbiol hnmunol. 1996; ~ 13 ( Pt
3):1 13-28.: Kairemo KJ.
Radioimmunotherapy of solid cancers: Acta Oncol. 1996; 35(3):343-55. Verhoeyen
ME, et al, Antibody
fragments for controlled delivery of therapeutic agents. Biochem Soc Trans.
1995 Nov; 23(4):1067-73.
Haagen IA. Performance ofCD3xCDl9 bispecitic monoclonal antibodies in B cell
malignancy. l~euk
Lymphoma. 1995 Nov; 19(5-6):381-93.
In another aspect the invention is directed to presenting antigen within the
lymphatic system (eg. in the
form of an anti-idiotype antibody) such as to facilitate a desired immune
response eg. vaccination type
responses). Optionally, adjuvants can be conventionally employed to assist
initial immune stimulation eg.
intradermally when appropriately delivered. Activatin~~ cytokines for example
as specified above, can also
be employed to enhance the immune response. Examples of antibodies having an
anti-idiotypic counterpart
or for which an anti-idiotypic counterpart could made by well known techniques
in the art (and that are
capable of exerting the desired anti-idiotypic effect) are numerous and
numerous such antiidiotypic
antibodies have application to immunization as well as applications relating
to tolerance (see for example
US patents: 6,146,627 Method for reducin~~ T cell-mediated cytotoxicity in HIV
using anti-idiotypic
antibody; 6,063,679 Anti-idiotypic monoclonal antibodies and compositions
includin~~ the anti-
idiotypic monoclonal antibodies; 6,060,049 Surrogate tolerogenesis for the
development of tolerance to
xenografts; 6,042,827Anti-idiotypic antibody induction of anti-tumor response;
6,007,815 Anti-idiotype
vaccination against diseases resulting from pathogenic responses by specific T
cell populations; 5,981,502
Methods and compositions for inducing apoptosis in tumor cells; 5,766,588
Tumor immunotherapy using
anti-idiotypic antibodies: 5.728,813 Anti-idiotypic antibody composition for
inhibitin~~ acute complement-
mediated cytotoxicity.
According to another aspect of the invention the multi-functional ligand
comprises a first portion which
binds to a lymph associated antigen and a second portion which binds to a
tumor cell infected cell or
infectious agent. This embodiment of the invention can be used for example, to
assess and affect the ability
of the tumor-binding portion to more advantageously inhibit metastasis.
Optionally, fi>r example, the
portion which binds to a lymph associated antigen has a lower affinity and'or
avidity so that the tumor cell
binding portion preferentially binds to the tumor cell and is therefore more
likely to accompany its passage
through the lymphatic system. This strategy also has application to bi-
specific antibodies of the invention in
which the second portion is for example targeted to an immune cell.
Optionally, multiple such muti-
functional ligands may permit sufticient tumor cell anchoring to permit the
tumor cell to be killed within
the lymphatic system via a toxic payload carried by the multifunctional ligand
or through the recruitment of
immune cells which accomplish this end (eg using the same or a different
multifunctic'mal ligand fused or
conjugated to a suitable cytokine (eg IL-2, IL-12). The prolonged presence of
these cells could be
advantageously used to assess methods of immunization directly against the
tumor cell using, for example,
cytokines including cytokines fused or conjugated in whole or functional part
to a lymph targeted Ab on the
same, or a different multifunctional ligand'delivered in a suitable dose (with
respect to ~Teneration of anti-
tumor antibodies and other antibody fragments for application herein as well
as important related
technologies see also WO 00/50008: WO 01!01 137; WO 97/37791; WO 99/37791; WO
97/10003;
Hoogenboom et al. Nat. Biotechnology 15(2) Feb 1997 p125-126: Fell H. et al.
Journal Of Immunolgy
Vol 146(7) Apr 1991 p2446-2452; Anderson D. et al Bioconjugate Chemistry 14(1)
Jan 1993 p10-18;
USP 6, 172,197; USP 6,171.782; Imrnunological Investigations 2000 29(2) entire
issue). Optionally the
tumor binding portion internalizes and!or delivers a toxic payload, for
example a radionuclide, or other
toxin, or a cytokine to the tumor cell (with respect to selection of tumor
internalizing human antibodies see
for example Pool M et al. J Mol Biol. 2000 Scp l; 301(5):1 149-61, see also
Kohl MD et al. J Mol. Biol.
Biotechniques (2000) Vol 28( 1 ) p 162 In this way the rnulti-functional
ligands of the invention, for
example, when provided in a sufficient dose to both target the tumor and line
a portion of the lymphatic
46
CA 02402930 2002-09-19
system to which the tar<~et tumor is likely to drain, acts as a cancer
treatment as well as a sentry system for
assessing / augmenting (for example as an adjunct therapy) the ability of the
tumor binding portion
with/without payload to inhibit metastasis. There are numerous examples of
functional cytokine and toxin
fusions used for example in cancer therapy that may have application to the
invention herein (for examples
and reviews see references herein cited as well as WO 99/37791: W099
WO00;'06605 ; WO
99/52562W0 99!37791 MULTIPURPOSE ANTIBODY; Proceeding ofthe IBC's 1 1'r'
Annual
International Conference on Antibody Engineering State of the Art Science,
Technology and Applications,
December 3-6, 2000; Amplification of T cell-mediated immune responsca by
antibody-cytokine fusion
proteins. Irnmunol Invest. 2000 May; 29(2): I 17-20: Cancer Res.1999 May l ;
59(9):2159-66.;
Pharmacokinetics and stability of the ch 14.18-interleukin-2 fusion protein in
mice. Cancer Immunol
Immunother. 1999 Aug; 48(5):219-29. Phase I study of single, escalating doses
of a superantigen-antibody
fusion protein (PNU-214565) in patients with advanced colorectal or pancreatic
carcinoma. J Immunother.
2000 Jan; 23( I ):146-53. Targeted toxin therapy for malignant
astrocytoma.Neurosurgery. 2000 Mar;
46(3):544-51 ; Targeting cytokines to tumors to induce active antitumor immune
responses by
recombinant fusion proteins. Hum Antibodies. 1999, 9( 1):23-36: Lode 14N, et
al. Tumor-tar~~eted IL-2
amplifies T cell-mediated immune response induced by y~ene therapy with single-
chain IL-12. froc Natl
Acad Sci U S A. 1999 Jul 20: 96(15):8591-6: CancerlVaccines and Immunotherapy
2000 (textbook) ;
Immunotherapy W ith Intravenous Immunoglobulins P. Imbach ( 1991 ) ,A~~ademic
Press: Molecular
Approaches to 'humor Immunotherapy (1997) World Scientific Publishing Company,
Incorporated;
Vaccines & Immunotherapy S. J. Crvz ( 1991 ) McGraw-Hill Ryerson, Limited
With respect to internalizing antibodies see eg Biological Effects of Anti-
ErbB2 Single Chain Antibodies
Selected for lnternalizin,4 Function.; Biochem Biophys Res Commun. 2001 Jan
12; 280( 1 ):27~t-279 and
references cited therein, Immunoconjugates ofgeldanarnycin and anti-HER2
monoclonal antibodies:
antiproliferative activity on human breast carcinoma cell lines J Natl Cancer
Inst. 200() Oct 4; 92( 19):1573-
81; Foulon CF, et al., Radioiodination via D-amino acid peptide enhances
cellular retention and tumor
xenograft targeting of an internalizing anti-epidermal growth factor receptor
variant I II monoclonal
antibody. Cancer Res. 2000 Aug 15; 60(16):4453-60. Poul MA, Becerril B,
Nielsen 11B, Morisson P,
Marks Selection oftumor-specific internalizing human antibodies from phage
libraries J Mol Biol. 2000
Sep I; 301(5):1 149-6l.Vrouenraets MB, et al..'fargeting of a hydrophilic
photosensitizes by use of
internalizing monoclonal antibodies: A new possibility for use in photodynamic
therapy. Int J Cancer.
2000 Oct 1; 88( I ):108-14.
In yet another aspect, the invention contemplates that the passage of tumor
cells can be inhibited within the
tumor vasculature using a bispecific ligand, optionally a bispecitic antibody,
which targets on the one hand
a well known vascular endothelial marker and one the other hand binds to a
ligand on the surface of the
tumor. Other aspects of the invention related to tumor cell targeting are
understood to described in
reference to this aspect of the invention as well. It is also contemplated
that markers which are present on
both the lymphatic endothelium and the tumor vasculature can be simultaneously
targeted with bispecitic
ligands of the invention to inhibit tumor metastasis and or immunize a subject
a~~ainst tumor cells.
It is contemplated that the multifunctional ligands of the invention when used
to inhibit metastasis, for
example, in the manner described above, could be advantageously employed in
combination with other
well known therapies foe example cytoxic drugs, other tumor targeted
antibodies and conjugate/fusions
therewith used or currently being evaluate for immunotherapies, angiogenesis
targeted dru;~s etc. (re
angiogenesis sec for example Ar~giogenesis in cancer and other diseases.
Nature. 2000 Sep 14:
407(6801 ):249-57).
Similarly, a bi-specific antibody of the invention could be used to bind to
antigens,'ligands on Ivmphocytes
which are known or become known to inhibit or enhance immune function or
mediate a disease e~~. CD45.
With respect to target receptors related to the inventions defined herein see
also USP 6,277,962.
As discussed above, as used herein the term "lymph associated antigen" refers
to antigens that are
expressed significantly on lymphatic endothelial cells but not significantly
expressed, if at all, on other
tissues. Examples of such antigen include LYVE-1 a CD44 receptor analo~~ue
which hinds to HA (February
22, 1999, Banerji et. al., Journal ofCell Biology Vol. 144. ~4, p789-801) and
which is expressed primarilyy
on lymphatic endothelial cells. LYVE-I specific antisera have been shown to
inhibit binding of HA. The
invention contemplates research and treatments using multi-functional lis;ands
ofthe invention with respect
to non-human mammals. including preferably agricultural animals. canine
species. primates and mice
47
CA 02402930 2002-09-19
having similar receptors;'antigens. For example, a murine counterpart to LYVE-
I (published in Prevo R.
et al. 2001 Feb 20, J. Biol. Chem.; Manuscript MO I 1004200) can be employed
to implement the various
methods and embodiments described herein in a mouse model, for example to
assess the extent of
inhibition of metastasis effected by a multifunctional ligand (optionally
comprising for example to a toxin,
cytokine T cell receptor etc) which has a first portion which binds to LYVE-I
and a second portion which
binds to, for example to Cil-101, a breast tumor which is known to metastisize
to the lung (see USP
6037520 and 5, 693, 533 see also US patents 5.643, 551, 5491284, 5569812,
5917124 and 6107540 and
references cited in these patents, particularly with respect to other
metastatic models and methods of
evaluating anticancer drugs in mice). LYVE-1 counterparts in other mammals can
be identified in the
manner described by Prevo R. et al. (see also Skobe M. et al. Induction of
tumor lymphangiogenesis by
VEGF-C promotes breast cancer metastasis Nat. Med. Feb; 7(2) 192-8.)
Other models of metastasis in animals are well known in the art (see for
example Chirgwin JM, Guise
TA.Molecular mechanisms of tumor-bone interactions in osteolytic metastases.
Crit Rev Eukaryot Gene
Expr. 2000;10(2):159-78. 3: Kobaek-Larsen M, et alReview of colorectal cancer
and its metastases in
rodent models: comparative aspects with those in humans. Comp Med. 2000
Feb;50(I):16-26. 5: Magnano
M, et al.A physical-based model for the simulation of neoplastic growth and
metastasis. J Sur<= Oncol. 2000
Jun;74(2):122-9. 6: Hol~fman RM. Orthotopic metastatic mouse models for
anticancer dru~~ d scovery and
evaluation:a bride to The clinic. Invest New Drugs. 1999:17(4):343-59.
Russo J, Russo IH.The pathway of neoplastic transformation of human breast
epithelial ceIIs.Radiat Res.
3001 Jan; 155( 1 Pt 2):15 I-154. Duffy MJ, McCarihy K.Matrix
metalloproteinases in cancer: prognostic
markers and targets for therapy(review).fnt J Oncol. 1998 Jun:12(6):1343-8.
22: Banerjee A, Quirke
P.Experimental models of colorectal cancer. Dis Colon Rectum. 1998
Apr;41(4):490-505.
Wu TT et aLEstablishin~~ human prostate cancer cell xenografts in bone:
induction of osteoblastic reaction
by prostate-specitic antigen-producin~~ tumors in athymic and SCID/b'~ rnice
using LNCaP and lineage-
derived metastatic sublines. Int J Cancer. 1998 Sep 1 1;77(6):887-94.61:
Molpus KL, et alCharacterization
of a xenograft model of human ovarian carcinoma which produces intraperitoneal
carcinomatosis and
metastases in mice. Int J Cancer. 1996 Nov 27;68(5):588-95.65: Pages JC,
Sordat B. Bautista D, Costa J,
Benhattar J. Detection of rare circulating human colon tumor cells in a nude
mouse xenograft model.
Cancer Lett. 1996 Au~~ 2p:106( I ):139-44.66: Sakakibara T, et al.Doxorubicin
encapsulated in sterically
stabilized liposomes is superior to tree drug or drub containing conventional
liposomes at suppressin~~
growth and metastases of human lung tumor xenografts. C ancer Res. 1996 Aug
15;56( 16):3743-6.
With respect to modifying an antibody to increase its affinity see also
Crystal structure of Fab 198, an
efficient protector of the acetylcholine receptor against myasthenogenic
antibodies. Eur J Biochem. 2001
Ju1;268( 13):3685-3693.
For example, in one embodiment the invention contemplates a bispecific
antibody comprisinv~ an antigen
binding component specific for a tumor cell associated antigen and a
relatively low affinity anti-IL-6
receptor antibody component. With respect to the anti-tumor role of IL-6 see
Wei LH et al. Interleukin-6 in
cervical cancer: the relationship with vascular endothelial growth factor.
Gynecol Oncol. 2001
Ju1;82( 1 ):49-56.
'The invention contemplates that TCRs and modified TCRs (see for example, WO O
1/48145) may be used
as ligands, in place of antibody fragments for bindin<~ to target ligands such
as peptide~MHC li~~ands.
Techniques for generating antibodies, and methods, for example of subtractive
screenin<~ useful to identify
other lymphatic vessel associated antibodies, including; those optionally
having smaller scFv, Fab and dAb
(single domain antibody or functional fragment thereot~ component (more easily
passa~Tin~~ to lymphatic
vessels from tissues particularly when constructed in the form of bispecitic
antibodies a«. diabodies etc.) by
phage or ribosome display are well known in the art (see for example Hoogenbom
HR et al. Immunol.
Today (Aug. 2000) Vol 8 p 371: Schaffitzel C. et al. J Imrnunol. Methods (Dec.
10, I 999) 331 ( 1-2) p.
1 19; Roberts RW et al. Curr Opin Chem Biol. 1999 Jun; 3(3):268-73; Winter G.
et al. Annu Rev
Immunol 1994 12:433-55; Kontermann RE et al. Nat Biotechnol. 1997 Jul; I
5(7):629-31: Phage
Display of Peptides and Proteins, A Laboratory Manual Kay BK et al. Eds 1996
Academic Press;
Immunology Methods Manual Lefhovits, I ed. 1997 Academic Press;Hoogenboom et
al.
Immunotechnolo~~y 4 (1998)1-20;
48
CA 02402930 2002-09-19
**With respect to making single domain antibodies see for example USP
5,824,520, USP 5622836, USP
5,702,892, USP 5,959,087, Unique single-domain antigen binding fragments
derived from naturally
occurring camel heavy-chain antibodies.J Mol Recognit. 1999 Mar-Apr; 12(2):131-
40. An antibody single-
domain phage display library of a native heavy chain variable region:
isolation of Functional single-domain
VH molecules with a unique interface. J Mol Biol. 1999 Jul 16; 290(3):685-98
and reterences cited in these
references.
Methods for making antibody fusion proteins and bi-specific antibodies
including diabodies etc. and fusion
proteins thereof are well established in the art (for reviews and particular
applications see for example
Adams GP et al. Journal of Immunological Methods 2 3 I ( 1999) 249-260; USP
6,121,424, 6,027,725 and
6,025.165; EP 0654085: Hudson P. Exp. Opin. Invest. Drugs (2000) 9(6): 1231-
1242; Antibody Fusion
Proteins Steven M Chamuvv , Avi Ashkenazi F:ds. ISBN 0471 18358X May 1999
Wiley; Antibody
Engineering, Carl A. Borrebaeck Oxford University Press. 1995; Antibody
Engineering:A Practical
Approach David J. Chiswell, Hennie R. Hoogenboom. John McCafferty
OxfordUniversity Press,1996;
Antibody Engineering Protocols, Sudhir Paul ( 1990 Humana Press; Antibody
Expression R Engineering
( 1998) Henry Y. Wang, Tadayuki Imanaka, American Chemical Society: Zhu Z.
Biotechnology (NY)
1996 Feb.; 14(2): 192-6: Nielsen UB et al. Cancer Res. 2000 Nov 15;
60(22):6434-40; Lawrence LJ.
Et al Febs Lett. 1998 Apr. 3; 425(3 ) 479-84; Hollin~~er et al., Cancer
Irnmunol Immunother 1997 Nov-
Decc 45 (3-4) 128-30; Immunotar~~etin~ of tumors: state of the art and
prospects in 2000 Bull Cancer.
2000 Nov; 87( 1 l ):777-91; Hellfrich Wet al Int. J. cancer 1998 Apr 13 76(2):
232-9; Wu AM, Q J Nuc
Med. 2000 Sep.; 44(3):?68-8 3 KrebsB. Et al. J Interferon cytokine Res 1998Sep
18(9): 783-9 l ; 'fakemura
Si, et al. Protein Eng, 2000 Aug.; 13(8): 583-8; Cochlovius B et al. J
Immunol. 2000 Jul I 5;
165(2):888-95; Atwelll Jl. et al. Protein En;~. 1999 Jul; 12(7) : 597-604;
kiprivanov SM et al. ,I. Mol
Biol. 1999 Oct 15, 293 ( I ): 41-56; Alt M. et al FEBS Lef-f. Jul 2 454 ()1-2)
90-4. Hudson P.I et al. J
Immunol Methods 1999 Dec 10; 231( 1-2):177-89 Ardnt MA et al. Blood 1999 Oct
15 94(8): 2662-8;
Lu D. et al. J Immunol. Methods 1999 Nov. l9; 230( 1-2):159-171; Santos AD et
al, Clin Cancer Res
1999 Oct 5 ( 10 supply: 3 I 185-31235 Kontermann RE et al. Nat Biotechnol.
1997 Jul: 15(7):629-31;
Dolez et al. Protein en's. (2000) Aug 13 (8): 565-74; Adams GP et al. Nucl.
Med. Biol (2000) May 27
(4); 339-46; Williams LE et al. Med phys 2000 may 27(5) 988-94; fitzgerald K.
Protein En~~ 1997 oct
10(10): 1221-5 and the various references cited therein) as are various
methods for identifying internalizing
antibodies and creating toxin, radionuclide and cytokine fusions ! conjugates
(see go Y et al Bioconj. Chem
1998 Nov-Dec; 9(6): 635-44) for fully exploiting various aspects ofthe
invention herein detmed (see for
example Becerril B et al. Biochem Biophys Res Comm 1999 Feb 16; 2'_~5(?):386-
93 see also additional
references blow.
Triabodies and other known multivalent antibodies etc. (see for example
Iliades P et al. fEBS I,ett. 1997
June 16; 409(3):437-41 ) etc. could advantageously be employed to provide
additional functionalities, as
well as variation in avidity etc. for the purposes of variously exploitin<~
the invention herein.
Methods of expressing and identifying new molecules like LYVE-I are also well
known in the art (see WO
98/06839)
Technologies for rendering the multifunctional ligands of the invention less
immunogenic (eg such as
employed by Biovation) are preferably applied to the multifunctional li~~ands
ofth a invention.
For recent progress in the treatment of lupus nephritis see Zimmerman R. Annu
Rev. Med. 2001; 52:63-
78.
With respect to targeting Fas-L see US6068841:Antibodies to Fas-L for
treatment of hepatitis.
The invention also contemplates usin~~ chemokines and variously targeted
antibodies and fragments thereof
fused or conjugated to chemokines or other molecules with for example,
lymphocyte or other immune cell
attractant properties (see for example Sun J. et al. l.,ymphology 32 ( 1999}
166-170; and Gerard C. et al.
Nature Immunology (20(11, Feb.) 2(2): p108; ImmunoloL=ical Reviews I~)99 Vol
170 p 5-197) to attract
immune cells into target tissues for eventual penetration into the lymphatic
vessels for activation,
signalling, binding to, inhibition, etc.. For example, for cancer treatment
antibodies that bind to
angiogenesis markers fused to such type such molecules a<~. 'FNF-a can be
advanta~~eously employed
optionally in conjunction with various vaccination strategies (including the
use of the muti-functional
ligands ofthe present invention) to attract innnune cells includin~~.
optionally, vaccination-activated tumor
targeting lymphocytes to the tumor site. In an indirectly related aspect
(havin~~ independent applications as
49
CA 02402930 2002-09-19
well as for combination therapy with a multifuctional ligand, the invention is
also directed to an antibody
that targets an angiogenesis marker fused/con,jugated to a cytokine or
antibody (ie a bispecific antibody)
which binds to a cytokine, which cytokine augments adhesion of immune cells to
blood vessels and method
of using same (by administration to a subject), alone, in combination with
multifunctional ligands of the
invention or with other vaccination strategies to increase immune cell
targeting to a solid tumor. In the case
of a bispecitic antibody it is contemplated that the cytokine binding portion
has a relatively low functional
affinity to the cytokine so as to compete unfavourably fc>r its bindings to
its natural receptor.
With reference to modulating binding of leucocytes to endothelial adhesion
molecules see for example US
Patent No. 6.123.915 and the references therein cited.
It is well known to those in the art to make bispecitic antibodies which are
adapted to bind two different
ligands on the same cell. for example so called antigen-forks as disclosed in
USP 6,705,614 (see also Shi '1'
et al. Murine bispecific antibody 1 A l0 directed to human transferrin
receptor and a 42-kDa tumor-
associated glycoprotein also Clin Immunol Immunopathol 1996 Feb;78(2):188-95;
Amoroso AR et al.,
Binding characteristics and antitumor properties of I A 10 bispecific antibody
recognizin~~ gp40 and human
transferrin receptor Cancer Res i 996 Jan 1;56( 1 ): l I 3-20; Ring DB et al.,
Antigen forks: bispecific reagents
that inhibit cell ~~rowth by binding selected pairs of tumor antigens, Cancer
Immunol Immunother 1994
Ju1;39( 1'):41-8; l~u D et al., Complete inhibition of vascular endothelial
growrth factor (VEGF) activities
with a bifunctional diabody directed against both VEGF kinase receptors. fms-
like tyrosine kinase receptor
and kinase insert domain-containing receptor. Cancer Res 2001 Oct
1;61(19):7002-8; Schmicdl A,
Breitlin'~ F. Dubel S. Expression of a bispecitic dsFv-dsFv' antibody fragment
in Escherichia call. Protein
En~~ 2000 Oct; I 3( 10):725-34 see also Park SS, et al.. Generation and
characterization <>f a novel tetravalent
bispecific antibody that binds to hepatitis B virus surface antigens Mol
Irnmunol 2000 Dec;37( 18'): I 123-30;
Kriangkum J et al., Bispecific and bifunctional single chain recombinant
antibodies Biomol Eng 2001
Sep; l 8(2):31-40; LJSPs 4.17-J,893, 5,989.830; VVO OOi2943 l ).
With respect to antibodies to autoantigens, ADEPT, use of anti-eotaxin
antibodies, Delmmunization,
antibody-cytokine fusions. ribosome display. xenomouse technology: cuttin;~
edge phage display
techniques. construction of human antibody fragment based pha~~e display
libraries, selection of
internalizin~~ antibodies by phage-display. cancer targeting antibodies.
antibody arrays, plantibodies, design
of mutant IGSF domains of CD2, CD58 and TCR; oligopeptide e~, paratope
mimetics, diabodies,
minibodies, triabodies, tetrabodies and related size:-kinetics issues, caspase
activatable pro-druts, delivery
of Bismuth-213 via scFv <rnd diabodies,anti-an_~ionenesis marker strategies,
immunoenzype therapy of
cancer (eg. with Rnases) pancarcinomic antigens like CEA (~~AG")-72; and
related technolo~~ies set the
papers and references in Proceedings of IBC's 1 I'~' Annual International
Conference on Antibody
Engineerin~~. State ofthe Ar-t. Science. Technology and Applications Dec :>-6
2000 f_a Jolla. CA.
With respect to biology of the lymphatic system havin~~ practical application
herein see Ikomi, F
lymphology (1999) 32:90-103; Shield JW. Lymphology 1999 32: 1 18-122 and
L.ymphology 33 (2000)
144-147, as well as the reterences cited therein.
The invention also contemplates control of such migration by inhibition of
receptors that mediate such
mibration (see far example Sun J. et a(. Eympholo~y 32 ( 1999) 166-170) for
controlled application of the
multitunctional ligands of the invention.
With respect to recent developments with respect to target ligands and~'or
immunotherapy having
application herein see also WO 01!12224, WO 01'14550, WO 01/11059, V'O Ol%
10205_ V1''O 01;00679,
W0029445 WO 01 / 14885, WO14564, WO 0114558. WO 01,'' 14234, WO 01;' 13945, WO
01:' 12840, WO
01 % 12781, Vv'o Oli 12674, w'0 0 1126?0, WO 0 112224. WO 0112646; VI!O
01'12223. WO 0I % 12218,
WOOI~'12217, WO01; 12216, WO01,'12154, WOOIi14557, VJOOli11059. WOOli10912,
WO01/11040.
WO 0 1!10888, Vv'O OI. 10=t60, WO O1 ' 10205. VfO 01'0961 I, WO 01!09328, WO
01 1'09186, WO 01 '0919?,
Vv'O Ol 108635. W'0 0('07481. V1~'O 011'07082. WO 01,''07084, Vv'O 01'07081,
WO 01!07484, WO 01107466,
'Triggering Fc alpha-receptor 1 (CD89) recruits neutrophils as etfeetar cells
for CD20-directed antibody
therapy. J Immunol. 2000 Nov 15; 165( 10):5954-61. CD47 engagement inhibits
cytokine production and
maturation of human dendritic cells. J Immunol. 2000 Feb 15; 164(4):2193-9.
The invention also contemplates that a multifunctional li~Tand that recognizes
an immune cell as a target in
virtue of a particular cell marker and will be able to deliver a toxic payload
to the cell. for example, in
virtue of its second portion comprising such toxic component fused or
conjugated thereto. The invention
CA 02402930 2002-09-19
also contemplates attracting or supplying other immune cells or molecules to
kill, or otherwise inactivate
the tartlet immune cell (eV~. lymphocytes eg. by TH cell modulation or CD4
cell modulation or usin~~
antibodies including anti-idiotypic antibodies. The invention therefore
contemplates that treatment of such
immune cells can be accomplished by a combination of different mechanisms or
drugs depending on the
disease so as to reduce immunosuppression due to immune cell ablation where
this is the dominant
consideration. Such interactions may require interaction with one or more
ligands on the surface of the
targeted immune cell, as facilitated via anchoring interactions of varying
affinity/avidity/duration. The
invention also contemplates using multifunctional ligands comprising or bound
to selectins and ICAMs etc.
to facilitate such targetin«, for example co-adminstering same in a proportion
which is for example 0.01%
to 25°,% of the tar;~etin~~ multifunctional ligand. The relative
amounts of the selectin!ICAM etc. (including
antibody mimics) bearin<,~ multifunctional ligand as compared with the
targeting multifunctional ligand can
be determined empirically by varying the proportions and assessing any
objective indicator of successful
targeting in a disease related or purely experimental context. For example
successful targeting leg. antibody
binding to eg. CD3, C D28, CD2) using multifunctional ligands of the invention
could be monitored by
evaluating levels of cytokines normally attributable to such binding (see for
example CD8 T cell activation
after intravenous administration of CD3 x CD19 bispecific antibody in patients
with non-Hodgkin
lymphoma. Cancer lmmunol lmmunother. 1995 Jun; 40(6):390-6. Definition of a
lamina propria T cell
responsive state. Enhanced cytokine responsiveness of T cells stimulated
throu~~h the CD2 pathway. J
lmrrrunol. 1995 ,Ian I 5; 154(2):664-75.
With respect to multifunctional ligands that are used to directly or
indirectly exert an immunization
function, other examples of disease associated peptides that can be presented
as imrnunogens or
inhibitor,%modulators of immune activity or disease pro~~ression in one of the
fashions su<~~~ested above
include. examples as well as technologies referenced in, for example, Knuth A,
Cancer Chemother
Pharmacol (2000): 46 suppl: 546-51; Engelhard VH, CancerJ Sci Arn 2000 May; 6
Suppl 3: 5272-80;
Pietersz GA et al, Cell Mol Life Sci. 2000 Feb; 57(2): 290-310; Algarra I et
al, Hum Immuno). 2000 Jan;
61 ( 1 ): 65-73; Tumour vaccines: a new immunotherapeutic approach in
oncolo~~y.Ann Hematol. ?000 Dec;
79(12):651-9; Human tumor-rejection antigens and peptides from genes to
clinical research Nippon Geka
Gakkai Zasshi. 2000 Sep; 1 Ol (9):612-7. Pinilla-Ibarz J, et al CML vaccines
as a paradi~Tm of the specific
immunotherapy ofcancer. Blood Rev. 2000 Jun; 14(2):1 I I-20).
In order to present an MHC-peptide complex in proximity to a B7 co-stimulatory
molecule. the invention
contemplates using, in addition to varying amounts (varying from a 50'50
proportion) of adjacent
multifunctional ligands (which may be a dAb, diabody etc.) preferably cross-
linked by an avidin
component. -- as a different strate~~y -- cross-linking with avidin or the
like adjacent arms of a single
diabody, triabody or tetrabody etc. which binds to or has been fused or
conjugated individually to
respective B7 and MHC peptide components (with respect to recombinant B7 and
MHC molecules and
fusion proteins thereof including antibody fusions and related technologies
see references above and EP
99/97477 WO 9y '42597. WO 97 28191. US 6, 197, 302, US6015884 US61401 13, US
(i,045,796, US
5580756, EP0935607, WO 9806749 W()9803552. EP I (154984. US 5869270,
Construction and
characterization of bispecitic costimulatory molecules containing a minimized
CD86 (B7-2) domain and
single-chain antibody fra~arnents for tumor targeting; method is useful for
cancer therapy Rohrbach F et al.,
CIin.Cancer Res.; (2000) 6. 1 I, 4314-22 WO 00!008057 17 Feb 2000; V~10
9921572 (i May 1999: WO
9913095 18 Mar 1999: VI~'O 9742329 13 Nov 1997; WO 9720048 5 Jun 1997; WO
9640915 19 Dec
1996; WO 00'023087: E:I' 610046 10 Au~r 1994, IJSP 6056952 as well as
references therein cited).
In a related aspect, the invention similarly contemplates using or more
antibodies (optionally biotinylated
and cross-linked by an avidin component) that bind to the same or different
epitopes on a tumor including,
where two such antibodies are used different proportions of MHC and B7 linked
lie fused, conjugated or
capable of bindings to) antibodies as well as different proportions of
different epitope-specific antibodies to
optimize the distribution of such cross-linked B7 and MF1C peptide complexes
for T-cell recognition. In
this way any strongly imrnunogenic peptide may be used in conjunction with
suitable vaccination strategies
to create a universal cancer antigen. Using a tumor unrelated peptide is
advantageous to avoid any
tolerization effects resulting front T-cell binding to the MHC-peptide alone
and does not preclude immune
system recognition of a difFerent epitope or other therapies. In a preferred
embodiment, a single
multifunctional li~~and or pair of multifunctional li~~ands optionally
biotinylated and cross-linked by an
avidin (or variants), is used to bind to both the lumen of the lymphatic
system and to a tumor cell. (using:
for example a trispecific antibody with monovalent linkage to both the cancer
cell and lymphatic
endothelial cell and a third antibody component having respective fusions try
one of MHC-peptide and B7
51
CA 02402930 2002-09-19
on heavy and light chain, or a trispecific or tetraspecific tetrabody having
an antibody component devoted
to each or the B7 and MHC linkages). This permits a single molecule to be used
for both the immunization
within the lymphatic system and the tumor targeted antigen display. However,
It will be appreciated that.
presentation of MHC-peptide complex on a tumor does not necessarily require
costimulatory B 7
presentation to induce a cytotoxic T cell response which is specific for the
peptide and that multiple such
presentations, preferably in a cross-linkable fashion may be preferable.
Accordin~~ly, strategies herein for
costimulatory presentation of MHC-peptide and B7 may be differently applied to
a lymphatic endothelial
cell surface for immunization purposes and and a tumor cell surface (primarily
for recognition purposes),
for example by using avidin facilitated cross-linking of in the former but not
the latter (tumor) context or
using different sets of molecules in each case or using modularly
reconstructing the tumor cell suface with
a bispefic antibody that binds to a separately administered MHC and: or B'J
component.
Subject to the latter proviso, in preferred embodiments, the invention
contemplates using as separate
counterparts 1) separate trispecific Abs, each including for example, one
antibody component which binds
to the each of the respective B7 and MHC molecules which are preferably
together, separately
administered. Such multifunctional ligands are preferably biotinylated for
cross-linking - both between
adjacent trispeeific Abs and adjacent T-cell stimulatorylco-stimulatory arms:
or ?) separate bispecific
pairs of Abs each respectively havin~~ I ) either <t B7 and lymphatic vessel
or B7 and tumor binding portion
or 2) a MHC peptide complex and a lymphatic vessel or MHC complex and tumor
binding, portions which
again are prefereably cross-linked by an avidin.streptavidin or a variant (ie.
using biotinylated antibodys)
This latter embodiment permits smaller size antibody molecules to be used for
better tumor targeting.
Antibody components which recognize the non-T cell interactive portion of the
B7 or MHC molecule can
be readily generated by phage display. for example in the case of a known
peptide specific antibody to an
MHC peptide complex (sec Chames et al, Proc Natl Acad Sci USA 97, 7969 and
Chames et. al. "Attinity
Maturation of TCR-Like MHC-peptide specific antibody: peptide specificity is
possible over a wide
affinity range" Proceedin~~s of IBC Conference on Antibody Engineeerin<~ Dec.
