U.S. patent application number 10/492117 was filed with the patent office on 2005-02-10 for immunoconjugates for the treatment of tumours.
Invention is credited to Corti, Angelo, Curnis, Flavio.
Application Number | 20050033026 10/492117 |
Document ID | / |
Family ID | 9935816 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050033026 |
Kind Code |
A1 |
Corti, Angelo ; et
al. |
February 10, 2005 |
Immunoconjugates for the treatment of tumours
Abstract
A pharmaceutical composition comprising a conjugate of a
cytokine and a tumor targeting moiety (TTM) and a pharmaceutically
acceptable excipient, wherein the cytokine is present in an amount
which does not induce a negative feedback mechanism.
Inventors: |
Corti, Angelo; (S. Paolo,
IT) ; Curnis, Flavio; (Milan, IT) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
9935816 |
Appl. No.: |
10/492117 |
Filed: |
September 17, 2004 |
PCT Filed: |
April 30, 2003 |
PCT NO: |
PCT/IB03/02187 |
Current U.S.
Class: |
530/351 ;
424/85.1; 424/85.2; 530/399 |
Current CPC
Class: |
A61K 51/0495 20130101;
A61K 47/6813 20170801; A61K 47/642 20170801; C07K 14/525 20130101;
A61K 47/64 20170801; A61K 38/191 20130101; C07K 2319/01 20130101;
C07K 14/57 20130101; A61K 49/0008 20130101; A61K 47/6887 20170801;
A61K 47/6851 20170801; A61P 35/00 20180101 |
Class at
Publication: |
530/351 ;
424/085.1; 424/085.2; 530/399 |
International
Class: |
C07K 014/53; C07K
014/54; A61K 038/20; A61K 038/19; A61K 038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2002 |
GB |
0209896.0 |
Claims
1. A pharmaceutical composition comprising a conjugate of a
cytokine and a tumor targeting moiety (TTM) and a pharmaceutically
acceptable carrier, diluent or excipient, wherein the cytokine is
present in an amount which does not induce a negative feedback
mechanism.
2. A pharmaceutical composition according to claim 1 wherein the
cytokine is present in an amount which does not induce soluble
cytokine receptor shedding.
3. A pharmaceutical composition according to claim 1 wherein the
conjugate is present in an amount to provide a dosage in the range
of 0.5 to 500 ng/kg.
4. A pharmaceutical composition according to claim 1 wherein the
conjugate is present in an amount to provide a dosage in the range
of 1 to 50 ng/kg.
5. A pharmaceutical composition according to claim 1 wherein the
conjugate is present in an amount to provide a dosage in the range
of 5 to 15 ng/kg.
6. A pharmaceutical composition according to claim 1 wherein the
conjugate is present in an amount such that the conjugate, or a
metabolite thereof, is provided to the blood plasma of a patient to
be treated in an amount of no greater than about 35,000 ng/day for
a 70 kg patient.
7. A pharmaceutical composition according to claim 1 wherein the
cytokine is an inflammatory cytokine.
8. A pharmaceutical composition according to claim 1 wherein the
cytokine is a chemotherapeutic cytokine.
9. A pharmaceutical composition according to claim 1 wherein the
cytokine is TNF.alpha., TNF.beta., IFN.alpha., IFN.beta.,
IFN.gamma., IL-1, 2, 4, 6, 12, 15, EMAP II, vascular endothelial
growth factor (VEGF), PDGF, PD-ECGF or a chemokine.
10. A pharmaceutical composition according to claim 1 wherein the
cytokine is TNF-.alpha., TNF-.beta. or IFN-.gamma..
11. A pharmaceutical composition according to claim 1 wherein the
TTM is a tumor vasculature targeting moiety (TVTM).
12. A pharmaceutical composition according to claim 11 wherein the
TVTM is a binding partner of a tumor vasculature receptor, marker
or other extracellular component.
13. A pharmaceutical composition according to claim 1 wherein the
TTM is a binding partner of a tumor receptor, marker or other
extracellular component.
14. A pharmaceutical composition according to claim 1 wherein the
TTM is an antibody or ligand, or a fragment thereof.