2000) eg. by first causing
binding of the "peptide specific" antibody and then doing the phage display
e~~. usin~~ an array of multiple
(eg. repeats of the same antibody) such peptide specific MHC antibodies.
applying the MHC peptide
complex to effect binding and then performing the phage or ribosome display.
Alternatively a TCR (eg
cell bound) or analo~,ue.-'mimotope could be used for the orientation.
Similarly antibodies could be
generated which in effect do not compete with CD28 or a mimotope thereof to
create suitable anti-B7 type
antibodies. Anti-B7 antibodies are known in the art. The invention also
contemplates that the MHC-peptide
binding function may be supplied using a linked superantigen (US 619729~a, WO
9601650 25 Jan 1996:
Proc.Natl.Acad.Sci.U.S.A.; (1994) 91, 19, 8945-49) in both the tumor and
lymphatic system binding sites.
Optionally, the tumor anti~:en or one or both of the antigens are a pan-
carcinomic antigen like TAG-72,
CEA, Ei I 1 (WO 97!44461 ). The invention also contemplates using one or more
phage display libraries to
optimize the development of MHC; B7 costimulatory bispecific antibodies, by
usin~~ cell sized latex spheres
coated with an antigen eg. C:EA in various surface dispersions (or a cell) and
usinG~ a array ofpreferably
biotinylated antibodies which recognize the antigen and have a "oppositely
located"portion fused,
conjugated or capable of binding to one or both of MHC and B7, the library
optionally also presenting
also variations and combinations of lengths (truncations) of one or more
constant reunions or for example
the CDR2 generated by phage display, dependin~~ on the choice of antibody',
and with microarray
technology, using a signalling means to detect T-cell recognition and
evaluating cytotoxicity with for
example a CrSI release assay. (with respect to protein chip or microarray
technology see WO 00.-'63701
references, for example in the Proceedings of IBC's conference on Protein
Microarray Technology March
19-21 Santiago California
The invention also contemplates use of recently published antibodies in the
context of the invention (see
WO Ol! 19861, WO OI % 19990, WO Ol % 19860, WO 0119987, WO 01:'19990, WO
99.58678, WO 00.''i994 3,
WO 01/18014, WO 01/18016, WO 01:'18204, WO 01;'18043, WO 01/18021, WO
01/18014, WO 01/18046,
WO01-16166, W00115731, W00115728, WO01'16183. W001%16171), WO01!1573?.
The invention is also directed to a method of evaluating dosing, ligand
saturation, avidity effects of
simultaneous ligand bindin'a on prolonged anchoring and associated benefits
(eg. to delay a cancer cell for
targeted killing or facilitate transfer of the multifunctional ligand to the
targeted cell), cooperative
interactions, cross-linking interactions (see J lntnnnau! 2001 Mar 1; I 66(5):
3356-3265; Nippon Rinsho.
(999 Dec; 57 Suppl:4?8-3?: Harefuah. ?000 Jun 15: 138(12):1046-50. Leuk
Lymphoma. 1998 Mar; 29(1-
2):1-l~ ) and costimulatory interactions by administering to a test subject tw
~o different multifunctional
ligands of the invention with cooperating second portions.
52
CA 02402930 2002-09-19
With respect to the display of functional peptides on an antibody type
scaffold see Nuttal SD; et al..,
Proteins (1999) 36: 217-227; see also Skerra A., J. Mol. Recognition 2000 July-
Auk 13(4): 167-187. The
invention also contemplates bispecific multifunctional ligands in which the
immune function exerting
moiety exerts its function throw<~,h binding to an immunc'rgenic component or
carrier for such eornponent as
discussed above, for example an Fc domain fused to a peptide, a heat shock
protein (see for example Wany
XY, Immunol Invest 2000 May 29(2): 1 3l-7 and references cited therein as well
as US6168793:
US6071956 ; US05981706; US05948646 Methods for preparation of vaccines against
cancer
comprising heat shock protein-peptide complexes; US05830464 Compositions and
methods for the
treatment and growth inhibition of cancer using heat shock/stress protein-
peptide complexes in
combination with adoptive immunotherapy as well as patents. scientitic
atricles and patent applications
referenced in these patenta: with respect to MHC peptide complexes( see for
example WO 99/64597, WO
98103552, WO 98r06749 and references cited therein).
As described above, the invention also contemplates that the lower affinity
ligand binding arm of the
aforementioned multifunctional li~,and (1e. having a high aftinity targeting
arm and a lower affinity effector
arm) is constituted by a high affinity ligand, for example an high affinity
antibody or functional fra~,ment
thereof, which binds to a target biological effector (eg. a cytokine,
chemokine, growth factor. hormone or
other biological response modifier or drug) with high af~iinity, in a manner
which permits the effector to
continue to bind to its desired target receptor while bound to the antibody
(1e. the antibody binds to a
portion of the effector which is not critically involved in the effector
binding to its receptor) provided that
when bound to the effector the antibody or fragment thereof has. when
cornbined with the eflector, a
suitably lower affinity for the receptor than the li~~and bindin<,g arm which
functions as the hi<~h affinity
binder has for its target cell marker. In one embodiment the bindings moiet_d
which binds to the biological
effector binds to it with hi'=her affinity than the atfinity that the effector
has for the effector receptor. The
invention also contemplates that this bindings ann can bind to biolo~~ical
eFfector in a manner which permits
it to bind to one receptor but not a related receptor to which the effector
would otherwise bind (see
examples below). The invention also contemplates that antibody arrays are used
to screen for antibodies
which are capable of bindings to such biological eftectors, while bind in situ
to their receptors. The
invention also contemplates that such binders, when bound to the biolo<~ica1
effector, can be used to test
their ability to bind to related receptors. such as those within the same
family a<,. within the same family of
TNF like receptors. With respect to antibody microarrays see for example
Cahill DJ.Protein and antibody
arrays and their medical applications.) Immunol Methods. 2001 Apr:250( 1-2):81-
91. MacBeath G.
Proteomics comes to the surface.Nat Biotechnol. 3001 Sep:19(9):828-9. flewley
JP. Recombinant protein
arrays.Commun Dis Public Health. 2000 Dec:3(4):31 I-2; Holt LJ, Enever f, de
Wildt RM, Tomlinson IM.
The use of recombinant antibodies in proteomics.Curr Opin Biotechnol. 2000
Oct; l I (5):445-9. Walter G, et
al.Protein arrays for gene expression and molecular interaction sereening.furr
Opin Microbiol. 2000
Jun;3(3):298-302. de ~'ildt RM, Mundy CR, Gorick BD, Tornlinson IM.Antibody
arrays for hi~~h-
throughput screening of antibody-antigen interactions.Nat Biotechnol. 200,)
Sep; 18(9):989-94.FIolt LJ, et
aLBy-passin~~ selection: direct screening for antibody-anti~~en interactions
r.rsin~ protein arrays. Nucleic
Acids Res. 2000 Au'~ 1;28( 15):E72 and the references cited therein. Th a term
receptor as used herein for
greater certainty includes decoy receptors. Examples of decoy receptors
inc;ludeTRAIL decoy receptors
(APO-2L), CD44 decoy like receptors (hyaluronan), interleukin receptor like
protein (1l.-17) ( see J Biol
Chem 2001 Nov 12), CD~75-Fe decay receptor, -fRAMf. IL.-1 RII receptor,
osteoprotegerin (OPG),
IL 13 Ralpha2.
**Affinity Maturation
Techniques for affinity maturation using hi~~h throughput screening techniques
to evaluate mutants are well
known in the art. Femtomolar affinities have been achieved and it is quite
common to obtain nanomolar to
picomolar affinities as a result of an affinity maturation process. For
example it well known to use
techniques of parsimonious mutagenesis to engineer amino acid change at
selected whotspots".With respect
to affinity maturation see fbr example Coia G. Hudson PJ. lrvin~~ RA. Protein
aftinity maturation in vivo
using E. coli rnutator cells. J Immunol Methods. 3001 May 1;251(1-2):187-93.
Manivel V, Sahoo NC,
Salunke DM, Rao KV. Maturation of an antibody response is governed by'
modulations in tlexibility of the
antigen-combining site. Immunity. 2000 Nov; 13(5):61 1-20. Boder LT, Midelfort
KS, W'ittrup KD.
Directed evolution of antibody fragments with monovalent femtomolar antigen-
bindin~~ affinity.Proc Natl
Acad Sci U S A. 2000 Sep 26:97(20):10701-5. holler 1D, Holman PO, Shasta EV.
O'Herrin S, ~'ittrup
KD. Kranz DM.In vitro evolution of a T cell receptor with high affinity for
peptide 'MHf. Proc Natl Acad
Sci U S A. 2000 May 9:97( 10):5387-92. Daugherty PS, Chen G, lverson BL,
Georgiou G.Quantitative
analysis of the effect of the mutation frequency on the affinity maturation of
single chain Fv antibodies.
53
CA 02402930 2002-09-19
Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2029-34. VanAntwerp JJ, Wittrup
KD. Fine affinity
discrimination by yeast surface display' and flow cy'tometry.Biotechnol Prog.
2000 Jan-Feb; i 6( 1 ): 31-7.
Adams GP, Schier R.Generatin~~ improved sin~~le-chairs Fv molecules for tumor
tar~~eting. J Immunol
Methods. 1999 Dec 10;231(1-2):249-60. Dau'~herty PS. Chen G, Olsen MJ, Iverson
BL, Georgiou G.
Antibody affinity maturation using bacterial surface display. Protein En~~..
1998 Sep;1 1 (9):825-32. VVons1
YW, Kussie PH, Parhami-Seren B, Margolies MN. Modulation ofantibody affinity
by an engineered y
amino acid substitution. J Immunol. 1991 Apr 1;154(7):3351-8. Balint RF.
Larrick JVv.'.Antibodv
ensineerin~~ by parsimonious rnuta'~enesis. Gene. 993 Dec 27;137(1):1()9-18.
Schillbach JF, Near Rl,
Bruccoleri RE, Haber E, Jeffrey PD, Novotny J.Sheriff S, Mar~~rolies MN.
Modulation of antibody affinity
by a non-contact residue. Protein Sci. 1993 Feb:2(2):206-l4.Chantes P, Baty D.
Engineering ofan anti-
steroid antibody: amino acid substitutions chan~.;eantibody fine specificity
from cortisol to estradiol.Clin
Chem Lab Med. 1998 Jun;36(6):355-9. Kussie PH, Parhami-Seren B, Wysocki L.J,
Margolies MICA single
en~~ineered amino acid substitution changes antibody tine specificity. J
Irnmunol. 1994 Jan I ;152( I ):146-
52, as well as references cited therein.
With respect to generation of high affinity antibodies and affinity
rttaturation of antibodies see also Hanes J.
Nat. Biotechnol. 2000 Dec; 18(12): 1287-92; references in Hudson PJ Exp. Opin.
Invest. Drugs (2000)
9(6) 1231-1242; Toran JL et al Evr. J. Immunol. 2001 Jan; 31(1') 128-137.
Nieison VB et al. Cancer
Res 2000 Nov l5: 60 (22) 6434-40 Adams Gp, Journal of Innnunological Methods (
1999) 249-260;
Chowdhury PS et al (June 1999) Nature Biotechnology Vol 17 p. 568With respect
to strategies and recent
technologies which have application to the invention see references in Hudson
PJ Exp. Opin. Invest. Drugs
(2000) 9(6) 1231-1242 and in particular references relating to strategies to
achieve multivalency and
multispeciticity; recruitment of viruses, ADF,P'F, photoactivation of
cytcrtoxic radionuclides; surface
receptor cross-linking: (see also Eur. J. Immunol 2000 30( 10) 3006), use of
anti-B antibodies:
imrnunocytokines (see also Lode HN lmmul. Res. 2000, ? I (2-3) 279-88; Gillies
SD Cancer Research 59
2159-2166 May 1999; (,ode HN et al Drugs of Today 2000 36(5) 3221-.i36).
With respect to practicial size limitations and pharmacokinetics of various
types of antibodies and
fragments see Colcher D. et al. G.J. Nucl. Med (1999) 43: 132-139: Wu AM et al
G.J. Nucl. Med 2000
Sep; 44(3): 268:83; Williams 1_E et al Med Phys 2000 May 37(5): 988-941 lkomi
F L_vrnphology 32
(1999)90-102.
With respect to the construction of diabodies see also Takemura SI et al.
Protein hng. 2000 Aug; l 3(8) 583-
8; Biomol. Eng. 2001 Sept;l8(2):31-40.
With respect to anti-cancer antibodies see also 6,180,357.
With respect to technolo~~ies to produce multivalent and'or multispecific
antibodies see also USI' 6, 172,
197; WO 92!01047; WO 93!1 1 161: WO 94!07921: WO 94.'13804: Helfrieh W. et al.
Journal of
Immunological Methods ?37 (2000) 131-14>. Proceeding's of 1 1'r' IBC
Conference on Antibody
Engineering; WO01%8579i
Monoclonal antibodies rnay be routinely produced as taught by Harlow, E. and
D. Lane, ( 1988)
ANTIBODIES: A Luhormor_v :l~antral, Cold Sprin~~ Harbor Laboratory, Cold
Spring Harbor N.Y.
Humanized antibodies ma_v be routinely produced as taught, for example, by
U.S. Pat. No. 5,585,089 and
U.S. Pat. No. 5,530,101. l echniques for engineering antibodies are well known
and described in Winter
and Millstein ( 1991 ) Nature 349:293, and Larrich and Fry ( 1991 ) llum.
Antibod. and Hybridorttas 2:17.
One having ordinary skill in the art may use well known techniques and
startin~~ materials and'or
commercially available expression vectors and systems that are readily
available and known in the art. See
e.g., Sambrook et al., Molecular Cloning a Lcrhururorw !Llanucrl, Second Ed.
Cold Sprin4~ Harbor Press
( 1989).
Examples of radionuclides useful as toxins in radiation therapy are well
known. Some examples are
referred to below. Auger emitters rnay be preferred for internalizing
antibodies. As sugy~ested above, the
teen antibody is used interchangeably with antibody fragment and antigen
bindings fra~~ment and includes a
whole antibody: antibody ti~agment a portion of an antibody such as a scFV
F(ab') ~ Flab) ,. Fab', Fab,
dAb, microbodies ( WO00 ?9004) or the fike or multivalent such 1-ra~~ments.,
including those itemized or
referenced herein. Regardless of structure, an antibody ti~agment can be made
to bind with the same antigen
that is recognized by the intact antibody. More particularly, in addition to
fragments formed by enzymaic
digestion of an intact Ah the term antibody or "antibody tragment" unless
otherwise stated also includes
any synthetic or genetically engineered protein that acts like an antibody by
binding to a specific antigen to
54
CA 02402930 2002-09-19
form a complex including%as applicable, cysteine noose peptides and minimal
recognition units consistin~~
ofthe amino acid residues that mimic the hypervariable region. Although fully
human antibodies, for
example, antibodies generated via human-human hybridomas or through pha~,t:
display usin~~ human
antibody based libraries, are preferred, the invention does not preclude other
strategies to avoid a HAMA
type response.
A chimeric antibody is a recombinant protein that contains the variable
domains and complementary
determinin~~ regions derived from, for example, a rodent antibody, while the
remainder of ttte antibody
molecule is derived from a human antibody.
With respect to stability engineering of scFv fragments for enhanced
mulfunctional ligands comprising
scFvs see.l:h-Col L3io12001 Feb 3; 305(5):989-1010.
Humanized antibodies are recombinant proteins in which murine LDR's of a
monoclonal antibody have
been transferred from heavy and light variable chains of the murinc
immunoglobulin into a human variable
domain.
The term therapeutic agent as used herein, is a molecule or atom which is
conjugated etc. to an antibody
moiety to produce combination including a conjugate which is useful for
therapy. Examples of therapeutic
agents include drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes,
and radioisotopes.
The term "a naked antibody" may be used to refer specitically to an entire
antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic agent. Naked
antibodies include both
polyclonal and monoclonal antibodies, as well as certain recombinant
antibodies, such as chimeric and
humanized antibodies.
The term immunoconjugate may be used to refer a conjugate of an antibody
component with a therapeutic
agent.
As used herein, the term antibody fusion protein refers to a recombinant
molecule that comprises an
antibody component and a second functional component for example a therapeutic
went. Examples of
therapeutic agents suitable for such fusion proteins include immunomodulators
("antibody-
immunomodulator fusion protein") and toxins ("antibody-toxin fusion protein").
Production of Antigen - Specitic Monoclonal Antibodies, Rodent monoclonal
antibodies to antigen can be
obtained by methods known to those skilled in the art. See ~~enerally, for
example, Kohler and Milstein,
Nature 256:495 ( 1975), and Coli~~an et al. (eds.), Current Protocols in
Immunology, Vol. 1, pages 2.5.1-
2.6.7 (John Wiley & Sons 1991 ) ["Coligan"]. Briefly. monoclonal antibodies
can be obtained by injectin~~
mice with a composition comprising the antigen in a question (Ag), verifying
the presence of antibody
production by removing a serum sample, removin~~ the spleen to obtain B-
lymphocytes, fusing the B-
lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas.
selecting positive clones
which produce anti-Ag antibodies, culturing the clones that produce antibodies
to the antigen, and isolating
the antibodies from the hybridoma cultures. Transgenic mice having for example
engineered Immune
systems to create human antibodies such those used by Medarex and Abgenix are
also contemplated for use
herein to create suitably targeted antibodies.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by
a variety of well-
established techniques. Such isolation techniques include affinity
chromatography with Protein-A
Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
See. for rxample, Coligan
at pages 2.7. I-2.7.12 and pages 2.9.1-?.9.3. Also, see Baines et al..
"Purification of Immunoglobulin G
(IgG) ," in Methods in Molecular Biology. Vol. 10, pages 79-104 ( Che Humana
Press, lnc. 1992).
With respect to relevant molecular biology techniques See also, for example,
Ausubel et al., (eds.).
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 ( 1990)
["Ausubel"]. Also,
see Wosnick et al., Gene 60:1 15 (1987); and Ausubel et al. (eds.), Short
Protocols in Molecular Biology,
3rd Edition. pages 8--8 to 8-t) (John Wilev & Sons, Inc. 1995). Established
techniques using the
polymerase chain reaction provide the ability to synthesize ~~t:nes as large
as I .8 kilobases in len<~th. Adam
et al., Plant Molec. Biol. 21:1 131 (1993) Bambot et al., PC.'R Methods and
Applications 2:26(i ( 199;);
Dillon et al., "l)se of the Polymerase Chain Reaction for the Rapid
Construction of Synthetic Genes," in
CA 02402930 2002-09-19
Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and
Applications, White led.),
pages 263-268, (Humana Press. lnc. 1993).
Techniques for constructing chimeric antibodies are well-known to those of
skill in the art. As an example,
Leung et al., Hybridoma 13:469 ( 1994).
In yet another embodiment, an antibody of the present invention is a
"humanized" monoclonal antibody.
That is. mouse complementarity determining regions are transferred from heavy
and light variable chains of
the mouse immunoglobulin into a human variable domain, followed by the
replacement of some human
residues in the framework regions of their murine counterparts. Humanizc;d
monoclonal antibodies in
accordance with this invention are suitable for use in therapeutic methods,
General techniques for cloning
murine immunoglobulin variable domains are described, for example, by the
publication of Oriandi et al.,
Proc. Nat'l Acad. Sci. USA 86: 383 3 ( 1989). Techniques for producin;~
humanized monoclonal antibodies
are described, for example, by Jones et al., Nature 321:522 (1986), Riechmann
et al., Nature 332:323
(1988), Verhoeyen et al., Science 239:1 X34 (1988), Carter et al., Proc. Nat'1
Acad. Sci. USA 89:4285
(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J.
Immun. 10:28=14 (1993). T'he
publication of Leung et al., Mol. Immunol. 32:1413 ( 1990, describes the
construction of humanized LL2
antibody.
In a preferred embodiment of the invention the multifunctional li~~and has a
unique portion which
differentiates it from other antibodies and preferably other co-administered
different multifunctional
ligands, which unique portion, allows the multifunctional ligand to be
efficiently segregated on an
immunoaffinity column. In the case of differentiating a sin~,le
multifunctional ligand an anti-idiotype
(assuming the first portion consists of an antibody) or other antibody
uniquely recognizing the first portion
could be employed. Modifying a portion of the tirst portion. for example in
the case where it is antibody
component and creatin~~ a antibody thereto, for example by pha~~e display, is
a matter of routine skill in the
arts of antibody engineering and phage display.
In another embodiment, an antibody of the present invention is a human
monoclonal antibody. Such
antibodies are obtained from transgenic mice that have been "en~tineered" to
produce specific human
antibodies in response to antigenic challenge. In this technique, elements of
the human heavy and light
chain locus are introduced into strains of mice derived from embryonic stem
cell lines that contain targeted
disruptions of the endogenous heavy chain and light chain loci. The
trans<~enic mice can synthesize human
antibodies specific for human antigens, and the mice can be used to produce
human antibody-secreting
hybridomas. Methods for obtaining human antihodies from transgenic mice are
described by Green et al,.
Nature Genet. 7:13 ( 1994), Lonberg et al., Nature 368:86 ( 1994), and Taylor
et al., Int. lmmun. 6:579
1994).
Examples of Production of Antibody Fragments
Antibody fi-a~~ments can be prepared, for example, by proteolytic hydrolysis
of an antibody or by
expression in E. coli of the DNA coding for the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole
antibodies by conventional
methods. For example, antibody fragments can he produced by enzymatic cleavage
of antibodies with
pepsin to provide a 5 S fra~.:ment denoted F(ab')~. This fra~~ment can be
further cleaved using a thiol
reducing agent, and optionally a blocking ~~roup for the sulfhydryl <groups
resulting from cleava~~e of
disulfide linkages, to produce 3.5 S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment directhy.
These methods are described,
for example, by Goldenberg, tJ.S. Pat. Nos. 4,036,94 and 4,331,647 and
references contained therein.
Also, see Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter,
Biochem. J. 73:1 19 ( 1969),
Edelman et al., in Methods in Enzymology Vol I, page 422 (Academic Press
1967), and Coli';an at pages
2.8. I -2.8.10 and 2.10.-2.10.4.
Other methods of cleaving antibodies, such as separation of heavy chains to
form monovalent light-heavy
chain fragments, further cleava~~e of fragments, or other enzymatic, chemical
or genetic techniques may
also be used, so long as the fragments bind to the antigen that is recognized
by the intact antibody.
For example. Fv fragments comprise an association of V,~ and V,. chains. 'this
association can be
noncovalent, as described in mbar et al., Proc. Nat'I Acad. Sci. USA 69:2659
(1972). Alternatively, the
56
CA 02402930 2002-09-19
variable chains can be linked by an intermolecular disulfide bond or cross-
linked by chemicals such as
glutaraldehyde. See, for example, Sandhu, supra.
Preferably, the Fv fragments comprise V,r and V, chains which are connf:cted
by a peptide linker. 'these
single-chain antigen binding proteins (scFv) are prepared by constructing. a
structural gene comprising
DNA sequences encodin4.: the V" and V,_ domains which are connected by an
oli~~onucleotide. 'fhe
structural gene is inserted into an expression vector which is subsequently
introduced into a host cell, such
as E. coli. The recombinant host cells synthesize a single polypeptide chain
with a linker peptide bridging
the two V domains. Methuds for producing scFvs are described, for example, by
Whitlow et al., Methods:
A Companion to Methods in Enzymology 2:97 (1991). Also see Bird et al.,
Science 242:423 ('1988),
Ladner et al., U.S. Pat. No. 4,946,778, Pack et al., Bio~'Technology 1 I :1271
( 1993), and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a single
cornplementarity-determining region
(CDR). CDR peptides ("minimal reco~~nition units") can be obtained by
constructing genes encodin~~ the
CDR of an antibody of interest. Such ;genes are prepared, for example, by
using the polymerase chain
reaction to synthesize the variable region trom RNA of antibody-producing
cells. See. for example, Larrick
et al., Methods: A Companion to Methods in Enzymology 2:106 ( 1991 );
Courtenay-Luck, "Genetic
Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical
Application, Bitter et al. leds.), pages 166-179 ('Cambridge University Prf~ss
1995): acrd Ward et al.,
"Genetic Manipulation and Expression of Antibodies." in Monoclonal Antibodies:
Principles and
Applications, Birch et al.. (eds.), pages 137-185 (Wiley-Liss, Inc. (995).
**Preparation of lmmunoconjugates
The present invention contemplates immunoconjugates to assess and effect
treatment of various disease
conditions. Such immunoconjugates can be prepared by indirectly conjugating a
therapeutic agent to an
antibody component. For example, ~~eneral techniques are described in Shih et
al.. Int. J. Cancer 41:832-
839 (1988); Shih et al., Int. J. Cancer 46:1 101-1 106 (1990); and Shih et
al., U.S. Pat. No. 5,057,313. 'the
general method involves reacting an antibody component having an oxidized
carbohydrate portion with a
carrier polymer that has at least one free amine function and that is loaded
with a plurality of drug, toxin,
chelator, boron addends, or other therapeutic ag.=ent. This reaction results
in an initial Schiff base (imine)
linkage, which can be stabilized by reduction to a secondary amine to form the
final conjugate.
The carrier polymer is preferably an aminodextran or polypeptide of at least
50 amino acid residues.
although other substantially equivalent polymer carriers can also be used.
Preferably, the final
immunoconjugate is soluble in an aqueous solution, such as mammalian serum,
for ease of administration
and effective targeting for use in therapy. 'Thus, solubilizing functions on
the carrier polymer will enhance
the serum solubility of the: tinal immunoconjugate. In particular, an
aminodextran w ill be preferred.
The process for preparing an immunoconjugate with an aminodextran carrier
typically begins with a
dextran polymer, advantageously a dextran of averay.:e molecular weight of
about 10,000-100,000. The
dextran is reacted with an oxidizing agent to effect a controlled oxidation of
a portion of its carbohydrate
rings to generate aldehyde groups. The oxidation is conveniently effected with
~~lycolytic chemical reagents
such as NalO<sub>4</sub>, accordin~~ to conventional procedures.
The oxidized dextran is then reacted with a polyamine, preferably a diamine,
and more preferably, a monu-
or polyhydroxy diamine. Suitable amines include ethylene diamine, propylene
diamine, or other like
polymethylene diamines, diethylene triamine or like polyamines. 1.3-diamino-2-
hydroxypropane, or other
like hydroxylated diamines or polyamines, and the like. An excess of the amine
relative to the aldehyde
~~roups of the dextran is used to insure substantially complete; conversion of
the aldehyde functions to
Schiff base groups.
A reducing agent, such as NaBH,, NaBHCN or the like, is used to effect
reductive stabilization of the
resultant Schiff base intermediate. The resultant adduct can be purified by
passage throu~~h a conventional
sizing column to remove cross-linked dextrans.
Other conventional methods ofderivatizing a dextran to introduce amine
functions can also be used, e.g.,
reaction with cyanogen bromide, followed by reaction with a diamine.
The aminodextran is then reacted with a derivative of the particular drug,
toxin. chelator
57
CA 02402930 2002-09-19
immunomodulator, boron addend, or other therapeutic agent to be loaded, in an
activated form, preferably,
a carboxyl-activated derivative, prepared by conventional means, e.g., using
dicyclohexylcarbodiimide
(DCC') or a water soluble variant thereof, to form an intermediate adduct.
Alternatively, polypeptide toxins such as pokeweed antiviral protein or ricin
A-chain, and the like, can be
coupled to aminodextran by glutaraldehyde condensation or by reaction of
activated carboxyl groups on the
protein with amines on the aminodextran.
Chelators for radiometals or magnetic resonance enhancers are well-known in
the art, Typical are
derivatives of ethylenediaminetetraacetic acid (EDTA) and
diethylenetriaminepentaacetic acid (DTPA).
These chelators typically have groups on the side chain by which the chelator
can be attached to a carrier.
Such groups include, e.v~.. benzylisothiocyanate, by which the D'TPA or FDTA
can be coupled to the amine
group of a carrier. Alternatively, carboxyl groups or amine groups on a
clnelator can be coupled to a carrier
by activation or prior derivatization and then couplings, all by well-known
means.
Boron addends, such as carboranes, can be attached to antibody components by
conventional methods. For
example, carboranes can be prepared with carboxyl funcaions on pendant side
chains, as is well known in
the art. Attachment of such carboranes to a carrier, e.~~., aminodextran, can
be achieved by activation of the
carboxyl groups of the carboranes and condensation with amines on the carrier
to produce an intermediate
conjugate. Such intermediate conjugates are then attached to antibody
components to produce
therapeutically useful immunoconjugates, as described below.
A polypeptide carrier can be used instead of aminodextran, but the polypeptide
carrier must have at least 50
amino acid residues in the chain, preferably 100-5000 amino acid residues. At
least some. of the amino
acids should be lysine residues or glutamate or aspartate residues. The
pendant amines of lysine residues
and pendant carboxylates of glutamine and aspartate are convenient for
attaching a dru~~, toxin,
immunomodulator, chelator, boron addend or other therapeutic absent. Examples
of suitable polypeptide
carriers include polylysine_ polyglutamic acid. polyaspartic acid, copolymers
thereof, and mixed polymers
of these amino acids and others, e.g., serines, to confer desirable solubility
properties on the resultant
loaded carrier and immunocon,jugate.
Conjugation of the intermediate conjugate with the antibody component is
effected by oxidizing the
carbohydrate portion of the antibody component and reacting the resulting
aldehyde (and ketonel carbonyls
with amine groups remaining on the carrier after loadin;~ with a drug, toxin,
chelator, innnunomodulator.
boron addend, or other therapeutic agent. Alternatively. an intermediate
conjugate can be attached to an
oxidized antibody component via amine groups that have been introduced in the
intermediate conjugate
after loadin~~ With the therapeutic agent. Oxidation is conveniently effected
either chemically, e.~~.. w ~ith
NalOr or other glycolytic reagent, or enzymatically, t.~~., with
neuralninidase and ~~alactose oxidase. In the
case of an aminodextran carrier, not all of the amines of the aminodextran
;are typically used for loading a
therapeutic agent. 'The remaining: amines of aminodextran condense with the
oxidized antibody component
to form Schiffbase adducts, which are then reductively stabilized, normally
with a borohydride reducing
agent.
Analogous procedures are used to produce other immunoconjugates according to
the invention. Loaded
polypeptide carriers preferably have free lysine residues remaining for
condensation with the oxidized
carbohydrate portion of an antibody component. Carboxyls on the polypeptide
carrier can, if necessary, be
converted to amines by, e.g., activation with DCC and reaction with an excess
of a diamine.
The final immunoconjugatc is puritied using conventional techniques. such as
sizing chrornato~graphy on
Sephacryl S-300.
Alternatively. immunoconju~,~ates can be prepared by directly conjugating an
antibody' component with a
therapeutic agent. The L~eneral procedure is analo<Vous to the indirect method
of conjugation except that a
therapeutic agent is directly attached to an oxidized antibody component.
For application to linking MHC 1'1l peptide~~B7 molecules to a latex which has
previously conjugated to
biotin, for avidin assisted linking to a multifunctional li~and, it will be
appreciated that biotin can be
conju~~ated to a part of a latex sphere which is then linked to MHC peptide
and B7 molecules by placing the
spheres in a confluent layer or in the spheres in a microwells such that only
part of the sphere is exposed for
conjugation and then coatings the spheres onto avidin coated plates for the B7
and MI-1C linkage.
58
CA 02402930 2002-09-19
It will be appreciated that other therapeutic absents can he substituted for
the chelators described herein.
Those ofskill in the art will be able to devise conjugation schemes without
undue experimentation.
As a further illustration, a therapeutic agent can be attached at the hinge
region of a reduced antibody
component via disulfide bond formation. For example, the tetanus toxoid
peptides can be constructed with
a single cysteine residue that is used to attach the peptide to an antibody
component. As an alternative, such
peptides can be attached co the antibody component using.: a
heterobifunctional cross-linker, such as N-
succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al~. Inf. J. Cancer
56:244 ( 1994). General techniques
for such conju<~ation are well-known in the art. See, for example, Wong, (.
HEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991 ); Upeslacis et al.,
"Modification of
Antibodies by Chemical Methods," in MONOC.'t.ONAL ANTIBODIES: PRINCIPLES AND
APPLICATIONS. Birch ct al. (eds.). pages 187-230 (Wiley-Liss, Inc. 1995);
Price, "Production and
Characterization of Synthetic Peptide-Derived .Antibodies," in MONOCLONAL
ANTIBODIES:
PRODUCTION, ENGINEERING AND CLINICAL. APPLICATION, Ritter et al. (eds.), pages
60-84
(Cambridge LJniversity Press 1995).
As described above, carbohydrate moieties in the Fc reunion of an antibody can
be used to conju~~ate a
therapeutic agent. However. the Fc region is absent if an antibody fragment is
used as the antibody
component of the immunoconjugate. Nevertheless, it is possible to introduce a
carbohydrate moiety into the
light chain variable re~_ion of an antibody or antibody fra=ment. See, for
example, Leung et al., J. Immunol.
154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,95 3 ( 1995). The
engineered carbohydrate moiety is
then used to attach a therapeutic agent.
In addition, those of skill in the art will recognize numerous possible
variations of the conjugation methods.
For example, the carbohydrate moiety can be used to attach polyethyleneglycol
in order to extend the half-
life of an intact antibody, or anti~~en-binding fragment thereof, in blood.
lymph, or other extracellular
fluids. Moreover, it is possible to construct a "divalent immunoconjugate" by
attaching therapeutic agents
to a carbohydrate moiety and to a free sulthydryl ~~roup. Such a free
sulfhydryl ~~roup may be located in the
hin~~e rev~ion of the antibody component.
Methods for determining the binding specificity of an antibody are well-known
to those of skill in the art.
General methods are provided, for example, by Mole. "Epitope Mapping." in
METHODS IN
MOLECULAR BIOLOGI'. VOLIIME 1(): 1MMUNOCLIEMICAL PROTOCOLS, Manson led.),
pages
105-1 16 (The Humana Press, lnc. 1992). More specifically, competitive
blocking assays for example to
determine CD22 epitope specificity are described by Stein et al., Cancer
Immunol, lmmunother. 37:293
( 1993), and by Tedder et al., U.S, Pat. No. 5,484,892 ( 1996).
In another aspect the invention is directed to a bispecitic ligand, preferably
a bispecifc antibody,
comprising at least a first ligand, preferably an antibody component, which
binds specifically to a first cell
surface associated ii~~and and at least a second ligand, preferably a second
antibody component which binds
specifically to a second cell surface associated ii~~and on the same cell, and
wherein the functional at4inity
of at least one and preferably both of said antibody components is selected so
as to substantially limit
functional bindin<~ unless both of said first and second antibody components
are substantially
contemporaneously bound to said cell. It is known to provide bifunctional
ligands wherein functional
binding, for example. to accomplish si~~nal transduction, is predicated on
both ligands being bound or
cross-linking. However this effect is not contemplated to be predicated on
differentially controlling the
functional affinity of the respective li~~ands. Accordin~~ to a broad aspect
of this invention (in which
inclusion of a ligand which binds to a lymphatic vessel associated marker is
optional). the invention
excludes known such bispecitic ligands which inherently have a suitable
differential functional affinity.
Such bispecitic ligand are mentioned herein. By controlling the at4inity of at
least one of said li<~ands, for
example where the functional affinity of one said ligands is substantially
less than that of the other li~~and
the invention contemplates that a substantially greater percenta;.;e of the
administered dose of the bispecitic
ligand will affect cells in which only both ligands are present, and;'or that
a reduced percentage ofthe dose
administered will functionally bind to the cells in virtue only of the reduced
functional affinity ligand. The
invention also contemplates that functional affinity of one ligand is greatly
increased to establish the
functional affinity differential and that the functional affinity of both
ligands is reduced relative to that of a
standard, for example relative to that of a comparable li~~ands in hand or
known in the art or identified by
phage display, ribsome display or other comparable techniques using a single
such ligand. 'The invention
also contemplates that a microarray (or library) of bispecitic li~~ands~in
which for esamp(v, the bispecitic
59
CA 02402930 2002-09-19
ligand is "tethered" (ie. immobilized) directly or indirectly in virtue of one
or more amino acid residues
which are positioned within the molecule to preferably minimally interfere
with any binding, and in which
the signal (e~~ its intensity) associated with a single li~~and bindiny~
interaction can be differentiated from a
two or more li'~and interactions, for example cell surface binding
(alternatively the ligands or cell may be
immobilized) and that ribsome and phage display could be adapted to bispecitic
single domain antibodies
constitutin~~ a single chain (see references herein) by elon~,ating the end of
the chain from which the
molecule is tethered. The invention contemplates that the affinity of one such
li~.;and may be fixed and that
the variability in members of the library lies in the permutations of certain
key residues to which binding is
attributable which can readily be identified by persons skilled in the art.
The invention also contemplates
assessing sin'=le ligand binding capability of successful bi-ligand binders
for example by blocking the other
(non-assessed at that time) ligand (cg. with correlative ligand or a mirnotope
thereon and for example
determining limited or non-existent such binding to as well as using inclined
ligand testing surfaces fbr
washing over the correlative ligand, for example of defined surface area,
including preferably defined
lengths and widths and concentrations % distributions :' amounts of the bound
li'~and, where the de~~ree of
incline is selected to rou~~hly sunulate the micro-environment of the
comparable in viva target, he it a
stationary cell with a rou4;hly defined avera~~e shear force of bathing fluids
a<.;. within a tumor or in the
lymphatic system, or a nubile cell within a vein, artery, or lymphatic
vessel,~including those of different
sizes. The invention is also directed to a mthod of generating a target ligand
or improving:, the tar;~et
specificity of any ligand by using a population of variants of that ligand
within a micro-environment
simulated microarrav system in w which the at least one of the follw~in~,
factors is sirnulat~d: concentration or
amount or distribution of correlative ligand, shear force and shape using
ie~n~~th and width parameters to
simulates intraluminal diameter and Iength.The invention also contemplates in
the case of a multifunctional
li~.:and or ire the case of a hispecitic or rnultispecfic li~,?and (as herein
described) that the affinity of its
component binding ligands may be selected for venous or arterial tagetin~~ or
to accommodate lymphatic
system targeting or targeting within or through tissues or combinations of the
aforementioned cg. median,
average or or wei<~hted compromises to improve desired tarwetin~~. In a
preferred embodiment the first
ligand is selected on the basis of its ability to at least partially
discriminate between a target population of
cells (cg. a li~and that is "associated" with a target population of cells)
and a non-target population of cells
(in one embodiment it is selected so as to have no other effect other than
binding for tar~~eting purposes)
and the second ligand is selected for its ability to modulate the activity
ofthe targeted cell, optionally in
virtue of binding alone cg. without delivering a payload )the term modulate
referring broadly to any desired
effect on the cell or its functionality) In this case the functional affinity
for the ligand which is tarv~eted for
modulating the activity of the cell is selected so as to reduce the likelihood
of binding unless bindings has
first or contemporaneously occurred to the first ligand targeted for
selectivity (cg, the second ligand~worrld
have rnonovalent as opposed to divalent binding to the ligand required for
selectivity andior from 0.20 to
10-~' fold reduction in affinity (for example as measured by Biacore) relative
to the bindings affinity for the
first ligand. This reduction in affinity is preferably ~~reater than a
100°,~ reduction in affinity (multiply by
O.l ), preferably greater than a 200°% reduction in affinity,
preferably ~~reater than a 300°o reduction in
affinity, preferably greater than a 400% reduction in affinity, preferably
~~reater than a S00°~o reduction in
affinity, preferably greater than a 600% reduction in affinity, preferably
greater than a 700°-~i, reduction in
affinity, preferably ~~reater than a 800°ro reduction in affinity,
preferably grceater than a 900°o reduction in
affinity, preferably greater than a 1000°io reduction in affinity,
preferably ~~reater than a ?000°~ reduction in
affinity, preferably greater than a 3000°'o reduction in affinity.
preferably greater than a 4000° o reduction in
affinity, preferably greater than a 5000°io reduction in affinity.
preferably <greater than a (i000°,!° reduction in
affinity, preferably ~~reater than a 7000% reduction in affinity, preferably
greater than a 8000% reduction in
affinity, preferably greater than a 9000% reduction in affinity, preferably
~~reater than a 10000°'o reduction
in affinity, preferably- greater than a ?0000°o reduction in aftmity.
preferably ~~reater than a 30000°io
reduction in affinity. preferably greater than a ~t0000°ro reduction in
affinity, preferably greater than a
50000% reduction in affinity, preferably ~_reater than a 60000°r
reduction in affinity, preferably greater
than a 70000% reduction in affinity, preferably greater than a 80000°%
reduction in affinity, preferably
greater than a 90000°,% reduction in affinity, preferably greater than
a 100000°ir reduction in affinity,
preferably ~~reater than a 500000°%b reduction in atlinity, preferably
greater than a 10000()0°~o reduction in
affiinity, preferably greater than a 10000000~~o reduction in affinity,
preferably greater than a ?0000000%
reduction in affinity, preferably a ;greater than 3000000° o reduction
in affinity, preferably a greater than
40,000.000% reduction in affinity, preferably a ~_reater than
50000000°,~u reduction in affinity. preferably a
greater than 60000000% reduction in aff7nity, preferably ar greater than
70000000% reduction in affinity_
preferably a ~~reater than 80000000°-~ reduction in affinity,
preferably a greater than 90000000°ro reduction
in affinity preferably a greater than 100,000,000°r reduction in
affinity, preferably a reduction in affinity of
between one and two orders of magnitude, preferably a reduction in affinity of
between two and three
orders of magnitude, preferably a reduction in affinity of between three and
tour orders of magnitude,
CA 02402930 2002-09-19
preferably a reduction in affinity of between four and five orders of
magnitude, preferably a reduction in
affinity of between five and six orders of magnitude, preferably a reduction
in affinity of between six and
seven orders of magnitude preferably a reduction in affinity of between seven
and eight orders of
magnitude, preferably a reduction in affinity of between eight and nine orders
of magnitude, preferably a
reduction in affinity of between nine and ten orders of magnitude.
It will be appreciated that a suitable reduction in affinity, if any, will
depend on the valency of the
respective first and second ligands and the selected affinity of the tirst
ligand, which for example may have
been augmented. The invention also contemplates a trispecific (and triavalent)
ligand in which two ligands
differently define its speciticity to reduce the likelihood of an undesired
effect attributable to the function
exerting moiety binding alone. In terms of the physical constitution of a
ligand having a trispecitie binding
capability, the invention also contemplates linking three monovalent dabs,
MRUs or the like or mixed
combinations thereof or two bivalent dabs, MRUs or the like or mixed
combinations thereof (see Vv'O
99'42077, (JS 6174691, W00029004. Camel single-domain antibodies as modular
building units in J Biol
Chem. 2000 Oct 25, & Mulligan-Kehoe U.S. patents including US 5702892, US
5824120; se also US
6040136 ) (in the latter case optionally one or both may be bispecific and
linked by well known methods in
the art (see WO 99/42077, Celltech's TFM, leucine zippers, US 5,910,573.
US5892020, f~,P 0654085B, see
also EP 0318554B). The teen functional binding is used to refer to binding
which yields the desired effect,
for example a therapeutic et"tect on a target cell population attributable to
the binding to one or both
ligands. Using the previous example, one ligand, eg. the first ligand. may be
used to target activated
immune cells, and the second liaand may be different and may upon being bound
to, for example result in
inactivation, anergy, apoptosis or reduced capacity for endothelial adhesion
of the immune cell. In this
case, the invention contemplates that the functional affinity of the antibody
component which binds to the
second ligand is selected such that binding is unlikely to occur without
binding to the specificity dictating
ligand, for example the ratio of targeted relative non-targeted cells affected
by the dose administered is
approximately 1.10 to I, preferably approximately 1.1 > to I, more preferably
approximately 1.20 to I,
more preferably approximately 1.25 to I, more preferably approximately 1.30 to
1. more preferably
approximately 1.35 to I, more preferably approximately 1.40 to 1, more
preferably approximately 1.45 to
I, more preferably approximately 1.50 to l, more preferably approximately 1.55
to l, more preterably
approximately 1.60 to 1, more preferably approximately 1.60 to l, more
preferably approximately 1.65 to
l, more preferably approximately 1.70 to l, more preferably approximately 1.75
to 1, mare preferably
approximately 1.80 to I, more preferably approximately 1.85 to l, more
preferably approximately 1.90 to
I, more preferably approximately 1.95 to 1, more preferably approximately 2 to
I, more preferably greater
than 2 to I, more preferably approximately greater than 3 to 1, more
preferably approximately greater than
4 to l, more preferably greater than 5 to 1, more preferably greater than 6 to
1, more preferably greater than
7 to 1, more preferably greater than 8 to 1 . more preferably greater than 9
to 1 , more preferahl~~ greater
than 10 to I , more preferably greater than 20 to I , more preferably greater
than 30 to I , more preferably
greater than 40 to I , more preferably ~~reater than 50 to 1 , more preferably
;greater than 60 to I , more
preferably greater than 70 to I, more preferably ~~reater than 80 to l, mare
preferably greater than 90 to I,
more preferably <greater than 100 to l, more preferably greater than 500 to 1
. more preferably ~~reater than
1000 to 1, more preferably ~~reater than 10,000 to I, more preferably greater
than 100,000 to l, more
preferably ~~reater than 500,000 to 1 more preferably greater than 1,000.000
to 1.
It will be appreciated by persons skilled in the art that the foregoin~~
aspects of the invention apply to a
variety of different combinations of immune function or other therapeutic
function exerting ligands and
specificity dictating ligands includin~~ those involved in immune signaling;,
stimulatory, eo-stimulatory.
inhibitory, adhesion or other interactions, including without limitation,
cytokine receptors, ligands
associated with immune cell adhesion. ligands to which bindings results in
stimulation, activation, apoptosis,
anergy or costimulation, or li<~ands which differentiate between different
populations or subpopr.rlations or
immune cells (see eg. US 6135941, WO 00/63251. WO 00/61 132, US 61:?0767).
including sub-populations
of B cells and T cells (see for example US 6197524) activated versus non-
activated lympocytes, diseased or
disease-causing cells versus non-diseased 1 disease causing lymphocytes (see
for exampleW'O
Ol/13945A1, US 6132980, ) and specific immune cell clones for example those
having specific Ig type and
MHC-peptide type ligands and correlative ligands. Examples of such iigands
include CC R5, CTLA-4,
LFA-1. LFA-.>. ICAMs e~~. /CAM-1. CD2, CD3, CD4 (e~~ see US 6,136,310), CD18,
CD22. CD40, CD44;
CD80, CD86, CD134 and CD154, to name only a few (see also US60874'75: PF4A
receptor) (see also
Glennie MJ et al. Clinical Trial of Antibody Therapy. Inumunology Today Aug
2000. Vol. 2 ! (no. 8)
p.406).
The invention also contemplates that the therapeutic function or immune
flmction effecting ligand is also a
61
CA 02402930 2002-09-19
specificity imparting ligand, which in the case of for example, an antigen
presenting cell may be an
antibody which recognizes and binds to a specific MHC peptide complex, as is
established in the art (see
pertinent Chames et al. references herein, see also WO 97!02342 , Direct
selection of <r human antibody
fragment directed against the tumor T-cell epitope HLA-A 1-MACE-A I from a
nonimmunized phage-Fab
library. Proc Natl Acad Sci U S A. 2000 Jul 5: 97( 14):7969-74). In this case
it will be appreciated that the
APC targeting ligand assist the particular MHC peptide bindings antibody to
bind to its target.
See also WO 97107819 which is hereby disclaimed with respect to all relevant
aspects of the invention
herein insofar as inherently disclosed therein. See also US 5,770,403 with
respect Co antibodies which bind
to cytokines.
In one embodiment, the respective antibody components of the multispecific
ligand recognize a
substantially different subset of non-targetted tissues so that functional
binding to a non-targetted tissue is
substantially precluded. It will be appreciated that this strategy can be
accomplished with two different
antibodies have differing and preferably non-overlapping normal 1e. non-
targeted tissue distributions. In a
preferred embodiment the target cell is a cancer cell and the respective first
and second cell surface
associated ligands are expressed on different subsets of normal cells, which
are non-overlapping subsets, so
as to minimize deleterious normal cell targeting and distibute the undesired
effects or normal call targeting
(eg. with a toxin), to different cell populations. For example in the case of
tumor cell targeting one or both
ligands may be expressed exclusively on a single tumor type (e~~. a human
sarcoma or carcinor~~a, e.g.,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer,
ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma. sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma, hepatoma, bile
duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Vv~ilms'
tumor, cervical cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma, bladder
carcinoma. epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma,
acoustic neuroma, oligodendrov~lioma, meningioma, melanoma. neuroblastoma,
retinoblastoma;
leukemias, e.'~., acute lymphocytic leukemia and acute myelocytic leukemia
(mycloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic
myelocytic (granulocytic)
leukemia and chronic lymphocytic leukemia): and polycythemia vera, lymphoma
(Hodgkin's disease and
non-Hodgkin's disease). multiple myeloma, Waldenstrom's macroglobulinemia, and
heavy chain disease)
or a particular category of tumor types (eg. adenocarcinomas, tumors of
neuroectodennal origin, or on
multiple different tumor types or categories of tumor. One or both components
(they may be the same or
different) may be a dAb, a scFv. an Fab, a minibody moiety or a substantially
intact antibody. for example
both may be scFvs and the resulting product may be a diabody, triabody, or
tetrabody. For example in a
preferred embodiment the bispecific antibody comprises two dAb components
comprisin~~ linked via a
linker (see above) and having at least at least part of a constant region for
fusion for example to a toxin (eg.
at least a partial hinge region, and preferably also at least a partial CH2
domain (optionally also at least a
partial CH3 domain). In another embodiment, a trispecific antibody or
tevtraspecific antibody with at least
two different and preferably 3 or 4 subsets (preferably at least one or more
of such subsets beings non-
overlapping subsets) of non-targeted cell reactivities may be employed in the
form of a trispecitic or
tetraspecific antibody respectively whereby up to three or four different
pairs of ligands are targeted, so as
further minimize normal cell targeting and also preferably tartlet a
heterogenous population of cells within
the same tumor. Ligands with distributions on normal tissues are well known,
some being referenced
herein, for example CEA. CD-20, P53, epidermal growth factor, including known
multicarcinomic and
pancarcinomic ligands (eg. see USS, 171.665, US 4349528.
The teen functional binding is used to mean binding for the purpose of
accomplishing the object of the
binding, for example binding for a sufficient duration to inhibit or enhance a
particular effect, such as cell
killing, for example in the case where one both antibody components arc'
selected for their ability to
internalize, binding for :~ sufficient duration to permit internalization, for
example to deliver a toxic
payload. As discussed above. the term substantially in reference to
therapeutic advantage is used to refer to
a degree which provides a si'~niticant benefit from a therapeutic standpoint.
Examples of tumor associated antigens (eg. WO O 1:21828) and targets a.nd
related antibodies <tre
referenced throughout the disclosure and the foregoing aspect of the invention
is for greater certainty
directed to bispecific antibodies (including trispecific and tetraspecific
antibodies, optionally including a
62
CA 02402930 2002-09-19
component which also binds to a lymphatic vessel associated ligand), which
target each of the
combinations and permuations of the target cell (diseased, disease causing or
immune) associated antigens,
ligands, epitopes or receptors well known to those skilled in the art, herein
directly or indirectly referenced
or referenced in the materials herein incorporated by reference (ie.
permutations and combinations of pairs
or where a tri-or tetra- specific antibody is used possibly permutations of (3
or ~) groups of pairs including
for example pairs in which one member is used for targetin_~ and the second is
used for modulation puposes
such modulation ineludiny= without limitation, simple binding eg. to deliver a
payload, apoptosis inducin'1
(eg. anti-fas), modified vascular adhesion properties (eg. anti-CU44'),
modified eytokine binding (anti-
CCRS) etc.(re: relevant ligands,!markers see also USP 6,010,902 and the
references cited therein. Samter's
Immunologic Diseases, f=fifth and Sixth Edition, Lippincott, Frank Austen, MD
Michael M. Frank, MD
John P. Atkinson, MD Harvey I. Cantor, MD (6'~'-ISBN: 0-7817-2120-.'.):
Fundamental Virology, 'fitird
and Fourth Edition, Lippincott Uavid M. Knipe. PhD Peter M. Howley, MU Uiane t-
;. Griftin, MD, PhD
Robert A. Lamb, PhD, ScD Malcolm A. Martin, MD Bernard Roizman, ScD Stephen E.
Straus. MD (4'h-
ISBN: 0-7817-I 833-3 ): Arthritis and Allied Conditions - A Textbook of
Rheumatology, 'thirteenth and
Fourteenth Editions, William J. Kooprnan, MD 14'~':ISBN: 0-7817-2240-3.
November2000; Cancer-
Principles and Practice of Oncology, Fifth and Sixth Editions, Lippincott,
Vincent T. DeVita. .1r.. MU
Samuel Hellman, MD Steven A. Rosenber~~, MD, PhD ISBN: 0-7817-2229-2; Dubois'
Lupus
Erythematosus. Fifth Edition. Daniel J. Wallace, MD ISBN: 0-683-086Ei~-0,
December 1996: Cytokine
Therapeutics in Infectious Diseases, Steven M. Ftolland, MD, PhD, Lippincott,
ISBN: 0-7817-1625-X, US
6054561 ), in each of their permuatations of size;'valency (ie. dabs, scFv,
diabodies etc herein referenced) as
applied to each of the applicable disease conditions herein referenced or
otherw rise known to those skilled
in the art.
With respect to recombinant techniques for producing Fv fragments see also WO
88!01649, ~'O 88.06630,
WO 88'07085, WO 88;07086, and WO 88!09344.
With respect to preparing ligands for specific MHC peptide complexes see also
WO 01 /22083; Direct
selection of a human antibody ti~agment directed against the tumor T-cell
epitope HL..A-A I-MAGE-A 1
from a nonimmunized phage-Fab library. Proc Natl Acad Sci U S A. 2000 Jul
5:97( 14):7969-74.
With respect to bispecific antigen binding constructs that are suitable for
for binding to more than one
antigen on the same cell see also Schmiedl A et al. Protein Eng 2000 Oct
13(10):725-34.
Preferred immunoconjugates include radiolabeled antibody components and
conjugates of an anti-Lyve-1
antibody component and an antbody component which comprises an
immunomodulator.
A radiolabeled immunoconjugate may comprise an .alpha.-emitting radioisotope,
a .B-emitting
radioisotope, a gamma emitting radioisotope, an Auger electron emitter, a
neutron capturing agent that
emits alpha-particles or a radioisotope that decays by electron capture.
Suitable radioisotopes include ~''~
Au, '. p,. ~-5 l, ~;~ 1, ''n' Y. n~ Re, ~~~ Re,"Cu, -n At. and the like.
As discussed above, a radioisotope can be attached to an antibody component
directly or indirectly, via a
chelating agent. for example, ~'' Cu. considered one ofthe more promising
radioisotopes for
radioimmunotherapy due to its 61.5 hour half=life and abundant supply of beta
particles and gamma rays.
can be conjugated to an antibody component usin~~ the chelating a;~ent, p-
bromoacetamido-benzyl-
tetraethylaminetetraacetic acid (TETA) . Chase, "Medical Applications of
Radioisotopes," in Remington's
Pharmaceutical Sciences, 18th Edition. Gennaro et al. (eds.), pages 624-6~2
(Mack Publishing. Co. 1990)
(see also 19'x' edition of Reminton's). Alternatively. "' Y, which emits an
ener~~etic beta particle, can be
coupled to an antibody component usin~~ diethylenetriaminepentaacetic acid
(DTPA). Moreover, a method
for the direct radiolabeling of the antibody component with ~'' I is described
by Stein et al., Antibody
lmmunoconj. Radiopharn. 4: 703 ( 1991 ) (see also LJSP 6, 080, 384).
Alternatively, boron addends such as carboranes can be attached to antibody
components, as discussed
above.
In addition, therapeutic immunoconjugates can comprise an immunomodulator
moiety suitable for
application for the purposes herein. Broadly speaking, the term
"immunomodulator" includes cytokines.
stem cell growth factors, lymphotoxins, such as tumor necrosis factor (1'NF),
and hematopoietic factors,
such as interleukins (e.~~., interleukin-1 (1L-1 ), 1L-2, Il.-3, IL6, II~-10
and I1.-12), colony stitnulatin'T factors
(e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte
macrophage-colony stimulating
63
CA 02402930 2002-09-19
factor (GM-CSF)), interferons (e.<~., interferonsalpha, -beta and gamma.), the
stem cell growth factor
designated "S I factor," erythropoietin and thrombopoietin. Examples of
suitable immunomodulator
moieties include 11.-2, Il.-6. IL-10, IL12, interferon-gamma., TNF-alpha., and
the like.
A related form of therapeutic protein is a fusion protein comprising an
antibody moiety and an
immunomodulator moiety.
Methods of making antibody-immunomodulator fusion proteins are known to those
of skill in the art as
discussed herein. For example, antibody fusion proteins comprising an
interleukin-? moiety are described
by Boleti et al., Ann. Oncol. 6:945 ( 1995), Nicolet et al., Cancer Gene ~
her. 2:161 ( 1995), Becker et al.,
Proc. Nat'I Acad. Sci. USA 93:7826 ( 1996). Hank et al., Clin. Cancer Rca.
2:1951 (1996), and Hu et al.,
Cancer Res. 56:4998 (1996). In addition, Yang et al.. Hum. Antibodies
Hybridomas 6:129 ( 1995). describe
a fusion protein that includes an F(ab')=fragment and a tumor necrosis factor
alpha moiety.
Such immunoconjugates and antibody-immunomodulator fusion proteins provide a
means to deliver an
immunomodulator to a target cell and are particularly useful against tumor
cells. The cytotoxic effects of
immunomodulators are well known to those of skill in the art. See, for
example. Kle et al.. "t_~~mphokines
and Monokines," in Biotechnology and Pharmacy, Pessuto et al. (eds.), pages 5
3-70 (Chapman R Hall
1993) as well as other references herein cited. As an illustration,
interferons can inhibit cell proliferation by
inducing increased expression of class I histocompatibility antigens on the
surface of various cells and thus,
enhance the rate of destruction of cells by cytotoxic T lymphocytes.
Furthermore, tumor necrosis factors,
such as TNF-alpha., are believed to produce cytotoxic effects by inducing DNA
fragmentation.
Moreover, therapeutically useful immunocon_jugates can be prepared in which an
antibody component is
conjugated to a toxin or a chemotherapeutic drug. Illustrative of toxins which
are suitably employed in the
preparation of such conjugates are ricin, abrin, ribonuclease. DNase 1,
Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtherin toxin, Pserrdomonas exotoxin,
and Pseudomonas endotoxin.
See, references herein as well as for example, Pastan et al., Cell 47:641 (
1986), and Golden berg, CA-A
Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins are known to
those of skill in the art.
With to respect to bispecific antibody constructs which are capable of biding
simultaneously to two
ligands on the same cell see also W096!32841. Various such constructs are
known in the art.An alternative
approach to introducin~~ the combination of therapeutic antibody and toxin is
provided by antibody-toxin
fusion proteins. An antibody-toxin fusion protein is a fusion protein that
comprises an antibody moiety and
a toxin moiety. Methods for making antibody-toxin fusion proteins are known to
those of skill in the art
(see references cited herein): antibody-Pseudomonas exotoxin A fusion proteins
have been described by
Chaudhary et al., Nature 339:394 ( 1989), Brinkrmann et al.. froc. Nat'1 Acad.
Sci. USA 88:8616 (1991),
Batra et al.. Proc. Nat'I Acad. Sci. USA 89:5867 ( 1992). Friedman et al., J.
Immunol. I 50:3054 ( 1993),
Wels et al., lnt. J. Can. 60:137 ( 1995), fominaya et al., J. Biol. Chen.
271:10560 ( 1996), Kuan et al.,
Biochemistry 35:2872 ( 1996), and Schmidt et al., Int. J. Can. 65:538 (1996).
Antibody-toxin fusion
proteins containing a diphtheria toxin moiety have been described by Kreitman
et al., Leukemia 7:553
(1993), Nicholls et al., .l. Biol. Chem. 268:5302 (1993), Thompson et al., J.
Biol. Chem. 270:28037 (1995),
and Vallera et al., Blood 88:2342 ( I 996). Deonarain et al., Tumor Tar~etin~~
1:177 ( ( 995), have described
an antibody-toxin fusion protein having an RNase moiety, while l_.inardou et
al., Cell Biophys. 24-25:243
( 1994), produced an antibody-toxin fusion protein comprising a DNase I
component. Gelonin was used as
the toxin moiety in the antibody-toxin fusion protein of Wane et al.,
Abstracts of the 209th ACS National
Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part I, BIOT005. As a further
example, Dohlsten et al., Proc.
Nat'I Acad. Sci. USA 91:8945 ( 1994), reported an antibody-toxin fusion
protein comprising Staphylococcal
enterotoxin-A. Numerous other examples have been reported in the literature.
Useful cancer chemotherapeutic drugs for the preparation of immunoconjugates
include nitrogen mustards,
alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine
analogs, purine analogs, antibiotics,
epipodoph_yllotoxins, platinum coordination complexes. hormones. and the like.
Suitable chemotherapeutic
agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and
in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 7th Ed.
(MacMillan Publishing Co.
1985). Other suitable chemotherapeutic agents, such as experimental drugs, are
known to those of skill in
the art.
In addition, therapeutically useful immunoconju;ates can be obtained by
conjugating photoactive agents or
dyes to an antibody composite. Fluorescent and other chromogens, or dyes, such
as porphyrins sensitive to
64
CA 02402930 2002-09-19
visible light, have been used to detect and to treat lesions by directing the
suitable light to the lesion. In
therapy. this has been termed photoradiation, phototherapy. or photodynamic
therapy (Jori et al. (eds.),
Photodynamic Therapy of Tumors and Other Diseases (L.ibreria Progetto 1985);
van den Bergh, Chem.
Britain 22:430 (1986)). Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for
achieving phototherapy. Mew et al.. J. Immunol. 130:1473 ( 1983); idern.,
Cancer Res. 45:4380 ( 1985);
Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem., Photochem.
Photobiol. 46:83 (1987);
Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers
Surd. Med. 9:422 (1989);
Pelegrin et al., Cancer 67:2529 ( 1991 ). However, these earlier studies did
not include use of endoscopic
therapy applications, especially with the use of antibody fragments or
subfragments. Thus, the present
invention contemplates the therapeutic use of in nnunoconjugates comprising
photoactive agents or dyes.
With respect to a multifunctional ligand having a first portion that binds to
both lymphatic endothelial cells
and tumor vasculature, the invention contemplates using phage display or
ribosome display to generate an
antibody that binds to vef'~r-3 as well as one or both of of vegfr-2 or vegfr-
L, having regard to the
sequences of those respective receptors (see USPs 5,776.755, 5877020, 5952199,
6107046, 6130071,
6221839, 62357(3, 6245530; see also WO 00!21560, WO 95;'33772. WO 97!05250, WO
98133917).
Preferably the antibody does not internalize, particularly in the case where
the multifunctional ligand is
fused or conjugated to a toxic moiety. The invention also contemplates, for
example, fusin~~ the bindint,~
domain of VEGF-C or VEGF-D to antitumor antibody. The invention also
contemplates that the risk of
retargeting cancer cells to non-tumor sites of an;;iogenesis, can be minimized
by employing one or more of
the following strategies pre- and%or co-treatment with inhibitors of
angiogenesis, providing the
multifunctional ligand with an effector function, such as a toxic moiety,
cytokine or antibody component
which retargets immune cells capable of killing such cancer cells. The
invention also contemplates using in
combination or alone a multifunctional ligand having a second portion that
comprises an anti-VEGF
antibody portion which hinds to one or more of the VE:GF family of li~~ands in
order to inhibit
lyrnphangiogenesis and;'or angiogenesis. (see also for example, WO 00/.>7025,
WO 98/33917, USP
6130071, WO Oli 12669). With respect to angio~~enesis and particularly
lymphangiogenesis see also:l:
Shibuya M. Structure and function of VEGE%VEGF-receptor system involved in
angiogenesis. Cell Struct
Funct. 2001 Feb;26( 1 ):25-35: Yonemura Y, et al.Lymphan~~iogenesis and the
vascular endothelial growth
factor receptor (VEGFR)-Sin gastric cancer Eur J Cancer. 2001 May;37(7):918-
23.: Iljin K, et aIVEGFR3
gene structure, regulatory region, and sequence polymorphisms FASEB J. 2001
Apr;lS(6):1028-36.: Tang
RF, et alOverexpression of lymphangiogenic growth factor VEGF-C in human
pancreatic cancer. Pancreas.
2001 Apr:22(3):285-92: Kadambi A, Carreira CM, Yun CO, Padera TP, Dolmans DE',
Carmeliet I',
FukumuraD, Jain RK.Vascular endothelial growth factor (VEGF)-C differentially
affects tumorvascular
function and leukocyte recruitment: role of VEGF-receptor 2 and hostVEGF-
A.Cancer Res. 2()01 Mar
I 5;61 (6):2404-8. Karpanen T, et alVascular endothelial growth factor C
promotes tumor
lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 2001 Mar 1;61
(5):1786-90: Baldwin
ME, et al The Specificity of Receptor Binding by Vascular Endothelial Growth
Factor-D Is Different in
Mouse and Man. J Biol Chem. 2001 Jun I?76(22):19166-19171: Niki T, et al J
Pathol. 2001
Apr; 193(4):450-7: Veikkola T, et al Signallin~~ via vascular endothelial
~~rowth factor receptor-3 is
sufficient forlymphangiogenesis in transgenic mice. EMBO J. 2001 Mar
15;20(6):1223-3 I Achen MG, et al
Localization of vascular endothelial growth factor-D in malignant melanoma
suggests a role in tumour
angiogenesis. J Pathol. 2001 Feb; 193(2):147-54 Stacker SA, et aIVEGF-D
promotes the metastatic spread
of tumor cells via the lymphatics. Nat Med. 2001 Feb;7(2):186-91 Plate K. From
an~~iogenesis to
lymphangiogenesis. Nat Med. 2001 Feb;7(2):151-2. . Joukov V, et al; A novel
vascular endothelial growth
factor, VEGF-C. is a ligand for the FIt4 (VECiFR-3) and KDR (VEGI=R-2)
receptor tyrosine
kinases.EMBO J. 1996 Apr 1;15(7):1751. Lee J. et al. Proc Natl Acad Sci USA.
1996 Mar 5;93(5):1988-
92
Multimodal therapies are also contemplated within the present invention,
including particularly for cancer,
therapies which can be determined to be useful complementary therapies for the
anti-metastatic
embodiments of this invention such as anti-angiogenic Ab conjugates
In another form of multimodal therapy, subjects receive the multifunctional
ligands of the present
inventionand standard cancer chemotherapy. For example, "CVB" ( 1.5 ;~~m<sup>2</sup>
cyclophosphamide, ?00-
400 mg/mv etoposide, and I 50-200 mg/m- carmustinel is a regimen used to treat
non-Hodgkin's lymphoma.
Patti et al., Eur. J. Haematol. 5 I :18 ( 1993). Other suitable combination
chemotherapeutic regimens are
well-known to those of skill in the art. See, for example, Freedman et al..
"Non-I lodgkin's Lymphomas," in
Cancer Medicine, Volume 2, 3rd Edition. Holland et al. (eds.), pages 2038-2068
(Lea & Febiger 1993). As
CA 02402930 2002-09-19
an illustration, first generation chemotherapeutic regimens for treatment of
intermediate-grade non-
Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine, procarbazine
and prednisone) and
CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). A useful
second generation
chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin,
cyclophosphamide,
vincristine, dexamethasone and leucovorin), while a suitable third generation
regimen is MACOP-B
(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,
bleomycin and leucovorin).
Additional useful drugs include phenyl butyrate and brostatin-1.