15. A pharmaceutical composition according to claim 1 wherein the
TTM is contains the NGR or RGD motif, or is HIV-tat, Annexin V,
Osteopontin, Fibronectin, Collagen Type I or IV, Hyaluronate,
Ephrin, or is a binding partner to oncofetal fibronectin; or a
fragment thereof.
16. A pharmaceutical composition according to claim 1 wherein the
TTM contains the NGR motif.
17. A pharmaceutical composition according to claim 1 wherein the
TTM is CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC, linear or
cyclic CNGRC.
18. A pharmaceutical composition according to claim 1 wherein the
TTM contains the NGR motif.
19. A pharmaceutical composition according to claim 1 wherein the
TTM is targeted to VEGFR, ICAM 1, 2 or 3, PECAM-1, CD31, CD13,
VCAM-1, Selectin, Act R11, ActRIIB, ActRI, ActRIB, CD44,
aminopeptidase A, aminopeptidase N (CD13), .alpha.v.beta.3
integrin, .alpha.v.beta.5 integrin, FGF-1, 2, 3, or 4, IL-IR, EPHR,
MMP, NG2, tenascin, oncofetal fibronectin, PD-ECGFR, TNFR, PDGFR or
PSMA.
20. A pharmaceutical composition according to claim 1 wherein the
conjugate is in the form of a fusion protein.
21. A pharmaceutical composition according to claim 1 wherein the
conjugate is in the form of nucleic acid.
22. A pharmaceutical composition according to claim 1 wherein the
composition further comprises another antitumor agent or diagnostic
tumor-imaging compound.
23. A pharmaceutical composition according to claim 22 wherein the
further antitumor agent is doxorubicin, melphalan or cisplatin.
24. [canceled]
25. A method of treating or diagnosing cancer comprising
administering to a patient in need of the same a conjugate as
defined in claim 1 in an effective amount, wherein said amount does
not induce a negative feedback mechanism.
Description
FIELD OF THE INVENTION
[0001] Immunoconjugates for the treatment of tumours
BACKGROUND OF THE INVENTION
[0002] The antitumor activity of some cytokines is well known and
described. Some cytokines have already been used therapeutically in
humans. For example, cytokines such as IL-2 and IFN-.gamma. have
shown positive antitumoral activity in patients with different
types of tumors, such as kidney metastatic carcinoma, hairy cell
leukemia, Kaposi sarcoma, melanoma, multiple mieloma, and the like.
Other cytokines like IFN.beta., the Tumor Necrosis Factor (TNF)
.alpha., TNF.beta., IL-1, 4, 6, 12, 15 and the Colony Stimulating
Factors (CFSs) have shown a certain antitumoral activity on some
types of tumors.
[0003] In general, the therapeutic use of cytokines is strongly
limited by their systemic toxicity. TNF, for example, was
originally discovered for its capacity for inducing the hemorrhagic
necrosis of some tumors, and for its in vitro cytotoxic effect on
different tumoral lines, but is subsequently proved to have strong
pro-inflammatory activity, which can, in case of overproduction
conditions, dangerously affect the human body.
[0004] As the systemic toxicity is a fundamental problem with the
use of pharmacologically active amounts of cytokines in humans,
novel derivatives and therapeutic strategies are now under
evaluation, aimed at reducing the toxic effects of this class of
biological effectors while keeping their therapeutic efficacy.
[0005] Some novel approaches are directed to:
[0006] a) the development of fusion proteins which can deliver TNF
into the tumor and increase the local concentration. For example,
the fusion proteins consisting of TNF and tumor specific-antibodies
have been produced;
[0007] b) the development of TNF mutants which maintain the
antitumoral activity and have a reduced systemic toxicity.
Accordingly, mutants capable of selectively recognizing only one
receptor have already been prepared;
[0008] c) the use of anti-TNF antibodies able to reduce some toxic
effects of TNF without compromising its antitumoral activity. Such
antibodies have already been described in literature;
[0009] d) the use of TNF derivatives with a higher half-life (for
example TNF conjugated with polyethylene glycol).
[0010] The preparation of TNF derivatives capable of selectively
targeting the tumoral sites has recently been reported. For
example, a fusion protein has been described, obtained by fusing
the gene of the heavy chain of an anti-transferrin receptor mAb and
the TNF gene, or a fusion protein of INF with the "hinge" region of
a monoclonal antibody against the tumor-associated TAG72 antigen,
or a Fv-TNF fusion protein.