In general, the dosage of administered multifunctional ligands,
immunoconjugates, and fusion proteins will
vary depending upon such factors as the patient's age, weight, height, sex,
general medical condition and
previous medical history. Typically, it is desir<rble to provide the recipient
with a dosage of antibody
component, immunocon,jugate or fusion protein which is generally at least in
the range of from about I
pg/kg to 10 mg,~kg (amount of agent%body weight of patient), although a lower
or higher dosage also may
be administered as circumstances dictate, particularly to take advantage of
the depot effect of the invention.
Administration of the invention including, immunoconjugates or fusion proteins
to a patient can be
intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous,
intrapleural, intrathecal, by
perfusion through a regional catheter, or by direct intralesional injection.
When administering therapeutic
proteins by injection. the administration may be by continuous infusion or by
single or multiple boluses.
Those of skill in the art are aware that intravenous injection provides a
useful mode of administration due
to the thoroughness of the circulation in rapidly distributing antibodies.
Intravenous administration,
however, is subject to limitation by a vascular bawier comprising endothelial
cells of the vasculature and
the subendothelial matrix. Still, the vascular barrier is a more notable
problem for the uptake of therapeutic
antibodies by solid tumors. Lymphomas have relatively high blood flow rates,
contributin~~ to effective
antibody delivery. Intralymphatic routes of administratyion, such as
subcutaneous or intramuscular injection,
or by catherization of lymphatic vessels, also provide a useful means of
treating lymphomas.
With regard to "low doses" of''' I-labeled immunoconjugates, the invention
includes a dosage is in the
range of 15 to 40 mCi, 20 to 30 mCi. In contrast, a preferred dosage
of''° Y-labeled immunoconjugates is in
the range from 10 to 30 mCi, while the more preferable range is 10 to 20 mCi.
Immunoconjugates having a boron addend-loaded carrier for thermal neutron
activation therapy will
normally be effected in similar ways. However, it will be advantageous to wait
until non-targeted
immunocon_jugate clears before neutron irradiation is performed. Clearance can
be accelerated using an
antibody that binds to the immunoconjugate. See U.S. fat. No. 4,624,84Ei for a
description of this general
principle.
The immunoconjugates, and fusion proteins of the present invention can be
formulated according to known
methods to prepare pharmaceutically useful compositions. whereby the
therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A composition is said
to be a "pharmaceutically
acceptable carrier" if its administration can be tolerated by a recipient
patient. Sterile phosphate-buffered
saline is one example of a pharmaceutically acceptable carrier. Other suitable
carriers are well-known to
those in the art. See, for example, REMING'I"ON'S PHARMACELIT1CAL SCIENCES,
19th Ed. ( 1995).
For purposes of therapy. antibody components (or immunoconjugates/fusion
proteins) and a
pharmaceutically acceptable carrier are administered to a patient in a
therapeutically effective amount. A
combination of an antibody component, optionally with an
immunoconjugate/fusion protein, and a
pharmaceutically acceptable carrier is said to be administered in a
"therapeutically effective amount" if the
amount administered is physiologically significant. An absent is
physiologically significant if its presence
results in a detectable change in the physiology of a recipient patient. In
one aspect, an agent is
physiologically significant if its presence results in the inhibition of the
growth of target tumor cells.
Yet another therapeutic method included in the invention is a method of
treating cancer by administering to
an animal suffering from cancer a pharmaceutically effective amount of one or
more multifunctional
ligands capable of binding to cancer cells, wherein the compound is associated
with a substance capable of
damaging cancer cells.
Pharmaceutical compositions herein described or alluded to include
multifunctional ligands of the
invention or therapeutics used in combination therapy which may be
administered by a variety of routes of
66
CA 02402930 2002-09-19
adminstration.
By administration of an "effective amount" is intended an amount of the
compound that is sufficient to
enhance or inhibit a response, is some embodiments particularly an immune
response or cellular response
to a multifunctional ligand. One of ordinary skill will appreciate that
efitective amounts of a multifunctional
ligand can be determined empirically and may be employed in pure form or,
where such forms exist, in
pharmaceutically acceptable salt, ester or prodrug form. The multifunctional
ligand rnay be administered in
compositions in combination with one or more pharmaceutically acceptable
excipients. It will be
understood that, when administered to a human patient, the total daily usage
of the compounds and
compositions of the present invention will be decided by the attending
physician within the scope of sound
medical jud~~ement. The specitic therapeutically etfective dose level for any
particular patient will depend
upon a variety of factors including the type and de<~ree of the cellular
response to be achieved; activity of
the specific multifunctional ligand employed: the specific composition
employed; the age, body weight,
general health, sex and diet of the patient; the time of administration, route
of administration, and rate of
excretion of the agonist or antagonist; the duration of the treatment; drugs
used in combination or
coincidental with the specific monist or anta~xonist; and like factors well
known in the medical arts.
On administration parenterally, for example by i.v. drip or infusion, dosages
optionally at leasr on the order
of from 0.01 to 5 mg/k~~'day. optionally 0.05 to 1.0 mg/kg/day and more
preferably 0. I to 1.0 rn~= kg/day
can be used. Suitable daily dosages for patients are thus on the order of from
2.5 to 500 mg p.o., optionally
to 250 m~~ p.o., optionally 5 to 100 mj p.o., or on the order of from 0.5 to
250 mg i.v., optionally 2.5 to
125 mg i.v. and optionally 2.5 to 50 mg i.v.
Dosaging may also be arranged in a patient specific manner to provide a
predetermined concentration of an
agonist or antagonist in the blood, as determined by the RIA technique. Thus
patient dosaging may be
adjusted to achieve regular on-going trough blood levels, as measured b_~ RIA,
optionally on tyre order of at
least from SO to 1000 n«'ml, preferably 150 to 500 n~~,'ml.
From above, pharmaceutical compositions are provided comprising an agonist or
antagonist and a
pharmaceutically acceptable carrier or excipient, which may be administered
orally, rectally, parenterally,
intracisternally, intravaginally, intraperitoneally, topically (as by powders,
ointments, drops or transdermal
patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable
carrier" is meant a non-toxic
solid, semisolid or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type. The
teen "parenteral" as used herein refers to modes of administration which
include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
Optionally a composition for for parenteral injection can comprise
pharmaceutically acceptable sterile
aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well
as sterile powders for
reconstitution into sterile injectable solutions or dispersions just prior to
use. Examples of suitable aqueous
and nonaqueous carriers. diluents, solvents or vehicles include water,
ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like). carboxymethylceuulose
and suitable mixtures thereof,
vegetable oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper tluidity can be
maintained, for example, by the use of coatings materials such as lecithin, by
the maintenance ofthe
required particle size in the case of dispersions, and by the use of
surfactants.
Some compositions herein descibed may also contain adjuvants such as
preservatives, wetting agents,
emulsifying agents, and dispersing agents. Prevention of the action of
microorganisms may be ensured by
the inclusion of various antibacterial and antifungal agents. for example,
paraben, chlorobutanol. phenol
sorbic acid, and the like. It may also be desirable to include isotonic agents
such as sugars, sodium chloride,
and the like. Prolonged absorption of the injectable pharmaceutical fornn may
be brought about by the
inclusion of agents which delay absorption such as aluminum monostearate and
gelatin.
In some cases, in order to prolong the effect of one or therapeutic components
herein described, it is
desirable to slow the absorption from subcutaneous or intramuscular injection.
This may be accomplished
by the use of a liquid suspension of crystalline or amorphous material with
poor water solubility. The rate
of absorption of the drub= then depends upon its rate of dissolution which, in
turn, may depend upon crystal
size and crystalline form. Alternatively, delayed absorption of a parenterally
administered drug form is
accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug
in biode~~radable
67
CA 02402930 2002-09-19
polymers such as polylactide-polyglycolide. Depending upon the ratio of drug
to polymer and the nature of
the particular polymer employed, the rate of drug release can be controlled.
Examples of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are
also prepared by entrapping the drug in liposomes or microemulsions which are
compatible with body
tissues.
The injectable formulations can be sterilized, for example, by filtration
through a bacterial-retaining filter,
or by incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or
dispersed in sterile water or other sterile injectable medium just prior to
use.
The multifunctional ligand can also be administered in the form of liposomes.
As is known in the art,
liposomes are generally derived from phospholipids or other lipid substances.
Liposomes are formed by
mono- or multi-lameflar hydrated liquid crystals that are dispersed in an
aqueous medium. Any non-toxic,
physiologically acceptable and metabolizable lipid capable of forming
liposomes can be used. The present
compositions in (iposome form can contain, in addition to the agonist or
antagonist, stabilizers,
preservatives, excipients, and the like. The preferred lipids are the
phospholipids and the phosphatidyl
choaes (lecithins). both natural and synthetic. Methods to form liposomes are
known in the art. See, for
example, Prescott, Ed.. Methods in Cell Biology, Volume XIV. Academic Press.
New York. N.Y. ( 1976).
p. 33 et seq.
The present invention also contemplates a method of treatment in which
immunomodulators are
administered to prevent, mitigate or reverse radiation-induced or drug-induced
toxicity of normal cells, and
especially hematopoietic cells. Adjunct immunomodulator therapy allows the
administration of higher
doses of cytotoxic agents due to increased tolerance of the recipient mammal.
Moreover, adjunct
immunomodulator therapy can prevent, palliate, or reverse dose-limiting marrow
toxicity. Examples of
suitable immunomodulators for adjunct therapy include G-CSF. GM-CSF,
thrombopoietin, IL-I, IL-3, IL-
13, and the like. The method of adjunct immunomodulator therapy is disclosed
by Goldenberg, U.S. Pat.
No. 5,120,525.
For example, recombinant IL-2 may be administered intravenously as a bolus at
6 x 10' IU!kg or as a
continuous infusion at a dose of I 8 x 10G IU/m' /d. Weiss et al., J. Clin.
Oncol. 10:275 ( 1992).
Alternatively, recombinant 1l.-2 may be administered subcutaneously at a dose
of 12 x 10'' 111. Vogelzang
et al., J. Clin. Oncol. 1 1:1809 ( 1993). Moreover, INF-.'~amrna. may be
administered subcutaneously at a
dose of 1.5 x10' U. Lienard et al., J. Clin. Oncol. 10:52 ( 1992).
Furthermore, Nadeau et al., J. fharmacol.
Exp. Ther. 274:78 ( 1995), have shown that a single intravenous dose of
recombinant 1L-12 (42.5
µg/kilogram) elevated IFN-.gamma. levels in rhesus monkeys.
Suitable IL-3 formulations include PROLEUK1N (Chiron Corp./Cetus Oncology
Corp.; Emeryville,
Calif) and TECELEUK1N (Hoffmann-La Roche. Inc.; Nutley, N.J.). ACTIMMUNE
(Genentech, Inc.;
South San Francisco, Calif.) is a suitable INF-.gamma. preparation.
In the precedinu detailed description, reference was made to various
methodologies known to those of skill
in the art of molecular biology and irnmunolo~;y. Publications and other
materials setting forth such known
methodologies to which reference was made or is made below are incorporated
herein by reference in their
entireties along with references cited therein as though set forth in full.
Standard reference works setting forth the general principles of recombinant
DNA technology include
Watson, J. D. et al. Molecular Biology of the Gene, Volumes I and II, the
Benjamin/Cummings Publishing
Company, lnc., publisher. Menlo Park. Calif ( 1987), Darnell. J. E. et al.,
Molecular Cell Biology.
Scientific American Books, Ine.. Publisher, New York. N.Y. (1986); I~t~win. B.
M. Genes Il, .lohn Wiley
& Sons, publishers, New- York, N.Y. ( 1985); Old. R. W., et al., Principles of
Gene Manipulation: An
Introduction to Genetic Engineering. 2d edition. University of California.
Press, publisher, Berkeley, Calif.
(1981); Maniatis. T., et al.. Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor
Laboratory, publisher, Cold Spring Harbor, N.Y. ( 1989), and Current Protocols
in Molecular Biology,
Ausubel et al.. Wiley Press, New York, N.Y. ( 1989). Standard reference works
setting forth general
principles and techniques of immunology include Handbook of Experimental
Immunology Blackwell
Science, Incorporated, ISBN:0632009756; Antibody I~ngineering Black well
Science. Incorporated,
ISBN:0632009756; Therapeutic Immunology ISBN: 086542375X Blac:kwell Science.
Incorporated ;
Encyclopedia of Immunology (1998) Morgan Kaufmann Publishers, ISBN:0122267656;
Immunology
68
CA 02402930 2002-09-19
Mosby, Incorporated, ISBN:0723429189; Abbas AK. et al. Cellular & Molecular
Immunology 4'~' Ed.
2000 ISBN 0721650026: Breitling F. et al. Recombinant Antibodies 1999 ISBN 0-
471-17847-0;
Masseyeff R. et al. Methods of Immunological Analysis Wiley-VCI-i ISBN 3-527-
27906-7, 1992;
Mountain et al. Eds, Biotechnology 2°'' ed. Vol 5A 1998 ISBN 3-527-
28315-3 Wiley-VCIi; Campbell,
A. , "Monoclonal Antibody Technology," in, Burdon, R., et al., eds, Laboratory
Techniques in
Biochemistry and Molecular Biology, Volume 13, Elsevier, Publisher, Amsterdam
( 1984);
Although the foregoing refers to particular preferred embodiments, it will be
understood that the present
invention is not so limited. It will occur to those of ordinary skill in the
art that various modifications may
be made to the disclosed embodiments and that such modifications are intended
to be within the scope of
the present invention.
All publications referred to herein are indicative of the level of skill of
those in the art to which the
invention pertains. All publications are herein (as well as references cited
therein)are incorporated by
reference to the same extent as if each individual publications were
specifically and individdually indicated
to be incorporated by reference in its entirety.
The present invention, thus generally described, will be understood more
readily by reference to the
preceding and following examples, which are provided by way of illustr<rtion
and are not intended to be
limiting of the present invention.
With respect to making applications, methods of using. target cell-associated
markers etc. of multispecific
ligands and bispecitic antibodies see also ,~-Tethocls Wol l3iol 2001; 166: t
77-92; USP 6.071,517; USP
5,897,861; USP 6,096.31 l; USP 5,922,845. Journal of Immunological Methods
February 2001 Vol.
248(1-2) page 1-200; Mol Immunol 1999 May; 36(7):433-45; US6051227 US06143297
US05977318 US05968510 US05885796 US05885579 US05869050 Methods of blockinU T-
cell
activation using anti-B7 monoclonal antibodies: US05851795 US05747034 US061
13901 US05877021
B7-1 targeted ribozvmes; US05844095 US06090914 US05718883 Trans~~enic animal
model for
autoimmune diseases; US05855887 US0581 1097 US05770197 Methods for regulating
the immune
response using B7 binding molecules and IL4-binding molecules; US06084067
EP01073741 A2 US06130316 US06068984 Antibodies to lymphocyte activation
antigens uses
therefor;
US05766570 Lymphocyte activation anti~~ens and thereto; US05434131 US05316920
Lymphocyte activation antigen HB 15, a member of the immunoglobulin
superfamily; US061 1 1090
Mammalian cell surface antigens: US0608375 I Chirneric receptors for the
generation of selectively-
activatable TH-independent cytotoxic T cells;
US05977303 Mammalian cell surface antigens; US05738852 Methods of enhancing
antigen-specific T
cell responses US05714667
Bruhl H, et alDepletion of CCRS-Expressing Cells with Bispecific Antibodies
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M, Eisner W.
Fraunberger P, Reifenber;;er G.Schlegel U, Wiestlcr OD. Reulen HJ_ Wilmanns W.
local imrnunotherapy
of glioma patients with a combination of 2 bispecific antibody fragments and
restin<~ autolo~~ous
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Int J Cancer. 2001 Jan 15;
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G7 . Thioether-
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71
CA 02402930 2002-09-19
With respect to anti-CCRS antibodies used to kill CCRS-expressing cells, with
for example. bi-specific
antibody chemokine fusions see Bruhl H. et al. J Imrnunol. 2001 Feb 15 166(4):
2420-2426.
With respect to targeting IKAP proteins see for example US 6172195.
With respect to pertinent diseased cells, disease causing cells and other
suitable targets for immunotoxins,
as well as optional toxins and methods of making and using immunotoxins and
related technolo~~ies see for
example US05980895 Immunotoxin containing a disulfide-stabilized antibody
fragment joined to a
Pseudomonas exotoxin that does not require proteolytic activation: US05686072
Epitope-specific
monoclonal antibodies and immunotoxins and uses thereof: US04956453 Antihuman
ovarian cancer
immunotoxins and methods of thereof; US0614663 I Innnunotoxins comprising
ribosome-inactivating
proteins; US05756699 Imrnunotoxins comprisin;~ ribosome-inactivating Proteins;
US05744580
Immunotoxins comprising ribosome-inactivating Proteins; US06146850 Proteins
encoding gelonin
sequences; US05837491 Polynucleotides encoding gelonin sequences; LJS05578706
Methods and
compositions concerning homogenous immunotoxin preparations; US05185434
Prolony~ed-action
immunotoxins containin~~ a glycopeptide constituent which inactivates
rih~osomes, modified on its
polysaccharide units; US04958009 Anti-human ovarian cancer immunotoxins and
methods of use
thereof; US05980896 Antibodies reactive with human carcinomas; US06074644
Nucleic acids
encoding immunotoxins containing a disulfide-stabilized antibody fragment
replacing half or more of
domain IB of pseudomonas exotoxin. and methods of use of the encoded
immunotoxins;
US04981953 Immunotoxins, process for their preparation and pharmaceutical
compositions in which
they are present; US04980457 Cytotoxic conjugates which can be used in therapy
and process or their
preparation; US04545985 Pseudomonas exotoxin conjugate immunotoxins;
US06020145 Methods for
determining the presence of carcinoma using the antigen binding region of
monoclonal antibody BR96;
US05792458 Mutant diphtheria toxin conjugates: US05338542
US06051230 Compositions for targeting the vasculature of solid tumors; B3
antibody fusion proteins
and their uses; US05990275 Linker and linked fusion polypeptides; US05981726
Chimeric and
mutationally stabilized tumor-specific Bl, B3 and B5 antibody fragments; in
~munotoxic fusion proteins;
and uses thereof: US05965132 Methods and compositions for targetinf; the
vasculature of solid tumors:
US05889157 Humanized B3 antibody fragments, fusion proteins, and uses thereof
US06027725
Multivalent antigen-binding proteins;
EP00861091A1 1MMUNOTOX1N CONTAINING A DISULFIDE-STABILIZED ANTIBODY
FRAGMENTUS05776427 Methods for targetinV~ the vasculature of solid tumors;
US05772997
Monoclonal antibodies directed to the HER2 receptorUS05665357 Antibodies
recognizin~~ tumor
associated antigen CA 55.1; US05660827
Friedrich K, et al A two-step selection approach for the identification of
li'~and-binding determinants in
cytokine receptors.Anal Biochem. 1999 Mar 15; 268(2):179-86.
Krebs B, et al Recombinant human single chain Fv antibodies recognizin~~
humaninterleukin-6. Specific
targeting of cytokine-secreting cells. J Biol Chem. 1998 Jan 30; 273(5):2858-
65 Wilbur DS,et
alRelatedArticles Biotin reagents for antibody pretargeting. 2. Synthesis and
in vitroevaluation of biotin
dimers and trimers for cross-linkin« ofstreptavidin. Bioconjug Chem. 1907 Nov-
Dec; 8(6):819-32.
Ring DB. et alAntigen forks: bispecific reagents that inhibit cell growth by
bindingselected pairs of tumor
antigens. Cancer Immunol Immunother. 1994 Jul; 39( l ):41-8.
W009942597A1 MONOVALENT, MULTIVALENT, AND MULTIMERIC MHCB1NDING DOMAIN
FUSION PROTEINS AND CONJUGATES, AND USES THEREFOR; EP00935607A2 SOLUBLE
MONOVALENT AND MULTIVALENT MHC CLASSII FUSION PROTEINS, AND USES
THEREFOR;
72
CA 02402930 2002-09-19
W009811914A1 TARGETING ANTIGENS TO THE MHC CLASS IPKOCESSING PATHw'AY WITH
ANTHRAX TOXIN FUSION PROTEINW009728191A1 MHC COMPLEXES AND USES THEREOF;
US05580756 B71G fusion protein; US06143298 Soluble truncated forms of ICAM-l;
US05852175 P-selectin glycoprotein ligand blocking
antibodiesLJS05800815Antibodies to P-selectin and
their uses;
US06037454 Humanized anti-CD1 la antibodies; US06020152 Lymphocyte-associated
cell surface
proteinUS05807734 Monoclonal antibodies and FV specitic for
CD2antigenUS05622701 Cross-reacting
monoclonal antibodies specificfor E- and P-selectin
US05622700 Method for treating a LFA-1-mediated disorder; JP06209'788A2
IMMUNOASSAY OF
HUMAN SOLUBLE ICAM-1. ANTIBODYAND KIT FOR MEASUREMENT THEREOF;
JP03072430A2 ANTIV1RAL AGENT BY USING FU'~ICTIONAI_DERIVATIVE OF INTERCELLULAR
ADHESIVE MOLECULE; JP01135724A2 TREATMENT FOR NONSPECIFIC INFLAMMATION;
US06123915 Methods for using agents that bind to VCAM-l; W009929706A2
DISAt_1CYLATE
ANALOG BASED SIAI_YL LEWISxMIME~I~ICS; W'009918442A1 DIAGNOSIS OF'THROMBOTIC
EVENTS BY DETEC'TINGP-SEL.ECTIN; US05877295 Antibodies which bind a
subpopulation of Mac-
1(CDI lb/CD18) molecules which mediate neutrophil adhesion to ICAM-land
fibrinogen; LJS05869460
Sulfated and phosphated saccharide derivatives,process for the preparation of
the same and use thereof;
US05858994 Carbohydrate conjugates as inhibitors of celladhesion; US0581 1405
Multiply fu cosylated
dicarboxylic acidspossessing antiadhesive properties; US05654282 Selectin
binding glycopeptides;
US05632991 Antibodies specitic for E-selectin and the usesthereof; US05599676
Method for isolating a
novel receptor for.alpha.4 integrins: US05580862 Sulfate ligands for L,-
selectins and methods
ofpreventing sulfate addition: US05508387 Selectin binding glycopeptides;
US06177547 Antibodies to
P-selectin glycoprotein ligandUS05827670 Methods of isolating and detecting
bone marrow stromal cells
with VCAM-1-specific antibodies; US05756095 Antibodies with specificity for a
common epitope on E-
selectin and L-selectin; US05565550 Antibodies to ICAM-3, and fragments
thereof;
US06099838 Pharmaceutical compositions comprising anti-CD45RB antibodies for
the inhibition of 1-
cell mediated immune responses;
US05595737 Methods for using monoclonal antibodies specific for cell-surface
bound LAM-1;
US05324510,
US06183988 Leukocyte-specific protein and gene, andmethods of use thereof;
US05998598
US05997865 Agonist antibodies against the tlk2/flt3 receptor and uses thereof;
1JS05993816 Methods to
inhibit humoral immune responses, immunoglobulin production and B cell
activation with 5c8-specific
antibodies:
US05869453 Cytotoxic T-cell epitopes; US05861 15l Soluble fusion molecules
with binding specificity
for cell adhesion molecules; US05843441 Use of endothelial-leukocyte adhesion
molecule-1 specific
antibodies in the treatment of asthmaUS05821332 Receptor on the surface of
activated CD4+ T-cells:
ACT-4; EP00868197A I ANTI-SELECTIN ANTIBODIES FOR PREVENTION OFMUL,T11'1_E
ORCi.AN
FAILURE AND ACUTE ORGAN DAMAGE; US05817515 Human B2 integrin alpha subunir
antibodies;
EP00528931B1 HUMANIZED CHIMERIC ANTI-ICAM-1 ANTIBODIES, METHODS OF
PREPARATION AND USE; US05776775 Anti-LAM 1-3 antibody and hybridoma;
US06063906 Antibodies to integrin alpha subunit; US05997865 Agonist antibodies
against the flk2/flt3
receptor and uses thereof; US05993816 Methods to inhibit humoral immune
responses, immunoglobulin
production and B cell activation with 5c8-specific antibodiesUS059519fi2
Methods to suppress an immune
response with variant CD44-specific antibodiesUS05843441 Use of endothelial-
leukocyte adhesion
73
CA 02402930 2002-09-19
molecule-1 specific antibodies in the treatment of asthma US05821332 Receptor
on the surface of
activated CD4+T-cells: ACT-4; US05821 123 Modified antibody variable domains;
EP00868197A I
ANTI-SELECTIN ANTIBODIES FOR PREVENTION OF MULTIPLE ORGAN FAILURE .AND
ACUTE ORGAN DAMAGE; US05817515 Human B2 integrin alpha subunit antibodies;
EF'00528931 B I
HUMANIZED CHIMERIC ANTI-(CAM-l ANTIBODIES, ME'T'HODS OF PREPARATION AND USE;
US05776775 Anti-LAM I-3 antibody and hybridoma; US05776755 FI_T4, a receptor
tyrosine kinase;
US05730978 Inhibition of lymphocyte adherence with alpha.4&tt2231; -specific
antibodies:
Examples of tumor specif7c antigens are numerous and are referred to in the
hereinabove cited references
andas well as the in the following referencesUS0613298010/17/2000 Antibodies
specific for TRP-2 a
human tumor antigen recognized by cytotoxic T lymphocytes
US06165464Monoclonal antibodies directed to the HE:R2 receptor
US0582431 (Treatment of tumors with monoclonal antibodiesagainst oncogene
antigens.
US0614005010/31/2000 Methods for determining breast cancer and melanoma by
assaying for a plurality
of antigens associated herewith; US06051226 MN-spexific antibodies and their
use in cancer treatment;
US06020145Methods for determining the presence ofcarcinoma using the antigen
binding region of
monoclonal antibody BR96.; US05980896 Antibodies reactive with human
carcinomasUS05955075Method of inhibitin«, tumor growth using antibodies to MN
protein
US05917124Transgenic mouse model of prostate cancer; US0591438906,'22/1999 E6
associated
proteinUS0591214306;' 15i i 999 Polynucleotides encodin<~ a human ma<~e
proteinhomologUS0591062606,''08..% 1999 Acetyl-CoA carboxylase compositions
and methodsof
useUS0587456002/23/1999 Melanoma antigens and their use in diagnostic and
therapeutic
methodsUS058722170' 16/1999 Antibodies which specifically bind a cancerrelated
antigenUS0586963602:09/1999 Immunoreactive peptide sequence from a 43 kDhuman
cancer
antigenUS0586904502(19;1999 Antibody conjugates reactive with human carcinomas
US0586612402/02/1999 Antiidiotypic antibodies for high molecularweight-
melanoma associated by
sameUS0584708312/08'1998 Modified p53 US05844075 Melanoma antigens and their
use in
diagnosticand therapeutic methods US0584368512/O l / 1998 Production of
chimeric mouse-human
antibodies with specificity to human tumor antigensUS05843648 Pl _5 and
tyrosinase melanoma antigens
and their use in diagnostic and therapeutic methods US05840854US05R~304701
1/03/1998 Humanized
antibodies to ganglioside GM2US0583046411/03/ 1998 US0580800SHuman carcinoma
Bispecific
molecules reco~~nizin<g lymphocyte anti~~en CD2 and tumor anti'~ensUS05792456
Mutant BR96 antibodies
reactive with humancarcinomas US0578368US05773579 Lung cancer marker
US05772997 Monoclonal
antibodies directed to the HER2 receptor 1JS05770374; US05705157 Methods of
treating cancerous cells
with anti-receptor antibodies US0569599412'09/1997 Isolated cytolytic T cells
specific forcomplexes of
MACE related peptides and HL.A moleculesUS0569376312021997 .Antibodies to
human carcinoma
antigen
Tumor rejection antigens which correspond toamino acid sequences in tumor
rejection antigen precursor
ba~~e, and uses thereof US05681701 Immortalized human fetal osteoblastic
ceIIsUSOS68156210;'28: 1997
US05(i7717110.'I:L,'1997 Monoclonal antibodies directed to the
FlER2receptorlJS0567448610/07~1997US0566535709/09!1997 Antibodies recognizing
tumor associated
antigen CA 55.1
Fonsatti E, et al En~er~~in~~ role of protectin (CD59) in humoral
irnmunotherapy of solid malignancies.Clin
Ter. 2000 May-Jun; 151(3):187-93 Knuth A, etalCancer immunotherapy in clinical
oncology. Cancer
Chemother Pharmacol. 2000; 46 SuppI:S46-51 : Sievers EL.Targeted therapy of
acute myeloid leukemia
with monoclonal antibodies andimmunoconjugates. Cancer Chemother Pharmacol.
2000; 46 SuppI:Sl8-22
74
CA 02402930 2002-09-19
.172 van Spriel AB,et allnnnunotherapeutic perspective for bispecific
antibodies.lmmunol Today. 2000
Aug; 21(8):391-7 273: Green MC, et alMonoclonal antibody therapy fur solid
tumors. Cancer Treat Rev.
2000 Aug; 26(4):269-86
Xiang J Targeting cytokines to tumors to induce active antitumor immune
responses by recombinant
fusion proteins. Hum Antibodies. 1999; 9( 1 ):23-Engberg J, et al Recombinant
antibodies with the antigen-
specific, MHC restricted specificity of T cells: novel reagents for basic and
clinical investigations and
immunotherapy. Immunotechnology. 1999 Mar; 4(3-4):373-8. O'Brien ~~J. 'fet
alMore than I 5 years of CA
125: what is known about the antigen, its structure andits function.lnt J Biol
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Shariti J, et allmprovin~T monoclonal antibody pharmacokinetics via chemical
modification.Q J Nucl Med.
1998 Dec: 42(4): 242-9.
Ligands on immune or other cells which may be targeted with bispecific ligands
in which one ligand of
the pair dictates specificity for a population of cells or particular sub-
population of those cells and a second
ligand with reduced functional affinity is used to effect a specific immune
function include those referenced
in the following patents and publications therein referenced: US06132992
Expression vectors encoding
bispecific fusionproteins and methods of producing biologically active
h~ispecitrc fusion proteins in a
mammalian cell: Antibody heteroconjugates and bispecificantibodies for use in
regulation of lymphocyte
activity;
W 009942077
COMPOSITIONS AND METHODS FOR REGULATINGLYMPHOCYTE ACTIVATION;
US059165600 Methods For inhibiting an immune response byblocking the GP39/CD40
and
CTLA4/CD28iB7 pathways and compositionsfor use therewith; US058~76718 Methods
of inducing T cell
non-responsiveness to transplanted tissues and of treating graft-versus-host-
disease with anti-gp39
antibodiesEP00445228BIMMUNO~fI-iERAPY INVOLVING CD28 STIMULATION;
US05709859 Mixed specificity fusion proteins; US05637481 Expression vectors
encoding bispecific
fusion proteins and methods of producing biologically active bispecific fusion
proteins in a mammalian
cell; W009720048 MODIFIED SFV MOLECULES WHICH MEDIATE ADHESIONBETWEEN CELLS
AND USES THEREOF;
EP00336379 Antibody heteroconjugates for use in regulation of lymphocyte
activity;
EP00537293 LIGAND FOR CD28 RECEPTOR ON B CELLS ANDMETHODS; US05182368 I_igands
and methods for augmenting B-cell proliferation; W009300431 CTL4A RECEPTOR.
FUSION
PROTEINS CONTAINING ITAND USES THEREOF; EP00445228 IMMUNOTHERAPY INVOLVING
CD28 STIMULATION:
Role of cellular adhesion molecules in HIV type 1 infection and their impact
onvirus neutralization.
AIDS Res Hum Retroviruses. 1998 Oct; 14 Suppl 3:S247-54 Cavenagh JD,et al
Adhesion molecules in
clinical medicine. Crit Rev Clin Lab Sci. 1998 Sep: 35(5):415-59 Vine:y JL,
Fong S. Beta 7 integrins and
their ligands in lymphocyte mi~~ration to the gut. Chem Inununol. 1998; 71:64-
76 Aplin AE, et alSi~~nal
transduction and signal modulation by cell adhesion receptors: the roleof
integrins, cadherins,
immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev. 1998
Jun; 50(2):197-263
With respect to ascertaining important amino acid residues for receptor
activation or binding see also Zang,
Q., Sprin~~er. T. A. (2001 ). Amino Acid Residues in the PSI Domain and
Cysteine-rich Repeats of the
Integrin beta 2 Subunit That Restrain Activation of the Integrin alpha xbeta
3. .l. Biol. C'hena. :?76: 6922-
6929; Bir~etinh site on the murine IFN-gamma reevptarr for IFN-gamma has been
identifiied using the
synthetic peptide approach,The Journal of Immunolo~~y, Vol 15 I. Issue 1 1
6206-6313; The Journal of
CA 02402930 2002-09-19
Immunology, Vol 143, Issue 1 1 3568-3579'fhe main immunogenic region of the
nicotinic acetylcholine
receptor. Identification of am ino acid residues interactin~~ with different
antibodies, M Bellone et al.;
Arend, W. P., Malyak, M., Guthridge. C. J., Gabay, C. (1998). INTERLEUKIN-1
RECf_P'fOR
ANTAGONIS'h: Role in Biology. .=lnnu. Rev. lmmtsnol. 16: 27-55: The Journal of
Immunology, Vol 155,
Issue 10 4719-4725, Mapping of receptor binding sites on IL-1 beta by
reconstruction of IL- Ira-like
domains; The Jourrmrl o/ lrrmnnoloy, 2000. 165: 6966-6974 Identification of
Fetal Liver Tyrosine Kinase
3 (FIt3) Ligand Domain Required for fZeceptor Binding and Function Using
Naturally Occurring Ligand
Isoforms Waithaka Mwangi'', Wendy C. Brown and Guy' H. Palmer.
The invention also contemplates multifunctional li~~ands comprising various
combinations and
permutations of such li,~ands including pairs and three different such ligands
including multifunctional
ligands including such combinations and a li~~and which binding to a lymphatic
vessel associated
Iigand.Additional pertinent references pertaining to formation of antibody
dimers, microarrays of (and
tissue microarrays) proteins including heterohunctional proteins and
recombinant, ligands having
application to the invention, and phage or ribosome display strate~~ies
havin<~ relevance herein include Zhu
H. et al. Protein arrays and microarrays.Curr Opin Chem Biol. 2001 Feb:
5(1):40-5, reterences in IBC's
conference on Protein Microarray Technology March 19-21 Santiago California;
WO 99/06834, WO
99/19506; WO 97!02343, WO 00/63701; WO 99%40434 ; US 6,127,127; US6146830, WO
00!075298,
US6165709 US0620402303!20i2001 Modular assembly of antibody genes, antibodies
prepared thereby and
use; US0584681812;08%1998 Pectate lyase si~~nal sequence; US0569843513/16;
1997 Modular assembly of
antibody genes, antibodies prepared thereby and use; US0569841712/ Ifiil997
Modular assembly of
antibody genes, antibodies prepared thereby and use: US0569349312/02!1997
Modular assembly of
antibody genes, antibodies prepared thereby and use; US055 t454805/07i 1996
Method for in viva selection
of ligand-binding proteins; US0564823707;'15/1997 Expression of functional
antibody fragments;
US0618034 In vitro scanning saturation mutanenesis of proteins; US06027933
Surface expression libraries
of heteromeric receptor; US0591057306i08i 1999 Monomeric and dimeric antibody-
fragment fusion
proteins; US061505831 1 Transgenic animals expressing artificial epitope-
tagged proteins; US06132992
Expression vectors encoding bispecific fusion proteins and methods of
producin<,~ biolo~~ically active
bispecific fusion proteins in a mammalian cell; US06127524Binding molecules
and computer-based
methods of increasing the binding affinity thereof;; US06071515 Dirner and
multimer forms of single chain
polypeptides_ L1S06054297 Humanized antibodies and methods for makingthem;
W000004 382 ARRAYS
OF PROTEINS AND METHODS OF USE THEREOF; US06008023 C'ytoplasmic expression of
antibodies. antibody fra~~ments and antibody fragment fusion proteins in E.
coli: Pha'.:emid for antibody
screening; US059808951 1!09/1999 Immunotoxin containing a disulfide-stabilized
antibody fragment
joined to a Pseudomonas exotoxin that does not require proteolytic activation:
IJS05962255 Methods for
producin~~ recombinant vectors; US05955341 Ileterodimeric receptor libraries
using phagemids;
W009939210A1 HIGLi DENSITY ARRAYS FOR PRO ~EOME ANALYSIS AND ME~I LiODS AND
COMPOSITIONS THEREFOR: W009931267 METHODS FOR THE SIMULTANEOUS
IDENTIFICA hIONOF NOVEL. BIOLOGICAL TARGETS AND LEAD STRUCTURES FOR
DRUGDEVELOPMEN f; US05869619 Modified antibody variable domains;
US058558851solation and
production of catalytic antibodies using phage technology; US05851801 12/22;
1998 Method of preparing
polypeptide binding compositions derived from immuno~~lobulin variable
regions; US05849500 Phagemid
for antibody screenin~~;binding composition; US058378461 1/17/1998
Biosynthetic binding proteins for
immuno-targeting; US0582133710/13% 1998 lmmunoglobulin variants: IJS05821
123Modilied antibody
variable domains; US0578965508/04/ 1998 'hransgenic animals expressin~~
artificialepitope-tagged
proteins; US05783384 Selection of bindinV~-molecules; US05780225 Method for
generating libaries of
antibodygenes comprising amplification of diverse antibody DNAs and methodsfor
using these libraries for
the production of diverge antigen combinin~~ molecules; US05770356 Phagemids
coexpressing a surface
receptor and a surface heterologous protein; WO09808603 ISOLATION OF
1MMUNOGI_OBULINS:
US05716805 Methods of preparing soluble, oli~omeric proteins: US05;s95898
Modular assembly of
antibody genes, antibodiesprepared thereby and use; US05582996 Bifunctional
antibodies and method of
preparing same; US05580717Recombinant library screenings methods; Haab BB.
Dunham MJ, Brown PO
.Protein microarrays for highly parallel detection and quantitation of
specific proteins and antibodies in
complex solutions.Genome Biol. 2001; 2(2):: Moch 1-l. Kononen T, Kallioniemi
OP, Sauter G. Tissue
m i c roarrays
Borrebaeck CA.Antibodies in diagnostics - from immunoassays to protein
chips.Immunol Today. 2000
Aug: 21 (8):379-82
76
CA 02402930 2002-09-19
Mendoza LG, et all-ligh-throughput microarray-based enzyme-linked
imrnunosorbent assay (EL,ISA).
Biotechniques. 1999 Oct; 27(4):778-80. 782-6, 788. Morozov VN, Morozova TYa
Electrospray deposition
as a method for mass fabrication of mono- andmulticomponent microarravs of
biological and biologically
active substances.Anal Chem. 1999 Aug 1; 71 ( 15):3 I l0-7.619: Lucking A, et
alProtein microarrays for
gene expression and antibody screening. Anal Biochem. 1999 May 15; 270( 1
):103-1 I . Silzel JW, et
alMass-sensing, multianalyte microarray immunoassay with imaging detection.
Clin Chem. 1998 Sep;
44(9):2036-43 Ekins RP.Ligand assays: from electrophoresis to miniaturized
microarrays.Clin C.'hem. 1998
Sep; 44(9):2015-30.
All publications referred to herein are indicative of the level of skill of
those in the art to which the
invention pertains.
With respect to lymphatic vessel associated li~~ands see also US 5, 776,755
(17t4), Mod Pathol 2000
Feb; 13(2):180-5; EMBO J 2001 Mar 15;20(6):1223-1231, Nat Med 2001
Feb;7(2):199-205 Inhibition of
lymphangiogenesis with resulting lymphedema in trans~~enic mice expressing
soluble VEGF receptor-3
(VEGFR-3). J Pathol 2001 Feb; 193(2):147-54 Localization of vascular
endothelial growth factor-D in
malignant melanoma suggests a role in tumour angio~~enesis.
With respect to technolo~~ies having application herein see also Immunity 2001
Apr; 14(4):437-46 The
immunological barrier to xenotransplantation.Cascalho M, Platt JL.; WO
01!43779; WO 01/42285; WO
98/10795: WO 01!40803: WO 00/14212; Gastroenterology 2001 May;l20(6):1330-8 An
engineered human
antibody to TNF (CDP571 ) for active Crohn's disease: a randomized double-
blind placebo-controlled trial.
Sandborn WJ: WO 01!44282: WO 01140309; WO O1i40274; WO 01/44300; Ann Rheum Dis
2001
May;60(5):433 Cancer and autoimmunity: autoimmune and rheumatic features in
patients with
mali~~nancies, Abu-Shakra M, et al.; WO 01;'40468; WO 01/40307; WO 01;42297;
WO 01!42294; WO
01/42296; WO 01!40456; WO 01;40308; WO 01'42306: Curr Opin Immunol 2001
Apr:l3(2):134-40
Immunity against cancer: lessons learned from melanoma. Houghton AN. Gold JS,
Blachere NEB.; WO
01!42288; WO 01/42288; WO 01!43771; WO 01/42308; WO 01!41804; 'vV0 01,'39722;
WO 01;44808;
WO 01/43770; WO 01'16166; WO 01/41803; WO O1/ 131 10; WO 00/32 7 52; WO
98'33528; WO
01/43695; J Am Pharm Assoe (Wash) 2001 May-Jun;41 (3):383-91 Magic: bullets
finally find their mark.;
Leukemia 2001 Apr; I 5(4 ):675-6; WO O 1''44301; Anticancer Res 2001 Jan-Feb;2
I ( 1 B):621-7
Immunotherapy for recurrent colorectal cancers with human monoclonal antibody
SK-I. Koda K et al.;
WO 01!1091 l; WO O1i42506: Int J Clin Pract 2001 Apr;55(3):211-6'Tumour
necrosis factor as a
therapeutic target in rheumatoid arthritis and other chronic inflammatory
diseases: the clinical experience
with infliximab; WO Ol /44472_ WO O 1/40302: WO 01 40305)
With respect to surface plasmon resonance measurements ofaftmity see LJS611
1652:High throughput
surface plasmon resonance analysis system 1JS06208422 Surface plasmon sensor
Ef01080365 SURFACE
PLASMON RESONAN(,E SENS012 FOR THE SIMULTANEOUS MEASUREMEN f OF A
PLURALITY OF SAMPLES IN FLUID FORM W000106236A HIGH THROUGHPUT ANALYSIS OF
MOLECULAR INTERACTION USING SURFACE PLASMON RESO1JANCE High throughput surface
plasmon resonance analysis system as well as Dimensions of antigen recognition
and levels of
immunological specificity. Adv Cancer Res. 2001;80:147-87. Use of optical
biosensors for the study of
mechanistically concerted surface adsorption processes. Anal Biochem. 2001 Jan
15;288(2):109-25
Experimental design for analysis of complex kinetics using surface plasrr~on
resonance. Methods. 2000
Mar;20(3):310-8. , and references cited in the foregoing references.
27: Skeie GO, Lunde PK, Sejersted OM, Mygland A, Aarli JA, Gilhus NE.
Autoimmunity against the
ryanodine receptor in myasthenia gravis. Acta Physiol Scand. 2001 Mar;171
(3):379-84. 28: Haufs MG.
Haneke E. Epidermolysis bullosa acquisita treated with basiliximab, an
interleukin-2 receptor antibody.
Acta Derm Venereol. 2001 Jan-Feb;81 ( I ):72. Woo EY, et al Regulatory
CD4(~)CD25(+) T cells in tumors
from patients with early-stage non-small cell lung cancer and late-stage
ovarian cancer. Cancer Res. 2001
Jtrn 15;61(12):4766-72. Barrera P, Joosten l~A, den Broeder AA, van De Putte
LB, van Riel Fl., van Den
Berg WB. Effects of treatment with a fully human anti-tumour necrosis factor
alpha monoclonal antibody
on the local and systemic homeostasis of interleukin I and TNFalpha in
patients with rheumatoid arthritis.
Ann Rheum Dis. 2001 Ju1;60(7):660-9. Nicholson JK, Browning SW. Hengel R1_.
Lew E, Gallagher 1..E,
Rimland D, McDougal JS. CCRS and CXCR4 expression on memory and naive T cells
in HIV-I infection
and response to highly active antiretroviral therapy. J Acquir Immune Defic
Syndr. 2001 Jun 1:27(2):105-
15. Kung SK. Su RC, Shannon J. Miller RG. Characterization of four new
monoclonal antibodies that
77
CA 02402930 2002-09-19
recognize mouse natural killer activation receptors. Hybridoma. 2001
Apr;20(2):91-101. 43: Bank I,
Dardik R, Levy V, Goldstein I, Shoham J. Differential expression and
regulation of CD6 on T-cell subsets
revealed by monoclonal antibody (MAb) CH I I . Hybridoma. 2001 Apr;20(2):75-
84.
With respect to making multifunctional ligands see also USP 5,731,168 and
5,821,333.
With respect to TN E' and TNFR variants, and functional fragments thereof, for
use as antibody targets and
binding moieties with respect to various aspects of the invention herein see
WO 00/67793, WO O l /30300,
WO 01/49321, WO 00/62790, WO 01!03720, WO 00/60079, WO 97/46686, WO 01/41803,
w'0 01/38526,
WO 01/37874, WO 01/12812, WO 01!12671, WO 01!05834, WO 01/03720, WO 00/77191
WO 00/73321,
WO 00/71 150, WO 00/67793, WO 00!67034, WO 00/66608. WO 00/66156., WO 012481
l, as well as
references cited therein. Many other TNFR variants and TNF analogs are known
in the art.
With respect to cytokines and cytokine receptors see also the latest editions
of Cytokine Reference: A
Compendium of Cytokines and Other Mediators of Host Defense by Joo,t J.
OppE:nhe im (F_ditor), J;nn
Vileck, Moos ~A. Nicoln (Editor); Cytokine Molecular Biology : A Practical
Approach by I=rarrcc~> R.
Balk4vill (Editor), ("ran I3alkwill (Editor); Guidebook to Cytokines and Their
Receptors by Nicos Nicola
(Editor); The Cytokine Network and Immune Functions by Jacqr~os ThL~:e; Novel
Cytokine inhibitors by
Gerrv ,1. C-li<~«s (Editor). 3rian I-Ienderson (Editor); Homology Folding of
Proteins : Application to
Cytokine Engineering by Sublr~rstrioi Srinivas~io; Cytokines and Cytokine
Receptors (2001 ); International
Review of Experimental Pathology : Cytokine-Induced Pathology, Pan E3 :
Inflammatory Cytokines,
Receptors, and Disease by C~.~~~. Richter, Kio~ Soiez (Editor).
With respect to antibodies that bind to CCRS see Mol Biol Cell 2002 Feb; 13(2)
: 723-737
With respect to variations in chemokine receptors, cytokine and other
receptors that can be exploited
according to one or more aspects of the invention herein see I : Csaszar A,
Abel T. Receptor polymorphisms
and diseases.Eur J Pharmacol. 2001 Feb 23;414(1):9-22. 2: Gibejova
A.(~hemokine receptors.Acta Univ
Palacki Olomuc Fac Med. 2000;143:9-18. 3: Nishimoto N, Kishimoto T, Yoshizaki
K.Anti-interleukin 6
receptor antibody treatment in rheumatic disease.Ann Rheum Dis. 2000 Nov;59
Suppl I:i21-7. 4: Aggarwal
BB.Tumour necrosis factors receptor associated signalling molecules an<i their
rolein activation of
apoptosis, JNK and NF-kappaB.Ann Rheum Dis. 2000 Nov;59 Suppl l :i6-16. 5:
Grignani G, Maiolo
A.Cytokines and hemostasis.Haematologica. 2000 Sep;85(9):967-72. 6: Idriss HT,
Naismith JH.TNF alpha
and the TNF receptor superfamily: structure-function relationship(s).Microsc
Res Tech. 2000 Aug
1;50(3):184-95. 7: van Deventer SJ.Cytokine and cytokine receptor
polyvmorphisms in infectious
disease. Intensive Care Med. 2000;26 Suppl 1:S98-102. 8: Gessner A,
Rollinghoff M.Biolo~~ic functions and
signaling of the interleukin-4 receptor complexes.lmmunobiology. 2000 Jan;201
(3-4):285-307 9: Platanias
LC, Fish EN.Signaling pathways activated by interferons.Exp Hematol. 1999
Nov;27( 1 1):1583-92. 10:
Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner
AJ.Hematopoietic,
immunomodulatory and epithelial effects of interleukin-1 l.Leukemia. 1999 Sep;
13(9):1307-15. 1 I: Blasi
F.The urokinase receptor. A cell surface, regulated chemokine.APMIS. 1999
Jan;107( 1):96-101. 12:
Izuhara K, Shirakawa T.Signal transduction via the interleukin-4 receptor and
its correlation with atopy.lnt
J Mol Med. 1999 Jan;3( I ):3-10. 13: Tsokos GC, Liossis SN.Lymphocytes,
cytokines, inflammation, and
immune trafficking.Curr Opin Rheumatol. 1998 Sep;lO(5):417-25. 14: Morishita
R, Nakamura S, Hayashi
S, Aoki M, Matsushita I I, Tomita N, YamamotoK, Mori~uchi A, Higaki J, Ogihara
T.Contribution of a
vascular modulator, hepatocyte growth factor (EIGF), to thepathogenesis of
cardiovascular disease.)
Atheroscler Thromb. 1998;4(3):128-34. 15: Kashiwamura S, Okamura Hf.[IL-18 and
IL-18
receptor].Nippon Rinsho. 1998 Ju1;56(7):1798-806. Japanese.l6: Paxton WA,
Kan<~ S.Chemokine receptor
allelic polymorphisms: relationships to HIV resistance anddisease
progression.Semin Immunol. 1998
Jun;lO(3):187-94. 17: Arend WP, Malyak M. Guthridge CJ, Gabay C.Interleukin-I
receptor anta;~onist:
role in biology.Annu Rev Immunol. 1998;16:27-55. 18: Camussi G, Lupia E.The
future role of anti-tumour
necrosis factor (TN F) products in the treatmentof rheumatoid arthritis.
Drugs. 1998 May;55(5):613-20. 19:
Taga T, Kishimoto T.Gp 130 and the interleukin-6 family of cytokines.Annu Rev
Immunol. 19~>7;15:797-
819. 20: Paul WE.fnterleukin 4: signalling mechanisms and control of T cell
differentiation.Ciba Found
Symp. 1997;204:208-16: discussion 216-9; 1.(W0 01!49321) TNF INHIBITORS FOR
THE
TREATMENT OF NEUROLOGICAL, RETINAL AND MUSCULAR DISORDERS2.(WO 01/46261)
METHOD FOR TREATING INFI_AMMATION3.(WO O1i40464) INTIRLEEJK1N-I-RECEPTOR
ASSOCIATED KINASI?-3 (IRAK3) AND IT S USE IN PROMOTION OR INHIBITION OF
ANGIOGENESIS AND CARDIOVASCULARI7ATION4.(WO 01/40464) INTERLEUKIN-I-
RECEPTOR ASSOCIATED KINASE-3 (IRAK3) AND ITS USE: IN PROMOTION OR INHIBITION
OF
78
CA 02402930 2002-09-19
ANGIOGENESIS AND CARDIOVASCULARIZATIONS.(WO 01!30850) UMLR
POI_YPEPTIDES6.(WO 00!77195) NUCLEIC ACID ENCODING NOVEL EGF-LIKE GROWTI-I
FACTORS7.(WO 00/74719) METHOD OF TREATING CARCINOMA USING ANTIBODY' THERAPY
AND AMELIORATING SIDE EFFECTS ASSOCIATED WITH SUCH THERAPYB.(WO 00/02582)
TREATMENT OF CELIAC. DISEASE WITH IN~TERLEUKIN-15 ANTAGONISTS9.(WO 99/47170)
PREVENTIVES OR REMEDIES FOR INFLAMMATORY INTESTINAL DISE?ASES CONTAINING
AS THE ACTIVE INGREDIENT IL-6 ANTAGONISTS 10.( WO 99/46376) RECEP~'OR FROM THE
SUPERFAMILY OF TNT-RECEPTORS FROM THE. HUMAN LUNG l 1.(W0 99/43809) PROTEASE-
AC;TIVATED RECEPTOR 4 AND USES THEREOF 12.( WO 98,148017) FAMIf.Y OF
IMMUNOREGULA'TORS DESIGNATED LEUKOCYTE IMMUNOCiI_OBULIN-LIKE RECEPTORS
(LIR)13.(WO 98/47923) II_-5R ANTAGONISTS FOR TREATMEN~h OF INFLAMMATION',
ASTHMA
AND OTHER ALLERGIC DISEASES14.(Vv'O 98/46620) A NOVEL HUMAN G-PROTEIN COUPLED
RECEPTOR15.(WO 98-46265) METHODS FOR USING ANTAGONISTIC ANTI-AVB3 INTEGRIN
ANTIBODIES16.(WO 98136767) MODULATION OF THE HYPOTHALAMIC-PITUI'TARY-
ADRENAL-ADIPOSE AXIS WITH LEPT1N RECEPTOR LIGANDS 17.(W0 98/31809) HC1MAN CC
CHEMOKINE SLC18.( WO 98;30706) COMPOUNDS. COMPOSITIONS AND METHODS FOR THE
ENDOCYTIC PRESENTATION OF IMMIJNOSUPPRESSIVE FACTORS19.(WO 98/24817) NOVEL
DNA, NOVEL PROTE IN, AND NOVEL ANTIBODY20.(WO 98/22499) NEURON AND NEURAL
TUMOUR GROWTH REGULATORY SYSTEM, ANTIBODIES THERETO AND USfS
THEREOF21.(W0 98/19706) IDENTIFICATION OF UNIQUE BINDING IN fERACfI'IONS
BETWEEN
CERTAIN ANTIBODIES AND THE HUMAN B7.1 .AND B7.2 CO-S~~IMULATORY
ANTIGENS22.(WO 98' 18456) PROTEASE-ACTIVATED RECEPTOR 3 AND USES THEREOF23.(WO
98!14480) G PROTEIN-COUPLED RECEPTOR ANTAGONISTS 24.( WO 98/02541 ) GAMMA-
HEREGULIN25.(WO 97/49818) G-BETA-GAMMA REGULATED PHOSPHATIDYLINOSIT'OL-3'
KINASE26.(WO 97/48804) TlE-2 RECEPTOR L1GANDS (TIE LIGAND-3; TIE LIGAND-4) AND
THEIR USES27.(WO 97/41225) MAMMALIAN MIXED I..YMPHOCYTE REC'EP fORS, CHEMOKINE
RECEPTORS [M.MLR-CCR]28.(WO 97/24373) MONOCLONAL ANTIBODY ANTAGONISTS TO
HAEMOPOIETIC GROWTH FACTORS29.(WO 97'21732) DESIGN OF HORMONE-L,IKI?
ANTIBODIES WITH AGONISTIC AND ANTAGONISTIC FUNCTIONS, 6,235,880 Human
sulfonylurea receptor 6.221,660 DNA encoding SNORF25 receptor 6, 214,797
Urocortin peptides, nucleic
acid encoding same methods for usingsame6,214,344 Hepatocyte growth factor
receptor antae.onists and
uses thereof 6,210,904 Anticoagulant test6,207, 152 Hepatocyte ~~rowth factor
receptor antagonists and
uses thereof6,204,017 folynucleotide encoding a histamine receptor 6,197,541
Recombinant thrombin
receptors and assays using them6, l 84,358 IP-10/Mig receptor designated
CXCR3, antibodies, nucleic
acids,and methods of use therefor6,177,079 Antagonists of interleukin-
156,177,078 Monoclonal antibody
antagonists to IL-36,177,077 TNT inhibitors for the treatment of neurological
disorders 6,171,815 Human
sulfonylurea receptor6,168,783 Antagonists of interleukin-156.166,185
Antibodies to human TIE-2
ligands6,165,466 Anta~~onists of interleukin-156,162,431 Serinelthreonine
protein kinase6,143,870
Thrombin receptor homolog6,136.957 Antibodies which bind granulocyte-
macrophane colony-
stimulatingfactor receptor6,124,101 Recombinant thrombin receptor and related
phannaceuticals6, I I 1,075
Protese-activated receptor PAR4 (ZCHEMR2) 6.103,874 Human KDEL receptor
6,096,873 Gamrna-
here'~ulin 6,086,874 Antitumor agent effect enhancer containing IL-6
anta~Tonist56,084.075 Agonist and
antagonist antibodies to the chemokine receptor-2(CCR2) 6,063,596 G-protein
coupled receptors
associated with immune response6,054,292 T-cell receptor protein6,04~~,212
Recombinant C140 receptor,
its agonists and antagonists, andnucleic acids encoding the receptor6,033.869
Polynucleotide encoding a
novel human cytokine; steroid receptor 6,024,936 Antibody-based method of
localizin~~ activated
thrornbinreceptors 6,017,763 G-beta-gamma regulated phosphatidylinositol-3'
kinase6.013,480
Antagonists of ir~terleukin-156,013,479 Human Emrl-like G protein coupled
receptor 5.994,097
Polynucleotide encoding human G-protein coupled receptor5,985,828 Mammalian
receptors for
interleukin-10 (IL-10) 5.985,583 Cloning and expression of gonadotropin-
releasin«
hormonereceptor5,977,072 High affinity immunoglobulin E receptor-like
protein5,976,852 K.kappa./µ-
like protein tyrosine phosphatase. PTP .lambda.5,976,815 Bioassay using ALK-7,
a novel serine threonine
kinase receptor 5,972,621 Methods of identifying compounds that modulate body
weightusing the OB
receptor 5.965,709 IgE antagonists5,965,365 Serine!threonine protein
kinase5,955,303 Human chemokine
receptor-like protein 5.952,175 DNA encoding a human progesterone receptor
complex p23-
Iikeprotein5,945,308 Human oxidized LDL receptor5.942,606 Viral receptor
protein 5.928.887
.kappa.!µ-Like protein tyrosine phosphatase, PTP .lambda.5,912, 144 Edb I-
receptor homolog5,902,585
Methods of inducing T cell unresponsiveness to donor tissue ororgan in a
recipient with GP39 antagonists
5,892,014 DNA encoding a protease-activated receptor 35,891,720 Isolated DNA
encoding a novel human
G-protein coupled receptor5,891,674 Insulin receptor tyrosine kinase substrate
5.891,638 Serine threonine
79
CA 02402930 2002-09-19
kinase receptor, alk-75,888,81 1 Corticotropin-releasin« hormone
receptor5,888,510 Chronic rheumatoid
arthritis therapy containing IL-6 antagonistas effective component 5,886,148
Parathyroid hormone
receptor5.874,400 Recombinant C 140 receptor, its agonists and antagonists,
andnucleic acids encoding the
receptor 5,874,273 G-beta-gamma regulated phosphatidylinositol-3'
kinase5,874,224 Growth factor
receptor binding protein 5,871,930 High affinity immunoglobulin E receptor-
like protein 5.869,633
Thrombin receptor homology polynucleotide5,869,609 G protein coupled glutamate
receptors5,869,271 G-
beta-gamma regulated phosphatidylinositol-3' kinase5,869,049 Methods of
inducin~~ T cell
unresponsiveness to bone marrow withgp39 antagonists 5,863,796 Antibodies
which specifically bind
mammalian receptors forinterleukin-10 (IL-10)5,863,766 Human sigma receptor
5,859,201 G-beta-gamma
regulated phosphatidylinositol-3' kinase5.856,448 Antibodies specifically
reactive with thrombin receptor
and itscomponents 5,856.133 G-beta-gamma regulated phosphatidylinositol-
3'kinase5,856, l3:>_ G-beta-
gamma regulated phosphatidylinositol-3' kinase5,851,797Tie ligand-3, methods
of making and uses
thereof5,840,853 Parathyroid hormone receptor and DNA encoding same5,837,499
DNA encoding C5A
receptor antagonists having substantially noagonist activity and methods of
expressing sarne5,834,240
DNA encoding a transforming growth factor-.beta. receptor associated protein
5,833,987 Treatment of T
cell mediated autoimmune disorders5,831,047 Oli~~onucleotide probes to L-AP4
sensitive glutamate
receptorsequences 5,830,678 Method for identifying a target peptide that
modulates thebinding of
epinectin ligand to inte'~rin receptors5,824,500 Nucleic acid encodin~~ novel
human KDEI_ receptor
5,817,480 DNA encoding a histamine H2 receptor5.814,507 .kappaJµ-like
protein tyrosine phosphatase,
PTP .Iambda.5,814,464 Nucleic acids encodin~~ TIE-2 li'~and-25.81 1,24:>
Antibodies that specifically bind
to ALK-7, a novel serinethreonine kinase receptor5,807.824C5 A receptor
anta~~onists having substantially
no agonistactivity5,795.966 Antagonists of interleukin-155,789,565 Serine
threonine kinase receptor,
ALK-7 5,789,192 Mammalian receptors for interleukin-10 (IL-10)5,763,575
Agonist and antagonist
peptides of the C140 receptor 5,759,994 Recombinant thrombin receptor and
related
pharmaceuticals5.750,366 Cloniny~ and expression of y~~onadotropin-releasing
hormonereceptor 5,747,279
Nucleic acid molecules encoding kappa<sub>3</sub> opioid receptors,receptors encoded
thereby, and uses
thereof5,747,267 Method for identifying a G protein coupled glutamate
receptoragonist and
antagonist5,738,999 L-AP4 sensitive glutamate receptors5,730,976 Method for
treatin~~ macrophage
pathogen infections by ( GF-Bantagonists5.726,036 Granulocyte-macrophage
colony-stimulating factor
receptor andderivatives thereof5,72 I ,107 Antibodies to G protein coupled
glutamate receptors5,716,804
Mammalian interleukin-10 (1I,-10) super-activating receptors:and
variants5,716,789 Method to determine
ligands, agonist and antagonist of C 140receptor 5,707,632 Receptors far
tibroblast growth
factors5,688,768 Recombinant thrombin receptor and related
pharmaceuticals5,686,597 Thrombin receptor
homology 5,686,292 Hepatocyte growth factor receptor antagonist antibodies
anduses thereof5,683,884
Methods for identifying modulators of human calcitoninmediated
metabolism5,683,693 Method for
inducing T cell unresponsiveness to a tissue ororgan graft with anti-CD=t0
li«and antibody or soluble
CD405,674.981 Human calcitonin receptor polypeptides5,674,689 Human calcitonin
receptor polypeptides
and methods of use5,646,036 Nucleic acids encoding hepatocyte growth factor
receptorantagonist
antibodies5,629,283 Granulocyte-macrophage colony-stimulating factor receptor
andderivatives
thereof5,622,839 Recombinant production of human calcitonin
receptorpolypeptides5,614,609 Serine
threonine kinase receptor5,556,780 CDNAS encoding: mouse and rat type-2
an~~iotensin II receptorsand
their expression in host cells5,543, 143 Method for activating macropha~;es-
monocytes5.516,894 A<sub>2b</sub> -
adenosine receptors5,514,555 Assays and therapeutic methods based on
lymphocytechemoattractants
5,506,107 Selecting ligand agonists and antagonists5,494,806 DNA and vectors
encodin~~ the parathyroid
hormone receptor,transfbrmed cells, and recombinant production of PTHR
proteins andpeptides5.451,658
Antagonists of human gamma interferon5,441,935 Growth factor
receptors5,385,831 Method for producing
a mammalian G protein coupled glutamatereceptor5,334,380 Anti-endotoxin,
interleukin-I receptor
antagonist andanti-tumor necrosis factor antibody with arginine-free
formulations forthe treatment of
hypotension5.256,766 Recombinant thrombin receptor and related
pharnaceuticals5,177,190 Purified C5a
receptor from human polymorphonuclear leukocytes4.857,637 Methods and
compositions for
immunologically modulating growthhormone receptor activity; and references
cited therein. (see also
1.(W0 01149744) MOUSE G-PROTEIN COUPLED RECEI''fOR MAS 2.(W0 01/49726) A NOVEL
POLYPEPTIDE-HUMAN NATRIURETIC PEPTIDE RECEPTOR 18 AND THE POI_YNUCLEOTIDE
ENCODING SAID POL,YPEPI~IDE 3.(W0 Ol~'49321) TNF INHIBI TGRS FOR TH1:
TREATMENT OF
NEUROLOGICAL,RF:fINAL ANDMUSCULARDISORDERS4.(WO01!00657)NOVEL.INDOLE
PEPTIDOMIMETICS AS THROMBIN REC.'EPTOR ANTAGONISTS 5.(W0 00!62790) SOLUBLE
'TUMOR NECROSIS FACTOR RECEPTOR TREA I'MI:NT OF MEDICAL DISORDERS 6.(W0
01/03720) PROMOTION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCUI,ARIZATION
BY TUMOR NECROSIS FACTOR LIGAND;'RECEI'TOR HOMOLOGS 7.(W0 01/46261) METHOD
FOR TREATING INFLAMMATION 8.(WOOL/46191)4-[ARYL(8-AZ.ABICYCLO[3.2.I~OCTAN-3-
CA 02402930 2002-09-19
YL)]AMINOBENZOIC .ACID DERIVATIVES 9.(W0 01;46176) NON PEPTIDE TACHYKININ
RECEPTOR ANTAGONISTS 10.(W0 01;45730) TWE?AK RECEPTOR I I.(WO 01;45703)
NITROSATED AND NITROSYLATED CYCLOOXYGENASE-2 INHIBITORS, COMPOSITIONS
AND METHODS OF USE 12.(W0 01!40464) 1NTERl,EUKIN-1-RECEPTOR ASSOCIA'T'ED
KINASE-3
(IRAK3) AND ITS USE IN PROMOTION OR INHIBITION OF ANGIOGENESIS AND
CARDIOVASCULAR1ZA'TION 13.(W0 01;'44213) NEW P2X7 RECE:F''fOR ANTAGONISTS FOR
USE
IN THE TREATMENT OF INFLAMMATORY. IMMUNE OR CARDIOVASCULAR DISEASES
14.(W0 01;'42268) DOG OREXIN 1 RECEPTOR IS.(WO 01;42208) Cl'CLOAMINE CCRS
RECEPTOR
ANTAGONISTS 16.(W0 01/41752) ISOFORM SPECIFIC INHIBITI01~I FOR TREATMENT OF
PAIN
AND REDUCTION OF ANESTHETIC THRESEiOI_f) 17.(W0 01!03730) PROMOTION OR
INHIBITION OF ANGIOGENESIS AND CARDlOV.4SCULARIZATION BY TUMOR NECROSIS
FACTOR LIGAND/REC'EPTOR HOMOLOGS 18.(W001''40464) INTI?RLEUKIN-I-RECEPTOR
ASSOCIATED KINASE=-3 (IRAK3) AND ITS tJSE IN PROMOTION OR INHIBI'T'ION OF
ANGIOGENESIS AND CARDIOVASCULARIZATION 19.(W0 01;4(1259) MONKEY OREXIN I
RECEPTOR 20.(W0 01.'40252) MONKEY CAL,CIUNI SENSING RECEPTOR 21.(W0 OIi04139)
HUMAN AXOR29 RECI?PTOR 22.(W0 01;36480) MOUSE 7-TRANSMEMBRANI: RECEPTOR,
AXOR45 23.(W0 01!00656) NOVEL INDAZOL.E PEPTIDOMIMET'ICS AS THROMBIN RECEPTOR
ANTAGONISTS 24.(W0 00;'67793) DEATH DOMAIN CONTAINING RECEPTOR 4 25.(W0
01/34645) MODULATING IL-13 ACTIVITY USING MUTATED II_-I 3 MOI_ECULE:S T'EiAT
ARE
ANTAGONISTS OR AGONISTS OF IL-13 26.(W0 O1%34138) COMPOSITIONS AND METHODS FOR
TREATMENT OF NEUROLOGICAL DISORDERS AND NE1.JRODEGENERATIVE DISE,~SES
27.(W0 01/32656) POLYMORPH1C FORM OF' A TACHYKININ RECEPTOR ANTAGONIST" 28.(W0
0(132166) NEW COMBINATION COMPRISING A 8#946;2-ADRENORECEPTOR AGONIST AND A
LEUKOTRIENE RECEPTOR ANTAGONIST 29.(W0 01!32163) NEVI'' COMBINA'flON
COMPRISING
A BETA ? (β)2 ADRENO RECEPTOR AGONIST AND A LENI<OTRIENE RECEPTOR
ANTAGONIST 30.(W0 01/01922) USE OF SUBSTANCE P AN'TAGON1STS FOR THE
TREATMEN'I~
OF ADENOCARCINOMA 31.(V1,'0 0('30850) IJMI.R POE,YPEPTIDES 32.(V1'0 01'27153)
A MURINE
SEVEN-TRANSMEMBRANE RECEPTOR, MUS MIJSCULLJS MHNEAA81 33.(WO01/25~69)
NOVEL HUMAN G-PROTEIN COUPLED RECEPTOR 34.(W0 01/24828) MODULA'T'ORS OF
CYTOKINE MEDIATED SIGNALLING PATHWAYS AND INTEGRIN αV&946;3
RECEPTOR ANTAGONISTS FOR COMBINATION T"FiERAPY 35.(W0 Ol/?4798) USE: OF
CENTRAL.
CANNABINOID RECEPTOR ANTAGONIST FOR PREPARING MEDICINES 36.(W0 O1i24797)
INTEGRIN RECEPTOR ANTAGONISTS 37.(V1'0 00,'68250) 7TM RECEPTOR RA r APJ
38.(V4'0
01/16121) I IETEROCYCLIC COMPOUNDS AND METHODS OF USE THEREOF 39.(W0 01!14406)
ANTIANDROGEN AGENTS 40.(W0 01; 12Ci71) HUMAN TUMOR NI~CROSIS FACTOR RECEPTOR
TRl6 41.(W0 01:'10891 ) IL-16 ANTAGONISTS 42.(Vv'O 01'10889) RAT-G-PROTEIN
COUPLED
RECEPTOR BRS3 43.(W0 01/10423) USE OF 5-HT3 RECEPTOR ANTAGONISTS FOR THE
TREATMENT OF INFLAMMATIONS OF 'fHE RESPIRATORY 'TRACT 44.(W0 01/07028) THE USE
OF RETINOID RECEPTOR ANTAGONISTS IN THI? TREATMENT OF PROSTATE CARCINOMA
45.(W0 01/05834) HUMAN TUMOR NECROSIS FACTOR RECEPTORS TR13 AND TR14 46.(W0
01/05783) BRADYKININ B I RECEPTOR ANTAGONISTS 47.(W0 01!04139)
POI.,YNUCL,EOTIDE
AND POLYPEPTIDE SEQUENCES OF HUMAN AXOR29 RECEPTOR AND METHODS OF
SCREENING FOR AGON(STS AND ANTAGONISTS OF TFiE INTERACTION BETVI-'EEN HUMAN
AXOR29 RECEPTOR AND ITS LIGANDS 48.(W0 01/03720) PROMOTION OR INHIBITION OF
ANGIOGENESIS AND CARDIOVASCULARIZATION BY TUMOR NECROSIS FACTOR
LIGAND%RECEPTOR HOMOLOGS 49.(W0 01'01922) USE OF SUBSTANCE P ANTAGONISTS IN
THE TREATMI~NT OF TEIE ADENOCARC.'INOMAS 50.(W0 01''00659) BENZIM1DAZOLONE
PEPT1DOMIMETTCS AS THROMBIN RECEPTOR ANTAGONISTS 51.(VVO 01!00657) NOVEL
INDOLE PEPT1DOMIMETICS AS THROMBIN REC.'EPTOR ANTAGONISTS 52.(W0 01:00656)
NOVEL INDAZOLE PI~PTIDOMIMET(CS .AS THROMBIN RECEPTOR ANT'AGONIS'TS 53.(W0
01!00576) INDOLE AND 1NDAZOLE UREA-PEPT'C)IDS AS TFIROMBIN RE:CEP'fOR
ANTAGONISTS 54.(V1'0 Ol %00198) COMPOSITIONS AND METHODS OF TREATING CANCER
USING COMPOSITIONS COMPRISING AN 1NHIB1'fOR OF ENDOTHELIN RECEI' fOR ACTIVITY
55.(W0 00/78317) INTEGRIN RECEPTOR AN TAG(7N1ST'S 56.(W0 00/77195) NUCLEIC
ACID
ENCODING NOVEL EGF-LIKE GROWTH FACTORS 57.(W0 00.'76502) ME'T'HODS AND
COMPOSITIONS FOR TREATING RAYNAUD'S PHENOME?NON AND SC'LERODERM.4 58.(W0
00/74719) METETOD OF TREATING CARCINOMA USING ANTIBODY TIiERAPY AND
AMELIORATING SIDE EFFECTS ASSOCIATED WITH SUCH THERAPY 59.(W000!73321)
HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR10 60.(W0 00;'72801 ) ALPHA V INTEGRIN
RECEPTOR ANTAGONISTS 61.(W0 00/71150) TUMOR NECROSIS FACTOR RECEI''fOR 5
62.(W0
81
CA 02402930 2002-09-19
00.!69831) SPIROIMIDAZOLIDINE DERIV,A'fIVES. THEIR PREPARATION, TIiEIR USE:
AND
PHARMACEUTICAL I'REPARA'fIONS COMPRISING THEM 63.(W0 00/69820) CYCLIC' AMINE
DERIVATIVES AND THEIR USES 64.(W0 00!69463) COMPOSITIONS AND METHODS FOR
TREATING CELL PROLIF'ERA'I ION DISORDERS 65.(W0 00/69459) TREATMENT OF
REFRACTORY HUMAN -TUMORS WITI~ EPIDERMAL GROWTH FACTOR RECEPTOR
ANTAGONISTS 66.(W'0 00/68250) 7TM RECEPTOR RAT APJ 67.(W0 00!68244) 7'fM
RI?CEPTOR
MOUSE APJ 68.(W0 00/67793) DEATH DOMAfN CONTAINING RE?CEPTOR 4 69.(W0
00/67034)
METHODS OF USE OF THE TACI/TACI-1. INTERACTION 70.(W0 00/67024) CANCER
TREATMENT WITFI ENDOTHELIN RECEPTOR ANTAGONISTS 71.(W0 00/66632) AGONISTS OR
ANTAGONISTS FOR IiAEMOPOIE'TIC GROWTH FACTORS 72.(W0 00/66522)
GLUCOCORTICOID RECEPTOR MODUC,ATORS 73.(W0 00/66156) DEATH DOMAIN
CONTAINING RECEPTOR 5 74.(W0 00/64465) DEATH DOMAIN CONTAINING RECEPTORS
75.(W0 00;'62790) SOLUBLE TUMOR NECROSIS FAC~I"OR RECEPTOR TREATMENT OF
MEDICAL DESORDERS 76.(W0 00/59532) THE USE OF DOMAINS OF 'TYPE IV COLLAGEN T
INHIBIT ANGIOGENESIS AN TUMOUR GROWTH 77.(W0 00!56862) HUMAN TUMOR
NECROSIS FACTOR RECEPTOR TR9 78.(W0 00/56405) HUMAN TUMOR NECROSIS FACTOR
RECEPTOR-LIKE 2 79.(W0 00/54772) AMYOTROPIC LATERAL SCLEROSIS TREATMENT WITH
A COMBINATION Of R1LUZOLE AND AN AMPA RECEPTOR ANTAGONIST- 80.(W0 00/53596)
IMIDAZOLE COMPOUNDS SUBS T(TUT'ED WI'fH A SIX OR SEVEN MEMBERED
HETE:ROCYCLIC RING CON'I AIMING TWO NI'I ROGEN ATOMS 81.(W0 00/5 3175)
COMPOUNDS
AND METHODS 82.(W'0 00/52028) TUMOR NECROSIS FACTOR RECEPTORS 6&agr; and
6&bgr;
83.(W0 00/51974) ALPHA-AMINOACETIC ACID DERIVA (IVES USEFUL AS ALPHA 4 BETA 7 -
RECEPTOR ANTAGONISTS 84.(W0 00/50459) HUMAN TUMOR NECROSIS FACTOR RECEPTOR-
L1KE PROTEINS TRI I, TRl 1SV1, AND TRl ISV2 85.(W0 00/49170) MURINE I lcby
RECEPTOR
86.(W0 00/48603) DIBE.NZO-AZEPINE DERIVATIVES AS &abr;V INTEGRIN RECEf''I~OR
ANTAGONISTS 87.(W0 00!48597) SYSTEMIC USE OF 5-f-I~I~ 3 RECEPTOR ANTAGONISTS
AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88.(W0 00148581) USE OF 5-HT3
RECEPTOR ANTAGONISTS 89.(W0 00/46343) SCREENING ASSAY FOR ANTAGONISTS OF
FGFR-MEDfATED MALIGNANT CELL TRANSFORMATION AND TUMOR FORMATION 90.(W0
00/46215) BENZAZEPINE DERIVATIVES AS ALPHA-V INTEGRIl\i RECEPTOR ANTAGONISTS
91.(W0 00/46197) INDOLE DERIVATIVES AND THEIR USE AS MCP-I RECEPTOR
AN~fAGONISTS 92.(W0 00!44763) COMPOSITIONS FOR TREATII'lG INFLAMMATORY
RESPONSE 93.(W0 00143031 ) TUMOR NECROSIS FACTOR AN~I~AGONIS TS AND THEIR USE
IN
ENDOMETRIOSIS 94.(W0 00%43852) COMPOUNDS AND ME'fIiODS 95.(W0 00/40716)
SOLUBLE
RECEPTOR BR43x2 AND METFIODS OF USING 96.(W0 00/40239) COMPOUNDS AND METHODS
97.(W0 00/39166) NOVEL FIYAI_URONAN-BINDING PROTF~INS AND ENCODING GENES
98.(W0
00/37462) NON-PEPTIDE NK I RECEPTORS ANTAGONISTS 99.(W0 00;35887) VITRONEC'TIN
RECEPTOR ANTAGONIST PF-IARMACELJTICALS 100.(W0 00/354'72) VITRONECTIN RECEPTOR
ANTAGONIST PHARMACEUTICALS 51.(W0 01'00657) NOVEL II'JDOLE PEPTIDOMIMETICS AS
THROMBIN RECEPTOR ANTAGONISTS 52.(W0 01!00656) NOVEL INDAZOI.E
PEPTIDOMIMETICS r1S TI-IROMBIN KECEPTOR AN'TAGONIS'TS 53.(W0 01/00576) INDOLE
AND INDAZOLE UREA-PEP'fOIDS AS THROMBIN RECEPTOR ANTAGONISTS 54.(W0
01100198) COMPOSITIONS AND METI-IODS OF TREA fING CANCER USING COMPOSITIONS
COMPRISING AN INE-IIBITOR OF ENDOTHELIN RECEPTOR ACTIVITY 55.(W0 00/78317)
INTEGR1N RECEPTOR ANTAGONISTS 56.(W0 00/77195) NUCLEIC ACID ENCODING NOVEL
EGF-LIKE GROWTFI I~AC fORS 57.(W0 00/76502) METHODS AND COMPOSITIONS FOR
TREATING RAYNAUD'S PHENOMENON AND SCI.ERODERMA 58.(W0 00!74719) MI?THOD OF
TREATING CARCINOMA USING ANTIBODY THERAPY AND AMELIORATING SIDE; EFFECTS
ASSOCIATED WITFI SUCH THERAPY 59.(W0 00 73331 ) HUMAN 'TUMOR NECROSIS FACTOR
RECEPTOR TRIO 60.(W0 00;72801 ) ALPHA V 1NTEGRIN RECEPTOR ANTAGONISTS 61.(W0
00/71 150) TUMOR NECROSIS FACTOR RECEPTOR 5 62.(W0 00169831)
SPIROIMIDAZOLIDINE
DERIVATIVES, THEIR PREPARATION, 'THEIR USE AND PHARMACEUTICAL PREPARATIONS
COMPRISING THEM fi3.(WO 00/69820) CYCLIC AMINE DERIVATfVES AND 'THEIR F:SES
64.(W0 00.'69463) COMPOSITIONS AND METHODS I~OR TREAT11~1G CELL, PROLIFERATION
DISORDERS 65.(W0 00/69459) TREATMENT OF KEFRAC'TORY I-IUMAN TUMORS WITFI
EPIDERMAL GROW'TI1 FACTOR RECEPTOR ANTAGONISTS 66.(W0 00/68350) 7TM RECEPTOR
RAT APJ 67.(W0 00~'68?44) 7TM RECEPTOR MOUSE APJ 68.(W0 00%67793) DE-:ATH
DOMAIN
CONTAINING RECEPTOR 4 69.(W0 00!67034) METHODS OF USE OF THE 'TACI/TACI-I.
INTERACTION 70.(W0 00;'67024) CANCER TREATMENT WITFI ENDOTHELIN RECEPTOR
ANTAGONISTS 71.(W000%66632) AGON1SI'S OR ANTAGONISTS FOR HAEMOPOIETIC
82
CA 02402930 2002-09-19
GROWTH FACTORS 72.(W0 00/66522) GLUCOCORTICOID RECEPTOR MODULATORS 73.(W0
00/66156) DEATH DOMAIN CONTAINING RECEPTOR 5 74.(W0 00/64465) DEATH DOMAIN
CONTAINING RECEPTORS 75.(W0 00/62790) SOLUBLE TUMOR NECROSIS FACTOR
RECEPTOR TREATMENT OF MEDICAL DESORDERS 76.(W0 0059532) THE USE OF DOMAINS
OF TYPE IV COLLAGEN T INHIBIT ANGIOGENESIS AN TUMOUR GROWTH 77.( WO 00/56862)
HUMAN TUMOR NE('ROSIS FACTOR RECEPTOR TR9 78.(W0 0(1/56405) HUMAN TUMOR
NECROSIS FACTOR RECEPTOR-LIKE 2 79.(W0 00/54772) AMYOTROPIC LATERAL SCLEROSIS
TREATMENT WITH A COMB1NA'TION OF RILUZOLE AND AN AMPA RECEPTOR
ANTAGONIST 80.(W0 00/53596) IMIDAZOLE COMPOUNDS SUBSTITUTED WITH A SIX OR
SEVEN MEMBERED HETEROCYCLIC RING CONTAINING TWG NITROGEN ATOMS 81.(W0
00/53175) COMPOUNDS AND METHODS 82.(W0 00/52028) TUMOR NECROSIS FACTOR
RECEPTORS 6&a~r; and 6&:b~r; 83.(W0 00/51974) ALPHA-AMINO.ACETIC ACiD
DERIVATIVES
USEFUL AS ALPHA 4 BETA 7 - RECEPTOR ANTAGONISTS 84.(W0 00/50459) I-IUMAN TUMOR
NECROSIS FACTOR RECEPTOR-LIKE PROTEINS TRI I, TR11SV1, AND TRl ISV2 85.(W0
00/49170) MURINE I lcby RECEPTOR 86.(W0 00/48603) DIBENZO-AZ.EPINE DERIVATIVES
AS
&agr;V 1NTEGRIN RECEPTOR ANTAGONISTS 87.(W0 00/48597) SYSTEMIC USE OF 5-HT 3
RECEPTOR ANTAGONIST"S AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88.(W0
00/48581) USE OF 5-H'I'3 RECEPTOR ANTAGONIS'T'S 89.(W0 00/46343) SCREENING
ASSAY FOR
ANTAGONISTS OF F(uFR-MEDIATED MALIGNANT" CELL TRAI'lSFORMATION AND TUMOR
FORMATION 90.(W0 00/46215) BENZAZEPINE DERIVATIVES AS ALPHA-V (NTEGRIN
RECEPTOR ANTAGONISTS 91.(W0 00/46197) IN DOLE DERIVATIVES AND THEIR USE AS
MCP-1 RECEPTOR ANTAGONISTS 92.(W0 00/44763) COMPOSITIONS FOR TREAT"ING
INFLAMMATORY RESPONSE 93.(W0 00/43031 ) TUMOR NECROSIS FACTOR ANTAGONISTS
AND THEIR USE IN F~NDOMETRIOSIS 94.(W0 00/42852) COMPOUNDS AND METH(>DS 95.(W0
00/40716) SOLUBLE RECEPTOR BR43x2 AND METHODS OF USING 96.(W0 00/40239)
COMPOUNDS AND METHODS 97.(W0 00/39166) NOVEL HYALCRONAN-BINDING PROTEINS
AND ENCODING GENES 98.(W0 00/37462) NON-PEPTIDE NK 1 RECEPTORS ANTAGONISTS
99.(W0 00/35887) VITRONECTIN RECEPTOR ANTAGONIST PHARMACEUTICALS 100.(W0
00/35492) VITRONECT IN RECEPTOR ANTAGONIST PHARMACEUTICALS 101.(W0 00/35488)
VITRONECTIN RECEI''TOR ANTAGONIST PHARMACEUTICALS 102.(W0 00.-35455)
HETEROARYL-ARYL UREAS AS IGF-l RECEPTOR ANTAGONISTS 103.(W0 00/32578)
BEN7.IMIDAZOLE COMPOUNDS THAT ARE VITKONEC1'IN RECEPTOR AN fAGONIS'F'S
104.(W0 00/28988) NI I ROSATED AND NITROSYLATED 1-12 RECEPTOR ANTAGONIST
COMPOUNDS, COMPOSITIONS AND METHODS OF USE 105.(W0 00/27421) FOCAL USE OF
SOLUBLE TUMOR NECROSIS RECEPTOR I (sTNFRI) FOR PROI'HYLAXIS AND TREATMENT
OF CORNEAL TRANSPLANT REJECTION AND OTHER DISORDERS OF'I"HE EYE 106.(W0
00/25805) VASCULAR ENDOTHELIAL GROWTH FACTOR-LIKE PROTEIN FROM ORE VIRUS
NZ2 BINDS AND ACT"IVATES MAMMALIAN VEGF RECEPTOR-2 107.(W0 00/25745]
IRRIGATION SOLUTION AND METHOD FOR INHIBITION OF PAIN AND INFLAMMATION
108.(W0 00/24395) NEW USE OF GLUTAMATE ANTAGONISTS FOR THE TREATMENT OF
CANCER 109.(W0 0023471 ) USE OF A CYTOKINE-PRODUCING LACT"OCOCCUS STRAIN TO
TREAT COLITIS I 10.(W0 00!23469) FRAGMENTS OF INSULIN-LIKE GROWTH FACTOR
BINDING PROTEIN AND INSULIN-LIKE GROW FFI FACTOR. AND USES THEREOF 111.(W0
00/23438 ) N-(IMIDAZOLYLALKYL)SUBSTI~I"U'1'ED CYCLIC AMINES AS HISTAMINE-H 3
AGONISTS OR ANTAGONISM"S I 12.(W0 00/231 13) PEPTIDE-BASED CARRIER DEV1C'ES
FOR
S'TELLATE CELLS 1 13.(W0 00/23066 ) IRRIGATION SOLUTION AND METHOD FOR
INHIBITION
OF PAIN AND INFLAMMATION I 14.(W0 00/23062) IRRIGATION SOLUTION AND METHOD
FOR INHIBITION OF PAIN AND INFLAMMATION I 15.(W0 00/2()578) A METHOD OF'
MODULATING CELL SURVIVAL AND REAGENTS USEFUL FOE: SAME I 16.(W0 00/20389)
NAPHTHALENECARBOXAMIDES AS TACHYKININ RECEPTOR ANTAGONISTS I 17.(W0
00/20371) PROSTAGLANDIN RECEPTOR LIGANDS 1 18.(W0 00!20003)
NAPHTHALENECARBOXAMIDES AS'TACHYKININ RECEPTOR ANTAGONISTS 119.(W0
00/14109) BASIC PRODUCTS HAVING ANTAGONISTIC ACTIVITY ON THE NK-I RECEPTOR
AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS 120.(W0 00/10391) THE USE OF
ADENOSINE A3 RECI~PTOR ANTAGONISTS TO INHIBIT TUMOR GROWTI-1 121.(W0 00/09503)
INTEGRIN RECEPTOR ANTAGONISTS 122.(W0 00!09152) TFIERAPEUTIC CHEMOKINE
RECEPTOR ANTAGONISTS 123.(W0 00/(18001 ) SUBSTITUTED ISOXAZOLE AS ESTROGEN
RECEPTOR MODULA'I~ORS 124.(W0 00/06169) INTEGR1N RECEPTOR ANTAGONISTS 125.(W0
00/03716)'TOP1CAL COMPOSITIONS COMPRISING AN OPIOID ANALGESIC AND AN NMDA
ANTAGONIST 126.(W0 00/02859) N-SUBSTITUTED NAPHTHALENE CARBOXAMIDI~S AS
83
CA 02402930 2002-09-19
NEUROKININ-RECEPTOR ANTAGONISTS 127.(W0 00/02582) TRI~ATMENT OF CELIAC
DISEASE WITH INTERLEUKIN-15 ANTAGONISTS 128.(W0 00!01802) PEPTIDE ANTAGONISTS
OF THE HUMAN L)ROKINASE REC.'EPTOR AND METHOD FOR SELECTING THEM 129.(W0
00/00194) OPHTHALMIC USES OF PPARGAMMA AGONISTS AND PPARGAMMA
ANTAGONISTS 130.(W0 99/65944) PEPTIDE INHIBITORS OF &agr;V&bgr;3 AND
&agr;V&bgr:5
131.(W0 99/62955) METHOD OF DESIGNING AGONISTS AND ANTAGONISTS TO EGF
RECEPTOR FAMILY 1 32.(W0 99!60015) IMIDAZOLIDINE DERIVATIVES, THE PRODUCTION
THEREOF, THF_IR USE. AND PHARMACEUTICAL PREPARATIONS CONTAINING ~'HE SAME
133.(WO 99/59635) USE OF A COX-2 INHIBITOR AND A NK-I REC.'EPTOR ANTAGONIST
FOR
TREATING INFLAMMATION 134.(W0 99'58142) IJSE OF ANTI-PR_OLACTIN AGENTS TO
'TREAT
PROL1FERATIVE CONDITIONS 135.(W0 99/58097) USE OF ANTI-PROI_ACTIN AGEN'TS'TO
TREAT PROLIFERATIVE CONDITIONS 136.(W0 99157245) METHODS OF SCREENING FOR
AGONISTS AND ANTAGONISTS OF THE INTERACTION BETWEEN THE FEUMAN KIAA0001
RECEPTOR AND L1GANDS THEREOF 137.(W0 99!51245) NON-PEPTIDE BRADYKININ
RECEPTOR ANTAGONISTS FOR I1SE~ IN TREA fINCi OPHTFiAl.MIC DISEASES AND
DISORDERS 138.(W0 99/50249) INTEGRIN ANTAGONISTS 139.(~1~0 99/49856 )
AN'FAGON1STS
FOR TREATMENT OF CDI 1lC.D18 ADHESION RECEPTOR MEDIATED DISORDERS 140.(W0
99/47170) PREVEN'LIVES OR REMEDIES FOR INFLAMMATORY INTESTINAL. DISEASES
CONTAINING AS THE ACTIVE INGREDIENT IL-6 ANTAGONIST S 141.(W0 99/47158)
THERAPEUTIC CHEMOK1NE RECEPTOR ANTAGONISTS 142.(W0 99/46376) RECEPTOR FROM
THE SUPERFAMILY OF TNT-RECEPTORS FROM THE HUMAN LUNG 143.(W0 99!45927)
VITRONECTIN RECEPTOR ANTAGONISTS 144.(W0 99/45905) PROPHYLAXIS AND
TREATMENT OF MIGRAINE I-iEADACHES W1TL1 THROMBOXA1~E SYNTHETASE INHIBITORS
AND/OR RECEPTOR ANTAGONISTS 145.(W0 99/44612) SUBSTITUTED QUINAZOLINES AND
ANALOGS AND THE L'SE THEREOF 146.(W0 99/43809) PROTEASE-ACTIVATED RECEPTOR 4
AND USES THEREOF 147.(WO 99/42464) SUBSTITUTED IMIDAZO[1,2-a:3,4-
a'~DIQUINOLINYLIUM INTERLEUKIN-8 RECEPTOR ANTAGONISTS 148.(WO 99/424(13)
SUBSTITUTED QUINOXAI,INE DERIVATIVES AS INTERLEUKIN-8 RECEPTOR ANTAGONISTS
149.(W0 99/42461) SUI3ST1TUTED QUINOXALINE DERIVATIVES AS INTE;RL.E1JKIN-8
RECEPTOR ANTAGONISTS 150.(W0 99/41257) GL.UC'OCORTICOID-SELECTIVE
ANTI INFLAMMATORY AGEN 1~S 151.(WO 99!41256) GI,UCOCORTICOID-SELEC~1~1VE ANT(-
INFLAMMATORY AGENTS 152.(W0 99/40192 ) EIUMAN RECEPTOR GPR14, AND A METHOD
OF FINDING AGONIST AND ANTAGONIST TO HUMAN AND RAT GPR14 153.(W0 99!40091)
BICYCLIC PYRIDINE AND PYRIMID1NE DERIVATIVES AS NEUROPEPTIDE Y RECEPTOR
ANTAGONISTS 154.(W0 99138532) METHODS FOR THE PREVENTION AND TREATMENT OF
FIBROSIS AND SCLEROSIS 155.(W0 99;36541) INTERLEUKIN-I RECEPTOR ANTAGONIST
BETA (IL-1 RA&bgr;) I 56.(W0 99%33806) 4-[ARYL(1'IPERIDIN-4-YL)]
AMINOBENZAMI17ES
WHICH BIND TO THI; DELTA-OPIOID RIJCEPTOR 157.(W0 99/31099) INTEGRIN R6'CEPTOR
ANTAGONISTS 158.(W0 99/3 1061) INTEGRIN RECEPTOR ANTAGONISTS 159.(WO 99/30713)
INTEGR1N RECEPTOR ANTAGONISTS 160.(W0 99!30709) INTEGRIN RECEPTOR
ANTAGONISTS 161.(WO 99129729) ANTAGONISTS OF NEUROPILIN RECEPTOR FUNCTIONAL
AND USE THEREOF 162.(W0 99.'27962) USE OF A FIBRINOGEN F;ECEPTOR-ANTAGONIST
FOR
PREVENTING DISSEMINATED INTRAVASCULAR COAGULATION 163.(W0 99/26945) 1,;,4-
THIADIAZOLES AND 1,3,4-OXADIAZOLES AS &agr; v &bgr; 3 A''dTAGONISTS 164.(V1'0
99'26943) THROMBIN RECEPTOR ANTAGONISTS 165.(W0 99/25857) TRANSGENIC MODELS OF
INFLAMMATORY DISL:ASE 166.(V1'0 99;24471) OPIATE, CANNABINOID. AND ESTROGEN
RECEPTORS 167.(W0 99,!24423) PIPERIDINE DERIVATIVES AND THEIR USE AS TACT-
IYKININ
ANTAGONISTS 168.(W0 99/24421) IMIDAZOYLALKYL SUBSTITUTED WITH A FIVE, SIX OR
SEVEN MEMBERED HETEROCYCLIC RING CONTAINING ONE NITROGEN ATOM 169.(W0
99/24406) PHENYL-ALKYL-1MIDAZOLES AS H3 RECEI'T'OR ANTAGONISTS 170.( WO
99/24405)
H 3 RECEPTOR LIGANDS OF THE PHENYL-ALKYL-IMIDAZOLES TYPE 171.(W0 99/21555)
ADENOSINE A3 RECEPTOR ANTAGONISTS 172.(W0 99/20758) HUMAN TUMOR NECROSIS
FACTOR RECEPTOR-LIKE PROTF;INS TR I I, TRI I SV1, AND TRI I SV2 173.(W0
99/19462)
ENHANCED IMMUNOCiENIC CELL. 1'OPUI.ATIONS PREPARED USING H2 RE:CEPTOR
ANTAGONISTS 174.(W0 99/17773) COMPOUNDS AND METHODS. 175.(W0 99!16465) METHOD
FOR INHfBfTING TUMOR ANGIOGENESIS IN A LIVING SUBJEC f 176.(W0 991 I 1790)
TUMOR
NECROSIS FACTOR RECEPTOR ZTNFR-6 177.(W0 99.%06049) fN T E:GR1N RECEPTOR
ANTAGONISTS 178.(WO 99,%04001 ) TUMOR NECROSIS FACTOR RECEPTOR ZTNFR-5 179.(W0
99/02499) QUINOLINE COMPOUNDS AND MEDICINAL USES THEREOF 180.(W0 99!01764)
METHOD FOR RECOGNIZING AND DETE:RMINING GNRIi RECEPTORS AND THE USE OF
84
CA 02402930 2002-09-19
GNRH AGONIS'TS AND GNRH ANTAGONISTS AND O-ft-lER GNF;H RECEPTOR LIGANDS FOR
THE TREATMENT WITH GNRH RECEPTORS OF TUMOURS ORIGINA~'ING IN THE BRAIN
AND/OR NERVOUS SYSTEM AND/OR MENINGE:S AND/OR OF KAPOSI SARCOMA 181.(W0
99/01444) POLYMORPHIC FORM OF THE; TACH~'KININ RECEI'~TOR ANTAGONIST 2-(R)-( I-
(R) -
(3,5-BIS(TRIFLUOROMETHYL) PHENYL)ETHOXY)-3-(S)-(4-FLUORO) PHENYL.-4-(3-5 (-0X0-
1 H.4H-1,2,4,-TRIAZOI-O) METHYLMORPHOLINE, 182.(W0 99-O1 127) COMPOUNDS AND
METHODS 183.(W0 99/00406) CYCLIC AGONISTS AND ANTAGONISTS OF C5a RECI~PT'ORS
AND G PROTEIN-COUPLED RECEPTORS 184.(W0 98/58674) ANTI-TUMOUR
PHARMACEUTICAL. COMPOSITIONS CAPABLE OF REDUCING DRUG RESISTANCE IN
TUMOUR CELLS 185.( WO 98'57647) COUP-TFII: AN ORPHAN NUCLEAR RECEPTOR REQUIRED
FOR ANGIOGENESIS 186.(W'0 98156892) HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR9
187.(W0 98!56779) 4-SUL,FINYL BENZAMIDES AS CALCITONIN GENE-RELATED PEPTIDE
RECEPTOR ANTAGONISTS 188.(W0 98155153) NON-STEROIDAL RADIOI_ABELED
AGONIST/ANTAGONIST COMPOUNDS AND THEIR USE IN PROSTATE CANCER IMAGING
189.(WO 98!54325) Hl~MAN FRP AND FRAGMENTS THEREOF INCLUDING METHODS FOR
USING THEM 190.(WO 98/54202) HUMAN TUMOR NECROSIS FACTOR RECE:P'TOR TR10
191.(W0 98/54201) LIUMAN TUMOR NECROSIS FACTOR RECEPTOR-LIKE PROTEIN 8 192.(W0
98/54187) SPIRO-AZACYCLIC DERIVATIVES AND THEIR L1SE AS THERAPEUTIC AGENTS
193.(W0 98!53069) GDNF RECEPTORS 194.(W0 98!49170) SPlRO-.AZACYCLIC'
DERIVATIVES
AND THEIR USE AS 1 HERAPEIJTIC AGENTS 195.(W0 98/48017) FAMILY OF
IMMUNOREGULATORS DESIGNATED LEUKOCYTE: 1MMLJNO~iLOBUL,IN-LIKE RECEPTORS
(L.IR) 196.(W0 98/47923) IL-5R ANTAGONISTS FOR TREATMENT OF INFLAMMATION,
ASTHMA AND OTIlE:R ALLERGIC DISEASES 197.(W0 98!46751 ) OSTEOI'ROTEGERIN
BINDING
PRO'fEINS AND RECI-:PTORS 198.(W0 98'46620) .A NOVEL HUMAN G-PROTEIN COUPLED
RECEPTOR 199.(W0 98/46265) METHODS FOR USING ANTAGOTJISTIC ANTI-AVB3 1NTEGRIN
ANTIBODIES 200.(WO 98/43962 ) HETEROCYCLIC INTEGRIN INHIBITOR PRODRUGS 251.(W0
97/44333) 1,2,4-OXADIAZOLE:S AS ADHESION-RECEPTOR ANTA.GONIS'TS 252.(W0
97/44329)
DIARYL,ALKYL CYCLIC DIAMINE DERIVATIVES AS CHEMOKINE RECEI'I OR ANTAGONISTS
253.(W0 97141225) MAMMALIAN MIXED LYMPHOCYTE RECEPTORS. CHEMOKINE
RECEPTORS [MMLR-C'CR] 254.(W0 97/37655) &agr;v&b~r;3 ANTAGONISTS 255.(W0
97/35969)
PEPTIDE LIGANDS OF THE UROKINASE RECEPTOR 256.(W0 97/34878) SUBS'1-ITUTED 2,3-
BENZODIAZEPIN-4-0NES AND THE LJSE T'EIEREOF 257.(W0 9733904) DEATH DOMAIN
CONTAINING RECEPTORS 258.(W0 97!33887) SPIROCYCLE IN'TE:GRIN INHIBITORS
259.(W0
97/33613) PARASITE-DERIVED ANTI-INFLAMMATORY IMMUNOMODULA~I'ORY PROTEIN
260.(W0 97%30991 ) NOVEL SUBSTITUTED N-METHYL-N-(4-(4-( I li-BENZIMIDAZOL-2-
YL)[1,4]DIAZEPAM-I-YL)-2-(ARYL)BUTYL)BENZAMII)ES USEFUL FOR THE TREATMENT OF
ALLERGIC DISEASES 261.(W0 97130990) NOVEL SUBSTITUTED N-MEl-HYI,-N-(4-
(P1PERIDIN-
1-YL)-2-(ARYL)BUTYL)BENZAMIDES USEFUL FOR THE ~fREA~fMEN f QF ALLERGIC
DISEASES 262.(W0 97'30989) NOVEL SUBSTITU fED N-METHYL-N-(4-(4-( I II-
BENZIMIDAZOL-
2-YL-AMINO)P IPER1DIN-1-YL)-2-(ARYI.)BUTYL.)BENZAMIDE'S USEFIII_ FOR THE
TREATMENT OF ALLERGIC DISEASES 263.(W0 97-30079) PEPTIDE ANTAGONISTS OF
CELLULAR MITOGENESIS AND MOTOGENESIS AND TIiEIR THERAPEUTIC USE 264.(W0
97130069) 17-BETA-Cl'CL.OPROPYL(AMINO/OXY) 4-AZA STEROIDS AS ACTIVE
INLTIBITORS
OF TESTOSTERONE 5-ALPHA-REDUCTASE AND C17-20-LYASE 265.(W0 97'29775)
COMfOSI'TIONS COMPRISING A CYCLOOXYGENASE-2 INHIBI fOR AND A LEUKOTRIENE B
4 RECEPTOR ANTAGONIST 266.(W0 97/29079) NOVEL COMPOUNDS AND PHARMACEUTICAL
USE THEREOF 267.(W0 97/28190) CYTOKINE ANTAGONISTS AI'JD AGONISTS 268.(W0
97/24373) MONOCLONAL ANTIBODY AN-fAGONISTS'fO HAEMOPOIETIC GROWTH FACTORS
269.(W0 97/23480) NOVEL INTEGRIN RECEPTOR ANTAGONISTS 270.(W0 97,'2260=I)
NOVEL.
SUBS'fITUTED 4-(lE1-BENZIMIDAZOL-2-YL)[1,4]DIAZEPANES USEFUL FOR THE TREATMENT
OF ALLERGIC DISEASES 271.(WO 97/21732) DESIGN OF HORMONE-LIKE ANTIBODIES WITH
AGONISTIC AND ANTAGONISTIC FUNCTIONS 272.(W0 97!21702) 3-
BENZYLAM1NOPYRROLID1NES AND -PIPERIDINES AS TACHYKININ RECEPTOR
ANTAGONISTS 273.(W0 97!21445 ) VASCULAR IRRIGATION SOLUTION AND ME HOD FOR
INHIBITION OF PAIN, INFLAMMATION. SPASM AND RESTENOSIS 274.(WO 97/20062) IL-12
P40 SUBUNIT FUSION POLYPEPTIDES AND USES THEREOF 275.(W0 97/19074 )
SUBSTfTUTED
4-(I H-BENZIMIDAZOI.-2-YL-AMINO)PIPERIDINI?S USEFUL FOF; THE TREATMENT OF
ALLERGIC DISEASES 276.(W0 97% 19059) NOVEL SUBSTITUTED ARYL COMPOUNDS USEFUL
AS MODULATORS Of ACETYLCHOLINE RECEPTORS 277.(W0 97/ 16442) SUBSTITUTED
PYRIDYL PYRROLES. COMPOSITIONS CONTAlNINCi SUCH Ci:)MPOUNDS AND METIiODS OF
CA 02402930 2002-09-19
USE 278.(W0 97/16302) CYTOKINES AND THEIR LJSE IN TREATMENT AND.'OR
PRC)PHYLAXIS
OF BREAST CANCER 279.(W0 97'1619) ENHANCED ANTI-INFLAMMATORY ORAL
COMPOSITION CONTAINING H 2 RECEPTOR AN'FAGONIS f APJD ANTIMIC ROBIAI. OILS
280.(W0 97/15298) COMBINATION OF LTD, RECEPTOR ANTAGONISMS WI'H
GLUCOCORTICOSTE:KOIDS 281.(W0 97114671 ) CYCLOPENTYL TACIIYKININ RECEPTOR
ANTAGONISTS 282.(W0 97% 137 I) 1NDOLE CARBAMATES AS I_EUKOTRIENE ANTAGONISTS
283.(W0 97!l3~ 14) NK-1 RECEPTOR ANTAGONISTS FOR PREVENTION OF NEUROGENIC
INFLAMMATION IN GENE THERAPY 284.(W0 97!09046) COMPOUNDS AND METHODS
285.(W0 97/07130 BINDING OF OSTEOGEN1C PROTEIN-1 (OI'-1 ) AND ANALOGS THEREOF
TO
THE CELL SURFACE RECEPTOR ALK-I AND ANALOGS THEREOF)
With respect to clinical and pre-clinical trial development see Antibody
Therapeutics Producaion, Clinical
Trials, and Strategic Issues, By Rathin C. Das, Ph.D.. M.B.A. & K. John
Morrow, Jr., Ph.D., D&MD
Publications October 2001, Chapter 6.
Entity Associated and Entity Specific Markers
The term marker is used broadly to refer to any' ligand or binding site for a
"targeting" or an
"effector" moiety of a multispecific ligand or antibody of the invention and
is primarily used herein
to refer to ligands which are the target of a "targeting" moiety (most often
though not exclusively
referred to herein as a first ligand binding moiety).
The literature is replete with examples of such markers, as well as antibodies
which recognize them.
With respect to cancer markers and immune cell markers, etc. many of these are
referred to in
Cancer: Principles and Practice of Oncology 6'~' Ed. De Vita et al. Eds
Lippincott 2001 and some are
summarized at pages 309-311, 3197.
With respect to markers for osteoclasts and antibodies that bind thereto see
for example
Endocrinology 1989 Aug; 125(2):630-7; Endocrinology 1990 Dec: 127(6): 3215-21;
Lab Invest Apr;
60(4):532-8; Calcif Tissue Int 1998 Aug; 63(2): 148-53. Such markers could be
used for example to
target RANK (associated with bone resorption etc.) on osteoclasts using a
relatively low affinity
second ligand binding moiety.
Other target applications of multispecific ligands of the invention include
particularly receptors
associated with angiogencsis (eg.VEGFRs 1,2,3) such as KDR, FLK-1 and FLT-1,
and various
cancers cell types eg. HER-2 and EGF-R, including FGF-R, PDGF-.R, Tek and
Tie2. Numerous other
examples are referred to specifically and through references to the literature
herein cited. Suitable
markers for many types of target entities eg. cells bearing such receptors are
referred to or
referenced herein or described in various subject reviews and texts herein
cited, in connection with
one or more aspects arid embodiments of the invention described in this
application, and many others
are known to those skilled in the art and desribed in the literature including
antibodies.
With respect to binding to biologic effector ligands the multispecific ligand
may comprise a
recombinatly produced receptor for such ligand.
As the invention contemplates that the multispectic li~~,ands herein may be
used for cancer, it is
contemplated that combination therapies with chemotherapeutic and
biotherapeutic agents may be used to
advantage. Such agents are well known to those skilled in the art and include
for example, alkvlating
agents, cisplatin anVd its analogues, antimetabolites, topoisomerase
interactive agents, antimicrotubule
agents, interferon s, interleukins, hormonal therapeutics, differentiation
agents, antiangiogenesis agents (see
Cancer: Principles and Practice of Oncology 6'" Ed. De Vita et al. Eds
Lippincott 3001 pp.335-~ 17)
With respect to ligands involved in mediating apoptosis see also W001~I4808
METHODS OF
DIAGNOSIS AND TREATMENT BY BINDING P73iAIRM 1 WOOl~I4282 BCL-G POLYPEPTIDES,
ENCODING NUCLEIC' ACIDS AND METHODS OF USE US6242S69 Regulators of apoptosis
EP1106183 Antibodies to erbB2 and their therapeutic uses WOO 13659~I Mcl-I
GENE REGULATORY
ELEMENTS AND A PRO-APOPTOTIC Mcl-1 VARIANT US2001001712 Monoclonal antibodies
having property of causing apoptosis W00134798 CLONING AND CHARACTERIZA-fION
OF VIRAL
IAP ASSOCIATED FACTOR (VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal
antibody
inducingapoptosis HU0003513 MONOCLONAL ANTIBODY INDUCING APOP'1'OSIS EP1094316
Method for the detection of DNA replicating cells US62074~2 Antibody of the
anti-proliferation domain
86
CA 02402930 2002-09-19
of human Bcl-2 W00123568 NOVEL MEMBERS OF THE LAP GENE FAMILY US6190661
Methods
and compositions for the use of apurinic/apyrimidinic endonucleases EP 1087993
FAS PEPTIDES AND
ANTIBODIES FOR MODULATING APOP'FOSIS WO01 19861 APO-.', RECEPTOR ANTIBODIES
US6184034 Deoxyribonuclease 1l proteins and cDNAS US617221 1 Nucleic acid
encoding tag?
polypeptide WO01 18042 APOPTOSIS PROTEINS Vv'001 16180 CD40 LIGAND AND CD40
AGONIST
COMPOSITIONS AND METHODS OF USE WO01 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION W00144808 ME'T'HODS OF DIAGNOSIS AND TRC:ATMENT BY
BINDING P7~-AIRM 1 W00144282 BCL-G POI_YPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Regulators of apoptosis EPl 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-I GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causing
apoptosis
W00134798 CLONING AND CHARACTERIZATION OF VIRAL LAP ASSOCIATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
I-IU0003513
MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of
DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human
Bcl-2 W00123~68
NOVEL MEMBERS OF 'THE LAP GENE FAMILY US6190661 Methods and compositions for
the use of
apurinic,%apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOSIS W00119861 APO-3 RECEPTOR ANTIE30DIES US(i184034
Deoxyribonuclease II proteins and eDNAS US617221 I Nucleic acid encodin~~
ta~~7 polypeptide
WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 L.IGAND AND CD40 AGONIST
COMPOSITIONS AND METHODS OF USE WO01 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULrI fION WO01=14808 METI-iODS OF DIAGNOSIS AND fRE:ATMENT BY
BINDING P75!AIRM1 WO0144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US(i242569 Regulators ofapoptosis EP1 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-1 GENE REGULA'I ORY ELEMENTS AND A PRO-
APOI'TOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causin~~
apoptosis
W00134798 CLONING AND CHARACTERIZATION Of VIRAL LAP ASSOCIA'T1D FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
HU0003513
MONOCLONAL ANTIBODY INDUCING APOPTOS1S EP1094316 Method for the detection of
DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human
Bcl-'? W00123568
NOVEL MEMBERS OF -CHE IAI' GENE FAMILY US6190661 Methods and compositions for
the use of
apurinic,!apyrimidinic endonucleases EP1087993 FAS 1'EP'FIDES AND ANTIBODIES
FOR
MODULATING APOPT(:)SIS WO01 19861 APO-2 RECEPTOR ANTIBODIES US6184034
Deoxyribonuclease 1I proteins and eDNAS US617221 t Nucleic acid enc~~ding tag?
polypeptide
WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIST
COMPOSITIONS AND ME FFIODS OF USE WO01 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULA CION W00144808 METI-fODS OF DIAGNOSIS AND 'TREATMENT BY
BINDING P7~lAIRM I W'00144282 BCL-G I'OLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US(i242569 Regulators of apoptosis EP1 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-I GENE REGULATORY ELEMENTS AND A PRO-APOf'TOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies havin<~ property of causing
apoptosis
W00134798 CLONING AND CHARACTERIZATION OF V1RAI. LAP ASSOCIATI:v:D FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducin~~ apoptosis
FiU0003513
MONOCLONAL ANTIBODY INDUCING APOP'TOSIS EP 1094316 Method for the detection of
DNA
replicating cells IJS6207452 Antibody of the anti-proliferation domain of
human Bcl-2 WO01:?3568
NOVEL MEMBERS OF TE-IE LAP GENE FAMILY (JS6190661 Methods and compositions for
the use of
apurinic;apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND .ANTIBODIES FOR
MODULATING APOPT()SIS WO01 19861 APO-3 RECEPTOR AN'FIEtODII=,S US6184034
Deoxyribonuclease 1l proteins and eDNAS US617221 l Nucleic acid encoding ta~~7
polypeptide
WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIST'
COMPOSITIONS AND ME'hHODS OF USE WO01 16170 NOVEL, CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION WO0144808 METHODS OF DIAGNOSIS AND'FREATMENT BY
BINDING P7~%AIRMI W'00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Regulators of apoptosis EPl 106183 Antibodies to
erbB2 and their
therapeutic uses W00136~94 Mel-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causin~~
apoptosis
W00134798 CLONING AND CHARAC1'ERIZA'I ION OF VIRAL, LAP ASSOCIATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
HU0003513
MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP 10943 l6 Method for the detection of
DNA
replicating cells US62074~2 Antibody of the anti-proliferation domain of human
Bcl-2 WO01:'3568
87
CA 02402930 2002-09-19
NOVEL MEMBERS OF THE IAP GENE FAMILY US6190661 Methods and compositions for
the use of
apurinic/apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOStS W00119861 APO-? RECEPTOR ANTIBODIES US6184034
Deoxyribonuclease II proteins and cDNAS U5617221 I Nucleic acid encoding tag?
polypeptide
WO01 18042 APOPTOS1S PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIST
COMPOSITIONS AND METHODS OF USE W001 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEA'fli REGULATION W00144808 METHODS OF DIAGNOSIS AND'TRfATMENT BY
BINDING P75!AIRMI Vv'0014428? BCL-G 1'OL.YPEPTIDES, ENCODING NUCI_f:IC ACIDS
AND
METHODS OF USE US6242569 Regulators of apoptosis EP 1 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENT'S AND A PRO-APOPTOTIC
Mcl-1 VARIANT US?001001712 Monoclonal antibodies having property of causin~~
apoptosis
WOOi34798 CLONING AND CHARACTERIZATION OF VIRAI_ IAP ASSOCIATf:D FACTOR
(VIAF) EN SEVERAL, ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
HU0003513
MONOCLONAL ANTlE3ODY INDUCING APOPTOSIS EP1094316 Method tbr the detection of
DNA
replicating cells US6207452 Antibody ofthe anti-proliferation domain ofhuman
Bcl ? W00123s68
NOVEL MEMBERS OF 'THE IAf GENE FAMILY US6190661 Methods and compositions for
the use of
apurinicapyrimidinic endonucleases EP1087993 FAS PEPTIDES AND.4NTIBODEES FOR
MODULA'hING APOPTOSIS Vl'001 19861 APO-2 RL:CEPTOR AN'fIBODIES US6184034
Deoxyribonuclease 1l proteins and cDNAS USC 17221 1 Nucleic acid encoding tag?
polypeptide
WOOL 18042 APOPTOS1S PROTEINS U1~'001 16180 CD40 LIGAND AND CD40 AGONIST
COMPOSITIONS AND METHODS OF USE W'001 Ifi170 NOVEL. CARD fRO'I EINS INVOLVED
1N
CELL. DEATH REGULATION W00144808 METHODS OF DIAGNOSIS AND T Rf:ATMENT BY
BINDING P75~'AIRMI W00144282 BCL-G POLY PEPTIDES. ENCODING NUCLEIC ACIDS AND
METI-IODS OF USE US6242569 Regulators of apoptosis EP 1 106183 Antibodies to
erbB2 and their
therapeutic uses W00131i594 Mcl-1 GENE RE:GUL.ATORY ELEMENTS AND A PRO-
APOP'fOTIC
Mcl-1 VARIANT' US2001001713 Monoclonal antibodies having property of causing
apoptosis
W00134798 CLONING .AND CHARACTERIZATION OF VIRAL IAP ASSOCIATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducin~~ apoptosis
HU0003513
MONOCLONAL ANTIBODY INDUCING AfOPTOSIS EP 1094316 Method for the detection of
DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human
Bcl-' W00123568
NOVEL_ MEMBERS OF THE IAP GENE FAMILY 1JS6190661 Methods and compositions for
the use of
apurinic.%apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTC)SIS W'001 19861 APO-2 RECEPTOR AN TI E3ODIES US6184034
Deoxyribonuclease 1l proteins and cDNAS US617221 I Nucleic acid encoding tag?
polypeptide
WO01 18042 APOfTOSIS PROTEINS WO01 16180 CD40 L1GAND AND CD40 AGON1ST
COMPOSITIONS AND METHODS OF LJSE WO01 16170 NOVEL CARD PRO~fEINS INVOLVED IN
CELL DEATH REGULATION V~'OO144808 METHODS OF DIAGNOSIS AND 'fREA'rMENT BY
BINDING P75/AIRM I WOO144282 BCL-G POLYPEfTIDES. ENCODING NLJCI.EIC ACIDS AND
METHODS OF USE US6242569 Re~~ulators of apoptosis EPI 106183 .Antibodies to
erbB2 and their
therapeutic uses W0013(i594 Mcl-1 GENE R.E:GLJLATORY ELEMENTS AND A I'RO-
AfOPTOTIC
Mcl-I VARIANT LJS200100171? Monoclonal antibodies having property of causing
apoptosis
W00134798 CLONING AND CHARACTERIZATION OF VIRAL. IAP~ASSOCIATED FACTOR
(VIAF) 1N SEVERAL ORGANISMS CZ?0000907 Monoclonal antibody induciny~ apoptosis
HU0003513
MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of
DNA
replicating cells US6207=453 Antibody of the anti-proliferation domain of
human Bcl-2 W001~3568
NOVEL MEMBERS OF'fHE IAP GENE FAMILY LJS6190661 Methods and compositions for
the use of
apurinic;'apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOSIS WO01 19861 APO-? RECEPTOR ANTIBODIES 1JS6184034
Deoxyribonuclease 11 proteins and cDNAS US617221 1 Nucleic acid encoding tag?
polypeptide
WO01 18042 APOPTOSIS PROTEINS W001 1618() CD40 L,IGAND AND CD40 AGON1ST'
COMPOSITIONS AND ME'ft-IODS Of USE WOOI 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION Vf00144808 METHODS OF DIAGNOSES AND'fRE.ATMENT BY
BINDING P75/AIRMI W00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC,' ACIDS AND
METHODS OF USE US6?42569 Regulators of apoptosis EP I 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTO'TIC
Mcl-1 VARIANT US200t001712 Monoclonal antibodies havin~~ property of causin~~
apoptosis
W00134798 CLONING AND CHARACTERIZATION OF VIRAL, IAP ,ASSOC.'IATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
HU0003513
MONOCLONAL ANTIBODY INDUCING APOPTOStS EP1094 316 Method for the detection of
DNA
replicating cells US6?07452 Antibody of the anti-proliferation domain of human
Bcl-2 W00123568
88
CA 02402930 2002-09-19
NOVEL MEMBERS OF THE IAP GENE FAMILY US6190661 Methods and compositions for
the use of
apurinic/apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOSIS WO01 19861 APO-2 RECEPTOR ANTIBODIES US6184034
Deoxyribonuclease II proteins and cDNAS LJS617221 1 Nucleic acid encoding tag?
polypeptide
WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIS-f
COMPOSITIONS AND METHODS OF USE WO01 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION W00144808 METHODS OF DIAGNOSIS AND TREATMf:N1' BY
BINDING P75!AIRMI W00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Re;.,~ulators ofapoptosis EPl 106183 Antibodies to
erbB2 and their
therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having propc;rty of causing
apoptosis
W00134798 CLONING AND CHARACTERIZATION OF VIRAL IAP ASSOCIA~CED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis
HU0003513
MONOCLONAL., ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of
DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human
Bcl-? W00123568
NOVEL MEMBERS Of THE IAP GC:NE FAMILY US6190661 Methods and compositions for
the use of
apurinie/apyrimidinic endonueleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOP'fOSIS Vl-'001 19861 APO-2 RECEPTOR ANTIBODIES US6184034
Deoxyribonuclease II proteins and cDNAS US617221 I Nucleic acid encoding tag?
laolypeptide
WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIS'f
COMPOSITIONS AND METHODS OF USE WO01 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL, DEATH REGULATION
As stated above, in a related but also independent aspect, the invention
contemplates a method of screening
for an antibody which preferentially binds to a ligand when bound to a first
receptor relative to another
second receptor by screening for antibodies leg. by phage display, ribosome
display, etc.) which bind to the
ligand eg. a cytokine, when bound in situ to the first receptor, and selecting
among them those that bind to
the ligand eg. cytokine but do not bind (substractive screening) or bind with
lesser affinity when bound to
the cytokine to the second receptor, as well as to antibodies and
multifunctional li~~ands created by this
method (see also tJSP 6.046,048 and WO 99/ 12973 and references cited therein
with respect to TNF
family of receptorsj. Variations in the extracellular domains of such
rect:ptors are known and can be
ascertained by methods known to those skilled in the art. Accordingly the
invention is directed to an
antibody characterized in that it binds to an epitope on the ligand which
permits the li~~and to bind, while
the antibody is bound to it. to a first receptor but not a second receptor. Yn
a preferred embodiment both are
cell surface receptors. In a preferred embodiment the li~~and is a natural
ligand, preferably a growth factor,
cytokine or chemokine. In another embodiment one of the receptors is a soluble
receptor. The invention is
also directed to a method of evaluating the pleitropic eti'ects of a natural
ligand by administering the said
antibody Including antigen binding fratiments thereof and MRUs) and monitoring
its etfiects. 1"he invention
contemplates that this antibody is a first or second moiety of a
multifunctional ligand disclosed herein.
Examples of receptors include the classes of VEGF receptors (see also l
:Sheppard D.Integrin-mediated
activation of transforming ~~rowth factor-beta( 1 ) in pulmonary
fibrosis.C'hest. 2001 Jul;120( I Suppl):549-
53. Chow D, Ho J, N~~uyen Pham Tl., Rose-John S, Garcia KC.In vitro
reconstitution of recognition and
activation complexes between interleukin-6 and gp130.Biochemistry. 2(101 .lun
2G;40('25):759i-603.
Kotenko SV, Izotova LS, Mirochnitchenko OV. Esterova E, Dickensheets H.
Donnelly RI', Pestka
S.Identification, cloning, and characterization of a novel soluble receptor
that binds I1.-22 and neutralizes
its activity.J lmmunol. 2001 Jun 15;166(12):7096-103. Gustin SE, Church AP,
Ford SC., Mann DA, Carr
PD, Ollis DL, Young IG.Expression, crystallization and derivatization of the
complete extracellular domain
ofthe beta(c) subunit of"the human IL-5, IL-3 and CiM-CSF receptors.Eur.l
Bioehem. 2001
May;268( 10):2905-1 1. MeCall AM. Shahied L, Amoroso AR, tJorak EM, Simmons
IiH, Nielson U,
Adams GP, Schier R, Marks JD, Weiner LM.Increasing the aftinity for tumor
antigen enhances bispecitic
antibody cytotoxicity.J Immunol. 2001 May 15;166( 10):61 12-7. Piehler J,
Roisman LC, Schreiber G.New
structural and functional aspects of the type I interferon-receptor
interacrion revealed by comprehensive
mutational analysis ofthe binding interface.) Biol Chem. 2000 Dec
22;27501):40425-33.DLINE]7:
Wiesmann C, Muller YA, de Vos AM.Ligand-binding sites in I~ like domains of
receptor tyrosine
kinases.J Mol Med. 2000;78(5):247-60. Review.DLINE)8: Born TL" Sn nith DE:,
Garka KE, Renshaw BR,
Bertles JS, Sims JE., Protein, Nucleotide Identification and characterization
of two members of a novel
class of the interleukin-I receptor (IL.-1 R) family. Delineation of a new
e:lass of IL-I R-related proteins
based on signaling.) Biol Chem. 2000 Sep 29;275('39):29946-54.DLINE)9: Xia XZ,
T'reanor J. Senaldi G,
Khare SD, Boone T, Kelley M, Theill LE, Colombero A, Solovyev l, Lee F, McCabe
S, Elliott R, Miner K,
Hawkins N, Guo J, Stolina M, Yu G, Wang J, Delaney J, Meng SY, Boyle WJ, Hsu
LI.. Protein, Nucleotide
89
CA 02402930 2002-09-19
TACI is a TRAF-interacting receptor for TALL-l, a tumor necrosis factor family
member involved in B
cell regulation.) Exp Med. 2000 Jul 3;192(1):137-43.DLINE] 10: Kumaran J,
Colamonici OR, Fish
EN.Structure-function study of the extracellular domain of the human type l
interferon receptor (IFNAR)-1
subunit.J Interferon Cytokine Res. 2000 May;30(5):479-8s.DLINEJ l I: I_u D,
Kussie P, Pytowski B,
Persaud K, Bohlen P, Witte L, Zhu Z.Identitication of the residues in the
extracellular region of KDR
important for interaction with vascular endothelial growth factor and
neutralizing anti-KDR antibodies.)
Biol Chem. 2000 May 13:275(19):14321-30.DLINE]12: Bowie A, O'Neill LA.T'he
interleukin-1
receptor/Toll-like receptor superfamily: signal generators for pro-
inflammatory interleukins and microbial
products.) Leukoc Biol. 3000 Apr;67(4):~08-14. Review. DL IN E] 13: Kumar S,
McDonnell PC, Lehr R,
Tierney L, Tzimas MN, Griswold DE, Copper EA, 'fal-Singer R, Wells Cil, Doyle
Ml., Young PR.. Protein,
Nucleotide. OMIM Identification and initial characterization of four novel
members of the interleukin-1
family.) Biol Chem. 2000 Apr 7;275(14):10308-14.DLINE]14: Fujio K, Nosaka T,
Kojima T, Kawashima
T, Yahata T, Copeland NG. Gilbert DJ, Jerkins NA, Yamamoto K, Nishimura T,
Kitamura T., Protein,
Nucleotide Molecular clcrnin~ of a novel type I cytokine receptor similar to
the common gamma
chain.Blood. 2000 Apr 1:95(7):2204-IO.DLINE] 15: Touw IP, De Koning; JP, Ward
AC. tiermans
MH.Signaling mechanisms of cytokine receptors and their perturbances in
disease.Mol Cell Endocrinol.
3000 Feb 2;160( 1-2); I-9. Review.DLINE] 11i: Chritton SL" Shen~, M.C'r'RL. a
novel cytokine receptor-
like protein expressed in testis, lung, and spleen.Biochem Biophys Res
C~~mmun. 2000 .lan 27;367(3):697-
703.DLINE] 17: 1.i H. Chen J, Huang A, Stinson J, lieldens S, Foster J, Dovvd
P. Gurney AL, Wood W1.
Free in PMC , , Protein, Nucleotide. OMIM Clonin~~ and characterization of IL.-
1713 and Il,-17C', two new
members ofthe IL-17 cytokine family.Proc Natl Acad Sci U S A. 2000 Jan
18;97(3):773-B.DLINEJ18:
Gary-Gouy H, Bruhns P, Schmitt C, Dallaul A, Daeron M, Bismuth G.The pseudo-
immunoreceptor
tyrosine-based activation motif of CDS mediates its inhibitory action on E3-
cell receptor signalin~'~.J Biol
Chem. 2000 Jan 7:27x( 1 ):~48-i6.DLINE] 19: Wiesmann C, LJltsch MH, E3ass SH,
de Vos AM., Protein,
Structure Crystal structure of nerve growth factor in complex with the ligand-
binding domain of the TrkA
receptor.Nature. 1999 Sep 9:401(6749):184-8.DLINEJ20: Liu Y, Cruikshank W W,
O'l,ou;,~hlin T, O'Reilly
P, Center DM. Kornfeld Il.ldentification of a CD4 domain required for
interleukin-16 binding and
lymphocyte activation.) Biol Chem. 1999 Aug 13;274(33):23387-95.DL.tNE]21:
Donnelly RP,
Dickensheets H, Finbloom DS.The interleukin-10 signal transduction pathway and
regulation of gene
expression in mononuclear phagocytes.) Interferon Cytokine Res. 1999 Jun;
19(6):563-7 3.
Review.DLINE]32: Kim H. Baumann H.Dual signaling role of the protein tyrosine
phosphatase SHP-2 in
regulating expression of acute-phase plasma proteins by interleukin-6 cytokine
receptors in hepatic
cells.Mol Cell Biol. 1999 Aug:19(8):~326-38.DLINE]2:i: Kernebeck T, Ptlanz S,
Muller-Newer G,
Kurapkat G. Scheek RM, Dijkstra K. Heinrich PC, Wollmer A, Grzesiek S,
Grotzinger J.. Protein,
Structure. The signal transducer ~p130: solution structure of the carboxy-
terminal domain of the cytokine
receptor homology region.Protein Sci. 1999 Jan:B(I ):5-12.DL,INE]24: Ernst M,
Novak LJ, Nicholson SE,
Layton JE. Dunn AR.The carboxyl-terminal domains of gp I _i0-related cytokine
receptors are necessary for
suppressing embryonic stem cell differentiation. Involvement of STAT3.J Biol
Chem. 1999 Apr
2;274(14):9729-37.DLINE]25: Hibi M, Hirano T'.Signal transduction through
cytokine rc:ceptors.lnt Rev
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interactions of the
cytokine-binding homolo~,y region and immunoglobulin-like domains of gp130
with oncostatin M:
implications for receptor complex formation. Protein En~~. 1998 Nov; I 1( I I
):1093-102).
With respect to markers that are useful for differentiatin« between various
populations and sub-populations
of cells. see also F-luman 11.-I 8 Receptor and ST2L Are Stable and Selective
Markers for the Respective
'type 1 and Type 2 Circulating Lymphocytes, Woon Ling C,'han, Nada Pejnovic,
C:hristinc A. Lee, and
Nadia A. AI-Ali, J lnnnunol 2001;167 1238-124=1; CD4+CD25 high Regulatory
Cells in Human Peripheral
Blood, Clare Baecher-Allan, Julia A. Brown, Gordon J. Freeman, and David A.
Ilatler. J Irnmunol
3001;167 12~t~- I 353.
In another aspect the invention is directed to multifunctional li~and
comprising at least a first moiety which
specifically binds to a ligand on the surface ofa virus particle that is
capable of infecting a mammalian and
particularly a human cell including a cancer cell (excludin<~ viruses which
are known for use in gene
therapy) and is preferably selected from the ~~roup consisting viruses which
infect substantial populations of
individuals including for example influenza virus and at least a second moiety
which speciticallv
reco~~nizes a cancer cell, in one embodiment preferably a marker present on
multiple different cancer types,
especially cancer types that are individually or collectively most prevalent
in the general population. In one
embodiment such multifunctional ligand is a bispecitic, trispecitic or
tetraspecitic antibody. The invention
contemplates that such a multifunctional ligand may be used to target such
viruses to tumors in a manner
which preferentially kills the cancer cells either through the action of the
virus and,'or by causing, the
CA 02402930 2002-09-19
immune response to the virus or virus infected cell to preferentially
(relative to non-cancer cells f target the
cancer cell for ablation. T'he invention is also directed to a method of
treating cancer by retar~~eting virus
with which an idividual is otherwise infected to the cancer cell eg.
int7uenza.~The invention also
contemplates that the multifunctional ligand includes one or more effector
moieties which assist in killing
the virus and/or cancer cell or directing immune cells to the virus and/or
cancer cell, if and when present in
the individual, for example a moiety which specifically binds to such immune
cell eg. a T cell. as discussed
above. Accordingly, the invention also contemplates that such multifunctional
ligand may be used to treat
infiluenza virus infections and secondarily to act prophylactically as a
sentinel against any cancer cells
which might develop during the course of the viral infection or a period of
immune suppression or
increased succeptibility to infection or cancer, including for example, as e-
rperienced by individuals with a
particular immune suppressive disorder or condition or under treatment with
immune suppressive drugs,
individuals at risk for cancer or recurrence of a cancer, individuals of a
particular ale group, individuals
experiencing a period of unusual stress which increases their succeptibility
to disease or infection. The
invention also contemplates that such a moiety is used in concert with prior
immunization a~~ainst the virus.
so that the augemented immune response to the virus benefits the treatment of
the cancer cells (see for
example USP 6169175 and the art cited therein). The invention also directed to
such a virus (excluding
viruses known for use in gene therapy applications e~,. adenovirus) which is
engineered to expresses on its
surface a cancer targeting moiety such as a scfv (see for example EP 1038967,
WO 94i (0323 and the art
cited therein). The invention is also directed to a method of identifying the
expression or over-expression of
cell surface markers associated with infection by such a virus, by
substractive screenin« relative to markers
also expressed on non-infected such cells, for example usin~~ pha~Te display
or the like. Such markers may
be used for vaccine-type or other imrnunotherapeutic strategies. Anti-virus,
markers includin~~ influenza
virus markers and methods of identitying new such markers are well known in
the art (see for example
USP 5589174) (see also The role of the antibody response in influenza virus
infection., Gerhard W.,Curr
Top Microbiol Immunol 2001;260:171-90, Fernandez-Sesma A, Schulman JL, Moran
TM.A bispecific
antibody recognizing influenza A virus M2 protein redirects effector cells to
inhibit virus replication in
vitro.) Virol. 1996 Ju1;70(7):4800-4; Todorovska A, Roovers RC. Dolezal O, Kor-
tt AA. Hoo~=enboom HR,
hfudson PJ. Design and application of diabodies. triabodies and tetrabodies
for cancer targeting. .l lmmunol
Methods. 2001 Feb 1;248( I-2):47-66.. Staerz UD, Yewdell .1W, Bevan MJ. Hybrid
antibody-mediated lysis
of virus-infected cells. Eur J Immunol. 1987 Apr; 17(4):571-4; Fernandez-Sesma
A, Schulman JL, Moran
TM.A bispecific antibody recognizing influenza A virus M2 protein redirects
effector culls to inhibit virus
replication in vitro. J Virol. 1996 Ju1;70(7):4800-4.)
The invention is also directed to a multifunctional ligand having at least a
tumor cell targeting moiety and
a moiety which binds to a tumor antigen which is shed from a cancer cell. In a
preferred embodiment, the
tumor antigen binding moiety preferably does not reco~~nize the portion of the
antigen which is most
imrnunogenic and leaves that portion exposed for reco~~nition by the immune
system. The invention
contemplates generating such preferred antibody or fragment thereof by using
an an immune complex
between an antibody that hinds to such immunogenic portion and the anti«en as
a target for phage display
or generation or polyclonal sera. The invention also contemplates identifying
antibodies which recognize
immunogenic portions of the antigen by screening patient sera for antibodies
which reco<rnize the antigen.
The invention also contemplates that such multifunctional ligand includes one
or more effector moieties
which assist in killing the cancer cell or directing immune cells to the
cancer cell, for example a moiety
which specifically binds to such immune cell egva T cell receptor, as
discussed above.
Without limiting the generality of or applicability of the foregoing, and
without being limited by or limiting
the scope of the claims, various embodiments of the invention may be
summarized for ease of reference as
follows: I. A multispecific li~~and comprising at least a first ligand binding
moiety which preferentially
binds with a first affinity to a first ligand having a first biodistribution*
and at least a second ligand
binding moiety which preferentially binds with a second affinity to a second
liaand having a second
biodistribution which is different' from that of the fwst ligand, and wherein
the affinity of first and second
ligand binding moieties are selected to bias the biodistribution of the
multispecific li~~and. 2. A
multispecific ligand according to paragraph I. wherein said multifunctional
ligand comprises one or more
ligand binding moieties which are antibodies. 3. A multispecfic ligand
according to paragraph I or 2,
wherein the affinity of said first li~~and binding moiety for the first ligand
is higher than the affinity of the
second ligand binding moiety for the second ligand so as to bias the
biodistribution of the multispecific
ligand in favor of the the first ligand. 4. A multispecific ligand accordin~~
to paragraph 3. wherein the first
'' Contrasted to functional affinity which may result from avidity
' see fn 8
91
CA 02402930 2002-09-19
and second ligands have overlapping biodistributions.' ~. A multispecfic
ligand according to paragraph 4,
wherein the first ligand is a target cell population associated ligand and
wherein said second ligand is
present on a broader population of cells and wherein the biodistribution of
the multispecific ligand is
skewed in favour of the target cell population. 6. A multispecflc ligand
according to paragraph I or >,
wherein said first ligand is a marker associated with* one or more specific
cell populations, infectious or
parasitic agents, diseased cells, or disease associated* cells, optionally one
of specific ligands herein
mentioned or referenced or known to those skilled in the art. 7. A
multispecific ligand according to
para~=raph 6, wherein said marker is a specific biological structure. 8. A
multispecific ligand according to
paragraph 6, wherein said marker is a specific receptor or receptor li~and. 9.
A multispecitic ligand
according to paragraph 6, wherein said marker is a specific antigen. 10. A
multispecific li~and accordin~~ to
paragraph 6, wherein said marker is a specific epitope. 1 1. A multispecific
ligand according to paragraph 6,
wherein said marker is a CD marker. 12. A multispecific ligand according to
paragraph 6, wherein said
marker is associated with a cancer cell or pre-cancerous cell. 13. A
multispecitic ligand according to
paragraph 6, wherein said marker is associated with an autoimmune disorder or
rheumatic disease. 14. A
multispecitic li~and according to paragraph 6, wherein said marker is
associated with a specific tissue type.
15. A multispecific ligand according to paragraph 6, wherein said
mark.°r is associated with a specific
organ. l6. A multispecific ligand accordin<~ to paragraph 6, wherein said
marker is associated with a cell or
tissue of specific origin or class. 17. A multispecitic li«and according to
paragraph 6, wherein said marker
is an MHC-peptide complex. 18. A multispecific ligand according to paragraph
6, wherein said marker is
associated with a cell surface immunoglobulin. 19. A multispecitic li~and
according to paragraph S or 6,
wherein said second li~~and is a receptor, family of receptors or one or more
particular receptor family
members, optionally one of those specific receptors herein mentioned or
referenced or known to those
skilled in the art. The invention contemplates targeting any receptor present
on any population of entities
for which there is an entity associated marker. 20. A multispecific ligand
according to para~~raph 19,
wherein said second ligand is a cell surface receptor chosen from a group
comprising tyrosine kinase type
receptors, serine kinase type receptors, heterotrimeric G-protein coupled
receptors, receptors bound to
tyrosine kinase, 'TNF family receptors, notch family receptors, guanylate
cyclase types, tyrosine
phosphatase types, decoy receptors. and adhesion receptors, optionally one of
the specific receptors herein
mentioned or referenced or known to those skilled in the art. 21. A
multispecific ligand according to
paragraph 19, wherein said receptor requires cross-linking for biological
activity. 22. A multispecific ligand
according to paragraph s or 6, wherein said second li'~and is a cell surface
receptor and wherein said second
ligand binding moiety blocks said receptor. 23. A multispecitic ligand
accordin<, to paragraph I, 5 or 6.
wherein said second ligand is a receptor ligand and wherein said second
li~~and binding moiet~~ blocks
interaction with the corresponding receptor. 24. A multispecific ligand
according to paragraph 5 or 6,
wherein said second ligand is a cell surface receptor which initiates a signal
transduction and wherein said
second ligand binding moiety effects a signal transduction. 3~. A
multispecific li~and according to
para<~raph >. 6 or 19, wherein said antibody comprises a first Vh* which
preferentially reco~~nizes said first
ligand and a second VH which preferentially reco~~nizes said second ligand.
26. A multispecific ligand
according to paragraph 25, wherein at least one of said first and second VHs
require the cooperation of a
VL for binding to their respective ligands. 27. A multispecitic ligand
according to paragraph .?5,
comprising a first VL associated with said first VI-1 and a second Vl,
associated with said second VH and
wherein both said first and second V Hs require the cooperation of a VL for
binding to the first and second
ligands, respectively, and wherein said first and second VLs are the same' or
functionally interchangeable*.
28. A multispecific ligand according to paragraph 2~ or 27, wherein said
bispecitic antibody is a tour chain
antibody. 29. A multispecific ligand according to paragraph 28, wherein said
bispecfic antibody is a
minibody, F(ab')= or antibody devoid of a CH3 domain. 30. A multispecifie
ligand according to paragraph
25, wherein said bispecitic antibody is a diabody. 31. A multispecific ligand
according to paragraph 25 or
27, wherein said bispecific antibody is devoid of light chains. 32. A
multispecitic ligand according to
paragraph 31. wherein said bispecific antibody comprises a pair of disulfide
linked heavy chains or heavy
chain portions each comprising at least a VH re~,~ion, a hinge region and
preferably. at least a portion of an
Fc region at the carboxy terminus ofthe hinge region. 33. A multispecific
liaand accordin~~ to paragraph
31, wherein said bispecitic antibody comprises a pair of VHs linked via a
polypeptidc linker. 34. A
' The term epitope though technically understood to be specific for a given
antibody, is used to refer to
antigenic determinants that are situated proximally to one another so that two
antibodies will be considered
to bind to the same epitope if one competivelv inhibits the binding of the
other throu~~h any probative
competitive inhibition experiment known to those skilled in the art.
have substantially the same amino acid composition ie. with possible exception
of one or more additions,
deletions or substitutions including conservative amino acid substitutions
which do not substantially affect
the specificity and amino acid composition of the paratope
92
CA 02402930 2002-09-19
multispecitic ligand according to paragraph 3, 5, 6 or 19 wherein the affinity
of the first ligand binding
moiety for the first ligand is at least approximately c', one, two, three,
four, five, six, seven or eight orders of
magnitude greater than the affinity of said second ligand binding moiety for
the second li~~and ~3~. A
composition comprising a multispecific ligand in a pre-determined dosage, said
multispecitic ligand
comprisinvt a first ligand binding moiety which preferentially binds with a
pre-selected first affinity to a
first ligand having a first biodistribution and a second ligand binding moiety
which preferentially binds
with a pre-selected affinity to a second ligand having a second
biodistribution, which is different from that
of the first ligand, and vaherein the affinity of the first and second ligand
binding moieties are selected to
bias the biodistribution of the multispecific ligand in favor of a selected
location of one or both of the
ligands' such that a desired proportion of the dosage is delivered to the
selected location. 36. A
multispecific ligand according to paragraph 1 or 35, wherein the
biodistributions of said first and second
ligands overlapx and wherein the affinities of the first and second ligand
binding moieties are selected to
bias the biodistribution of the multispecific ligand in favour of a target
cell population on which both first
and second ligands are bioavailable for recognition by the first and second
ligand binding moieties, relative
to one or more non-target cell populations. 37. A multispecitic ligand
according to paragraph 3ti, wherein
the affinity of first ligand binding moiety for the first ligand is at least,
approximately, one, two, three, four,
five, six, seven or eight orders of magnitude greater than the affinity of the
second ligand bindings moiety
for the second li~~and. 38. A multispecific ligand according to paragraph 3(i
or p7, wherein first and second
ligands are bioavailable fbr contemporanous* recognition by the first and
second ligand binding moieties.
39. A method of controlling the biodistribution of a ligand which interacts
with a tar~~tt li~~and present on a
hetero<~enous population of ligand bearing entities, said method comprising
using a multispecitic ligand
comprising at least a first ligand binding moiety which preferentially* binds
with a pre-selected* first
af~inity9 to at least a first ligand associated with a target sub-population
of said hetero~~eneous population
on which said first ligand and target (second) li'~and are bioavaible for
contemporaneous recognition and a
second ligand binding moiety which preferentially binds with a pre-selected
lesser af~tinity to said target
ligand, and wherein the affinity of first and second ligand binding moieties
are selected to bias the
biodistribution of the multispecific ligand in favor of said target sub-
population of ligand bearings entities.
40. A method of testing the biological effects of lirnitin'; the
biodistribution ofa ligand which interacts with
a target ligand present on a heterogenous population of lli~~and bearings
entities, said method comprising the
step of administering a multispecitic li<~and comprising at least a first
ligand binding moiety which
preferentially* binds with a pre-selected* first affinity "' to at (east a
first ligand associated with a target
sub-population of said population of ligand bearing entities on which said
first li~~and and target (second)
ligand are bioavaible for contemporanous recognition and a second ligand
binding moiety which
preferentially binds with a pre-selected lesser affinity to said tartlet
ligand, and wherein the affinity of first
and second li~~and bindiny_ moieties are selected to bias the biodistribution
of the multispecitic li~~and in
favor of said target sub-population of ligand bearing entities. 41. A
multispecitic li'.~and which
preferentially binds to a target li~~and on a selected sub-population of a
heterogeneous population of cells
bearing the target-ligand, the multispecific ligand comprising a first ligand
binding moiety which
preferentially binds to a cell sub-population associated ligand and a second
ligand binding moiety which
binds to the tar~~et-ligand. said first li~~and binding moiety having an
affinity for the sub-population
associated ligand that is higher than the affinity of the second ligand
binding moiety for the tartlet ligand.
G the term approximately in the context of orders of ma~~nitude variations in
affinity refers a variability that
is up to a half an order or magnitude.
' having regard to their respective bioavailabilities
R The term "overlap" connotes that notwithstanding the difference in
distributions of the first and second
ligands the first and second ligands are bioavailable for recognition on the
same entity. this term and
related terms, exemplified below, are intended to exclude a situation where
both ligands are preferentially
expressed on substantially the same entity, for example mvo different tumor
associated antigens associated
differentially with a differentiated population of cells within a tumor , most
paoicularly in the case where
they are individually suitable tartlets for delivery of a toxic payload, and
the terms wditterent" distributions
and "heterogeneous" population are similarly understood to exclude such a
common distribution, in the
appreciation that the invention primarily represents an improved strategy for
targeting two different
ligands, in which one li~~and has a broader distribution than the other or
both have distributions that may
overlap but are different from that of the target population. It will also be
appreciated that the invention has
particular application to a situation in which at least one of the non-target
populations is one on which one
of said first and second ligands is substantially represented (in contrast to
one on which it simply enjoys
limited expression).
'' Contrasted to functional affinity which may result from avidity
'° Contrasted to functional affinity which may result from avidity
93
CA 02402930 2002-09-19
42. A multispecific ligand according to paragraph 41, wherein the affinity of
said first ligand binding
moiety for the cell sub-population associated li<~and is approximately, one,
two, three. tour, five, six, seven
or eight orders of magnitude greater than the affinity of said second ligand
binding moiety for said target
ligand. 43. A multispecitic ligand according to para<graph 42, wherein sai<t
target ligand is a receptor or a
receptor ligand'r . 44. A rnultispecitic ligand according to paragraph 42.
wherein at least one of said first or
second ligand binding moieties comprises an antibody heavy chain or functional
portions) thereof
includin<~ a VH or fra~~ment thereof and an antibody light chain or functional
portions) thereof including a
VH or fragment thereof. 4s. A method of selectively exerting a biological
effect mediated through binding
a tar~~et-ligand on a selected sub-population of <r population of cells
bearings the target-ligand, the method
comprising the step of exposing the cells to a multispecitic ligand
comprisin<T a first ligand binding moiety
which preferentially binds to a cell sub-population associated ligand and a
second ligand bindin~7, moiety
which binds to the target ligand, said first ligand moiety having an affinity
for the sub-population
associated ligand that is hi;~her than the affinity of the second ligand
binding moiety for the target ligand.
46. A method accordin~~ to paragraph 4S, wherein the affinity of said first
ligand binding moiety for the cell
sub-population associated li~~and is at least approximately, one. two, three,
four, five, six, seven or eight
orders of magnitude ;greater than the affinity of said second li<~and binding
moiety for said target ligand. 47.
A method accordin~~ to para~~raph 4>, wherein said tar<~et ligand is a
receptor or a receptor ligand. 48. A
method according to para~,raph 4~ wherein at least one~of said first or second
ligand binding moieties is an
antigen binding firagment'v of an antibody . 49. A method of testing or
controllin~~ the biological effects of a
ligand binding molecule by circumscribin~~ its ability to bind to a diverse
population of cells bearing a
complementary target ligand, said method comprising usin<~ said ligand binding
molecule together with a
different li~~and binding,, molecule which preferentially binds to another
target li~,~and which is exclusively
or preferentially associated with one or more sub-populations) of said
population of cells, and wherein the
biological effects of said ligan d bindings molecule are controlled through
prior association of said ligand
binding molecule with said other li~~and bindin' molecule to form a
multispecitic li~.:and and through the
affinity of at least one of said li~~and binding molecules being pre-selected
to limit the propensity of said
ligand binding molecule to bind to cells within said population of cells which
do not preferentially express
said other target ligand. sU. A method according to paragraph 49, wherein the
affinity of said ligand
binding molecule for said complementary target ligand is less than the
affinity of the other ligand binding
molecule for the other target figand. 51. A method according to paragraph 49
or 50, wherein at least one of
said first and second ligand binding molecules is an antibody*. 52. A method
according to paragraph 49
wherein said first ligand bindings molecule is an entity which exerts a
biologic effect and said second ligand
binding molecule is a multispecitic ligand comprisin~~ a first li«and bindin;:
moiety that binds to a cell sub-
population associated li~~and and a second ligand bindings moiety which binds
to said entity. >3. A method
according to paragraph 52 wherein said second ligand bin<~in<~ moiety is an
antibody which binds to a pre-
selected epitope on said entity and wherein the epitope of said second ligand
bindings moiety is selected on
the basis of its proximity to a ligand binding porrtion of said entity such
that the entity when bound to said
multifunctional ligand has an affinity for said ligand which is less than the
affinity of said first li~:and
binding moiety for said cell sub-population associated ligand. ~4. A
multispecific ligand according to
paragraph 12, wherein said second ligand is a Il_,-8 receptor, a CCR7
receptor, a I=AS receptor, or a CXCR4
receptor. 55. A multispecific ligand according to para<~raph 6, wherein said
marker is associated with an
immune cell that is succeptible to viral infection. ~6. A multispecitic ligand
accordin~~ to paragraph 5>,
wherein said marker is CD4. ~7. A multispecific ligand accordin~~ to
para~:raph ~5 or 56, wherein said
second ligand is a CCRS or C XCR4 receptor. s8. A multifunctional ligand
accordin~~ to paragraph 52,
wherein said entity is a biologic effector li~~and. 59. A multispecific
ligand, according to paragraph 36,
Wherein the affinities of said first and second li~and bindings moieties are
both selected to limit their
individual ability to bind to the first and second li'~ands, respectively. and
wherein their combined
functional affinity biases the distribution of the multifunctional in favour
of said target cell population. 60.
An antibody which binds to an epitope on an entity which exerts a biologic
effect via a binding interaction
with a target ligand, said epitope being proximal to the binding site of said
entity for the target ligand, such
that the antibody bound to the entity reduces the affinity of the entity for
its ligand without precluding its
functional binding activity vis-a-vis said li'~and. 61. A multispecitic
ligand, comprisin~~ a first lig<md
binding moiety which binds with a pre-selected affinity to a target entity
associated ligand and a second
ligand bindings moiety which binds with pre-selected affinity to an epitope on
a biologic effector ligand
"The term "receptor ligand" means a target ligand which is a ligand for a
receptor, for example, ;r receptor
on a cell or infectious agent or a receptor which circulates independently of
another entity.
~' The term "antigen binding fra<'~rnent' refers to a polypeptide or a
pluralitw of associated polypeptides
comprising one or more portions of an antibody includin~~ at least one VI-1 or
VL or a functional fragment
thereof.
94
CA 02402930 2002-09-19
which exerts a biologic effect via a binding interaction with a target ligand
, said epitope being proximal to
the binding site of said biologic effector ligand for the target ligand, such
that the second ligand moiety
bound to the biologic effector ligand reduces the affinity of the molecule for
the target ligand without
precluding its functional binding activity vis-a-vis said ligand and wherein
said target ligand is present on a
diverse population of entities consisting of the target entity and one or more
non-target entities and
wherein the affinity of the first ligand binding moiety for the target entity
associated ligand is greater than
that of the biologic effoctor ligand for the target ligand when bound to
second ligand molecule, and
wherein the affinity of the first ligand binding moiety is selected to bias
the biodistribution of said biologic
effector ligand in favour of the target entity relative to the non-target
entit('ies). 62. A multispecific ligand
according to paragraph 61, wherein first ligand binding moiety comprises a
light chain linked to an
antibody heavy chain portion comprising at least a VH, CH 1 domain, hinge
region and preferably at least a
truncated Fc portion and said second ligand binding moiety comprises at least
a VL linked to a heavy chain
portion, optionally through a disultide bond'' and wherein the heavy chain
portion of said second ligand
binding moiety is devoid of CH I domain, and comprises a hinge region and
preferably at least a truncated
Fc portion, and wherein said heavy chain portions are linked via their
respective hinge regions and
optionally wherein the respective hinge regions are wholly or partially
substituted or supplemented by a
another linkage pair" eg. a leucine zipper. 63. A multispecitic ligand
according to paragraph 12, wherein
said second ligand is a marker associated with a lymphatic endothelial cell.
64. A multispecific li~~and
comprising a first ligand bindin<:,~ moiety which preferentially binds to a
lymphatic endothelial cell
associated marker and a second moiety which exerts a biologic function",
optionally a therapeutic function,
optionally an immune function*, optionally at least one of an immunizing"'
function, a tolerizin~~ function,
a neutralizing" function, an immune mediating function, and immune modulating
funetion'R, in relation*
to an independent "entity, preferably within the lymphatic system. 65. A
multispecific ligand accordin'= to
paragraph 62, wherein the marker is selected to limit the ability of said
endothelial cell to internalize said
multispecific ligand. 66. A multifunctional liaand havinvg, at least, a first
portion which binds to a
lymphatic vessel associated ligand and a second portion comprising an immune
function exerting moiety.
67. A multifunctional ligand as defined in paragraph 66, wherein said tirst
portion is an antibody. 68. A
multifunctional ligand as detined in para;raph 66, wherein said immune
function exerting moiety binds to a
target ligand. 69. A multifunctional ligand as defined in paragraph 67,
wherein said immune function
exerting moiety comprises an antibody. 70. A multifunctional ligand as defined
in para~~raph 68, wherein
said immune function exerting moiety comprises an antibody. 71. A
multifunctional ligand as defined in
paragraph 68, wherein said immune function exerting moiety binds to a ligand
selected form the ~~roup
consisting of CC RS, CTLA-4, LFA-l, ICAM-I. CD2, CD3. CD4, CD22, CD40, CD44:
CD80, CD86,
CD134 and CD1~4. 72. A multifunctional ligand as defined in paragraph 70,
wherein said first portion
binds to LYVE-1 or podoplantin. 73. A multifunctional ligand as defined in
paragraph 70, wherein said
immune function exertin<~ moiety comprises an anti-idiotypic antibody. ','~. A
multifunctional li~and as
defined in paragraph 73, wherein said anti-idiotypic antibody binds to an
autoimmune antibody~75. A
multifunctional ligand as defined in paragraph 73, wherein said anti-idiorypic
antibody mimics a cell
surface expressed tumor antigen or a viral antivgen. 76. A multifunctional
ligand as def fined in paragraph 70,
wherein said immune function exertin~~ moiety binds to a diseased cell. 77. A
multifunctional ligand as
defined in paragraph 70. wherein said immune function exerting moiety binds to
an infectious ;agent or
parasite. 78. A multifunctional ligand as defined in paragraph 76, wherein
said diseased cell is a cancer cell.
79. A multifunctional li~gand as defined in paragraph 76. wherein said
diseased cell is a virally infected cell.
'' For example of the disulfide stabilized type developed by the NCI
''' eg. fos-jun
's The maiety that exerts a biologic function is understood to be a biologic
effector in the sense that its
intended interaction with an entity in the lymphatic system or elsewhere in
the organism has a biological
consequence.
'~' For example using a toxin or immunogen fused or conjugated to (or having a
corresponding li~gand on
the second binding moiety to which it binds) to an antibody which recognizes a
lymphatic endothelial
marker, for example an anthrax toxin fusion
" The tern neutralizing is used broadly to refer to any interposition,
interference or impediment which
affects the function of the target entity
'k the terms modulating, mediating, neutralizing function etc. are not
intended to be mutully exclusive and
are each used broadly, for example the term modulating referring to effecting
a change, and the term
mediating preferably connoting an indirect effect achieved through the
instrumentality of another entity, for
example a cell, cytokine, chemokine etc..
''' 1e. an entity other than the lymphatic endothelial cell and other than any
cell to which the first moiety is
anchored.
CA 02402930 2002-09-19
80. A multifunctional ligand as defined in paragraph 68, wherein said immune
function exerting moiety
binds to an immune cell. 81. A multifunctional ligand as detined in paragraph
69, 76 or 80 wherein said
immune function exerting moiety binds with greater functional affinity co its
target li«and than said first
portion binds to its tartlet ligand. 82. A multifunctional ligand as detine<t
in paragraph 69, 76 or 80 wherein
said immune function exerting moiety binds with greater affinity to its target
ligand than said first portion
binds to its target ligand. 83. A multifunctional li~~and as defined in
paragraph 69. 76 or 80, wherein said
binds with greater avidity to its tartlet ligand than said first portion binds
to its tar~,et li~~and. 8~. A
multifunctional ligand as defined in paragraph 80, wherein immune cell is
associated with an autoimmune
reaction. 8~. A multifunctional ligand as defined in paragraph 80, wherein
said immune cell is a CC.'R5-
expressing cell. 86. A multifunctional ligand as defined in paragraph 78 or
80, wherein said second portion
comprises an internalizin~~ antibody and a cytotoxic component. 87. A
multifunctional li~and as detined in
paragraph 78 or 80, which is a bispecitic antibody having a monovalent first
portion and a monovalent
second portion. 88. A multifunctional ligand as detined in paragraph 78 or 80,
which is a bispecific
antibody havin« a divalent first portion and a divalent second portion. 89. A
multifunctional lit;and as
defined in paragraph 78 or 80, which is a trispecific antibody having a
monovalent tirst portion and a
second portion comprisin~~ a divalent immune function exerting moiety which
binds to one or more target
ligands on a target diseased cell or immune cell and a movovalent anti-('D3 or
anti-CD?8 antibody. 90. A
multifunctional li~~and adefined in para~~raph 78 or 80, which is a trivalent
trispecific antibody having a
monovalent first portion and a second portion comprising a divalent immune
function exerting moiety
which binds to a target ligand on a tartlet diseased or immune cell. 91. A
multifunctional ligand as defined
in paragraph 78, wherein said second portion comprises a cytokine component.
92. A multifunctional
li~~and as defined in para~~raph 78, wherein said second portion comprises a
cytotoxic component. 93. A
multifunctional ligand as defined in paragraph 78, w herein said second
portion comprises a ligand which is
capable of binding to T cells. 94. A multifunctional ligand as defined in
paragraph 87, wherein said ligand
is an antibody which binds to T cells. 9>. A multifunctional ligand as
delfined in paragraph 78, wherein said
second portion comprises an anti-CD3 antibody or anti-CD?8 antibody. 96. A
multifunctional ligand as
deFned in para~~raph 67. wherein second portion is a cytokine component. 97. A
multifunctional li~and as
defined in paragraph 67. wherein second portion is an anti-CD3 antibody or an
anti-CD28 antihody. 98. A
multifunctional ligand as defined in paragraph 1 3, wherein said second
portion further comprises one or
more components selected from the group consisting of a cytokine component, a
cytotoxic ec>mponent and
an anti-CD3'CD?8 component. 99. A multifunctional li~and as detined in
para~~raph 14, wherein said
second portion further comprises one or more components selected from the
group consistin<~ of a cytokine
component, a cytotoxic component and an anti-CD3.'CD28 component. 100. A
pharmaceutical composition
comprisin~u a multifunctional ligand as detined in para~~raph I OI. A
pharmaceutical composition
comprising a plurality of different multifunctional ligands. 102. A
pharnoaceutica) composition as defined
in paragraph 10i. wherein said plurality of different multifunctional ligands
exert a cooperative immune
effect. 103. A pharmaceutical composition as detined in para~~raph 101.
wherein said plurality of different
multifunctional ligands ecnnprise a multifunctional ligand as described in
para~~raph 76 and at feast one or
bath of the multifunctional ligands described in paragraph 95 or 96. 104. A
method of inhibiting the
formation of metastasis during the course of surgical removal of a tumor
comprising administering to a
patient prior to sur~~ieal treatment of the tumor site, a pharmacetical
composition comprisin~~ a
multifunctional IiYand as described in paragraph 78. 10~. An immunocytokine
comprisin« an anti-idiotypic
antibody which recognizes the paratope ofan antibody which binds to a
lymphatic vessel associated ligand
and a cytokine fused therewith or conjugated thereto. 106. An immunocytokine
as detined in paragraph
i O5, wherein said cytokine component comprises lL-2 or a functional fra;~ment
thereof and'or 11_-12 or a
functional fragment thereof 107. An immunocytokine as defined in paragraph 42,
wherein said cytokine
component comprises TNF-a or a functional fragment thereof 108. A bis,pecific
antibody comprising an
anti-idiotypic antibody which recognizes the paratope of an antibody which
binds specifically to a
lymphatic vessel associated li~and and an anti-CD3 antibody or an anti-CD28
antibody. 109. A
multifunctional ligand accordin~~ to paragraph 66 comprising one or more amino
acids that are substituted
for amino acids that contribute to an immunogenic epitope. I 10. A
multifunctional ligand having, at least, a
first portion which binds to a lymphatic vessel associated ligand and a second
porrtion comprising an
independent therapeutic function exerting moiety. 1 1 1. ~4 bispecitic ligand
comprising a tirst ligand which
binds to a tirst tarvuet ligand and a second li~,and which binds to a second
tartlet lir~and. and wherein the
affinity of said first li~~and is selected to enable binding to the first
tar~,ret ligand independently of the ability
of said second liaand to bind to the second target ligand and wherein the
e~ffinity of said second ligand is
selected to substantially reduce the probability of its bindings to the second
target ligand without the first
ligand bindin~a first or substantially contemporaneously to the first target
liv~and. I 12. A bispecific antibody
comprisin<,~ a first antibody component which binds to a first target ligand
and a second antibody
component which binds to a second target ligand, and wherein the affinity or
avidity or both the affinity
96
CA 02402930 2002-09-19
and avidity of said first antibody component are selected to enable binding to
the first target ligand
independently of the ability of said second antibody component to bind to the
second target li~and and
wherein the avidity or affinity or bout the affinity and avidity of said
second ligand are selected to
substantially reduce the probability of its binding to the second target
ligand without the first ligand binding
first or substantially contemporaneously to the first target ligand. 1 13. A
multispecific ligand comprising a
first moiety which binds to a first target li<~and and a second moiety which
binds to a second target ligand,
and wherein the affinity or avidity or both the affinity and avidity of said
first moiety are selected to enable
the first moiety to bind to the first tartlet li~~and independently of the
ability of said second moiety to bind to
the second target ligand and wherein the avidity or affinity or both the
affinity and avidity of said second
moiety are selected to substantially reduce the probability of its binding to
the second target ligand without
the first moiety, first or substantially contemporaneously, binding to the
first target ligand. 1 14. A
multispecitic ligand according to paragraph 1 13, wherein both moieties bind
to different target ligands on
the same cell. I I ~. A multispecitic ligand comprising a first moiety whi<:h
binds to a first target ligand and
a second moiety which binds to a second target ligand, and wherein the
affinity or avidity or both the
affinity and avidity of said first moiety and the avidity or affinity or both
the affinity and avidity of said
second moiety are selected to substantially reduce the probability of either
moiety binding for a sut~icient
duration or series of durations to its respective target ligand to a
accomplish a therapeutic function without
the other moiety. first or substantially contemporaneously, bindin<~ to its
respective tartlet ligand I I (i. A
multispecific ligand comprising a first moiety which specifically binds to a
first tartlet ligand on a first
entity and a second moiety which specifically binds to a second target ligand
on a second entity, and
wherein the affinity or avidity or both the affinity and avidity of said first
moiety are selected to enable the
tlrst moiety to bind to the first target ligand independently of the ability
oi'said second moiety to bind to the
second target li~~and and wherein the avidity or affinity or both the affinity
and avidity of said second
moiety are selected to enable the second moiety to bind to the second entity
in preference to the first moiety
binding to the first entity when both first and second moieties are
substantially contemporaneously bound
to the respective first and second entities. I 17. A multispecitic li<~and
comprising a first moiety which
specifically binds to a first target li~and on a first entity and a second
moicay which specifically binds to a
second target ligand on a second entity, and wherein the second entity binds
to a third target li~~and, and
wherein the affinity or avidity or both the affinity and avidity of said first
;~~oiety are selected to enable the
first moiety to bind to the first target ligand independently of the ability
of said second moiety to bind to the
second target ligand and wherein the avidity or affinity or both the affinity
and avidity of said first moiety
are selected to enable the first moiety to bind to the first entity in
preference to the second moiety binding
to the second entity when hoth first and second moieties are substantially
<;ontemporaneously bound to the
respective first and second entities, and wherein the avidity or affinity or
both the ai~inity and avidity of
said second moiety are selected to enable the third target ligand to bind to
the second entity in preference to
the second moiety bindings to the second entity when both said third target
ligand and the second moiety are
substantially contemporaneously bound to the second entity. I 18. A
multispecitic li~~and comprisin<~ at least
a first li~;and binding moiety which specifically binds to a first ligand
havin~~ a first biodistribution and a
second ligand binding moiety which specifically binds to a second li<~and
having a second biodistribution.
and wherein the affinity of the first and second ligand binding moieties are
different and selected to bias the
biodistribution of the multispecific ligand, and wherein the affinity of the
first ligand binding_ moiety for the
first ligand is at least, approximately, one order of magnitude greater than
that of the second ligand binding
moiety for the second ligand. The affinity of the first ligand bindin<~ moiety
for the first ligand is optionally
at least, approximately, two orders of magnitude ~~reater than that of the
second li~~and binding moiety for
the second ligand. The affinity of the first ligand binding moiety for the
first ligand is optionally at least,
approximately, three orders of magnitude ~~reater than that of the second
li,~and binding moiety for the
second ligand. The affinity of the first ligand binding' moiety for the first
ligand is optionally at least,
approximately, four orders of magnitude greater than that of the second
li~~and bindings moiety for the
second Iigand. The affinity of the first ligand binding moiety for the first
ligand is optionally at least,
approximately, five orders of magnitude greater than that of the second ligand
bindings moiety for the
second li~~and.'i'he affinity of the first ligand binding moiety for the first
lit;and is optionally at least,
approximately, six orders of magnitude ;realer than that of the second ligand
binding moiety for the second
ligand. The affinity of the first ligand binding moiety for the first ligand
is optionally at least,
approximately, seven orders of magnitude ~,~reater than that of the second
ligand binding moiety for the
second ligand The affiinity cof the first ligand binding moiety for the first
ligand is optionally at least.
approximately, eight orders of ma~~nitude greater than that of the second
li~~and binding moiety for the
second ligand. I 19. A multispecific ligand according to paragraph I 18,
wh~°rein the first ligand is present
on a first target cell population and wherein said second ligand is present on
a second target cell population
comprising the first target cell population and wherein the biodistribution of
the multispecific ligand
favours the first target cell population. 120. A multispecitis ligand
according to paragraph I 19, wherein
97
CA 02402930 2002-09-19
said multispecific li~~and is capable of contemporaneously binding the first
and second ligands on said
target population. 121. A host cell or cell free expression medium comprising
one or more polynucleotides,
said one or more polynucleotides comprising one or more DNA sequences, said
one or more DNA
sequences comprising one or more polypeptides which are sufficient to
constitute a multispecific ligand as
defined in any of the preceding para<.;raphs . 122. A kit comprisin~~ one or
more polynucleotides. said one or
more polynucleotides comprisin~~ one or more DNA sequences, said one or more
DNA sequences encoding
one or more polypeptides which are sufficient to constitute a multispecitic
ligand as defined in any of the
preceding paragraphs. 123. A liquid medium comprising comprising one or more
poly peptides which are
sufficeint to constitute a multispecific li~~and as defined in any of the
preceding paragraphs. 12~. A liquid
medium comprising one or more host cells, said one or more host cells
comprising one or more
polynucleotides, said one or more polynucleotides comprising one or more DNA
sequences. said one or
more DNA sequences encoding one or more polypeptides which are sutfc;eint to
constitute a multispecific
ligand as defined in any of the preceding paragraphs. 125. A substantially
isolated polynuceotide
comprising a DNA sequence encodin<~ a polypeptide porrtion of a second ligand
binding moiety as defined
in any of the preceding claims. said polypeptide portion comprising a VH or
VL, said second li~~and
binding moiety havin<~ a low affinity for said second ligand. 126. A
substantially isolated polynucleotide
according to paragraph 125, wherein said polynucleotide is a substantially
isolated expression or cloning
vector. 137. A method of making a multispecific ligand as defined in any of
the preceding para<=raphs
comprising expressin~~ at least one polynucleotide as defined in paragraph 122
or 135. 128. A y
pharmaceutical composition comprisin~~ a multispecitic li~~and as defined in
any of the preceding
paragraphs and a pharmaceutically acceptable excipient. 129. A therapeutic
composition comprisin~~ a
multispecific ligand as defined in any of the preceding para<~raphs and a
pharmaceutically acceptable
excipient. 130. A method of treating a disease in a mammal comprising
administerin~~ a therapeutically
effective amount of a multispecitic ligand according to any of the preceding
claims. 13 I . A kit comprising
a plurality of different multispecitic ligands as defined herein.
Notwithstanding any indication to the contrary, it will be appreciated that
the references herein cited have
application to multiple different subjects and any qualifying remarks as to
the applicability of the references
is to be understood as relating to each of the subjects tar which references
are herein provided, as limited
only by the title and subject matter of the reference.
All publications and references therein cited are herein incorporated by
reterence to the same extent as if
each ofthe individual publications were specifically and individually
indicated to be incorporated by
reference in its entirety.
98