[0011] EP 251 494 discloses a system for administering a diagnostic
or therapeutic agent, which comprises: an antibody conjugated with
avidin or streptavidin, an agent capable of complexing the
conjugated antibody and a compound consisting of the diagnostic or
therapeutic agent conjugated with biotin, which are administered
sequentially and adequately delayed, so as to allow the
localization of the therapeutic or diagnostic agent through the
biotin-streptavidin interaction on the target cell recognized by
the antibody. The described therapeutic or diagnostic agents
comprise metal chelates, in particular chelates of radionuclides
and low molecular weight antitumoral agents such as cis-platinum,
doxorubicin, etc.
[0012] EP 496 074 discloses a method which provides the sequential
administration of a biotinylated antibody, avidin or streptavidin
and a biotinylated diagnostic or therapeutic agent. Although
cytotoxic agents like ricin are generically mentioned, the
application relative to radiolabelled compounds is mostly
disclosed.
[0013] WO 95/15979 discloses a method for localizing highly toxic
agents on cellular targets, based on the administration of a first
conjugate comprising the specific target molecule conjugated with a
ligand or an anti-ligand followed by the administration of a second
conjugate consisting of the toxic agent bound to an anti-ligand or
to the ligand.
[0014] WO 99/13329 discloses a method for targeting a molecule to
tumoral angiogenic vessels, based on the conjugation of said
molecule with ligands of NGR receptors. A number of molecules have
been suggested as possible candidates, but doxorubicin only is
specifically described. No use of ligands of NGR receptors as
cytokines vehicles to induce immuno responses is disclosed.
[0015] In WO01/61017 the current inventor describes how
surprisingly he has found that the therapeutic index of certain
cytokines can be remarkably improved and their immunotherapeutic
properties can be enhanced by coupling with a ligand of the
aminopeptidase-N receptor (CD13). CD13 is a transmembrane
glycoprotein of 150 kDa which is highly conserved in various
species. It is expressed on normal cells as well as in myeloid
tumor lines, in the angiogenic endothelium and in some epithelia
The CD13 receptor is usually identified as the "NGR" receptor, in
that its peptide ligands share the amino acid "NGR" motif.
[0016] However, there remains a need for further and improved
pharmaceutical compositions and methods for the treatment and
diagnosis of cancer.
SUMMARY OF THE INVENTION
[0017] Drug delivery and penetration into neoplastic cells is
critical for the effectiveness of tumor chemotherapy. We have now
found that surprisingly targeted delivery of picogram doses of
cytokines enhances the penetration of chemotherapeutic drugs. In
more detail we have found that delivery of very low doses of
cytokines to tumors and the tumor-associated environment including
tumor vasculature represents a new approach to avoiding negative
feedback mechanisms and to preserve its ability to alter
drug-penetration barriers. The present invention thus represents a
novel and surprising strategy for increasing the therapeutic index
of chemotherapeutic drugs. In a preferred embodiment, we have found
using vascular targeting achieved by coupling TNF with CNGRC, a
peptide that targets the tumor neovasculature, that this treatment
enhanced 8-10 times the therapeutic efficacy of doxorubicin, with
no evidence of increased toxicity. Similarly, vascular targeting
enhanced the efficacy of melphalan, a different chemotherapeutic
drug. Synergy with chemotherapy was observed with 3-5 ng/kg of
targeted TNF (i.p.), 10.sup.6-fold lower than the LD.sub.50 and
10.sup.5-fold lower than the dose required for non-targeted TNF. In
addition, we have also found that targeted delivery of low doses of
TNF to tumor vessels does not induce the release of soluble TNF
receptors in circulation. We have also found that RGD-TNF and
IFN.gamma.-NGR are active in the picogram range. We have also shown
that NGR-TNF increases the effect of cisplatinum. These results
indicate that the delivery of minute amounts of cytokines to tumor
vessels represents a new approach to avoid negative feedback
mechanisms and to preserve its ability to alter drug-penetration
barriers. Vascular targeting in this way represents a novel
strategy for increasing the therapeutic index of chemotherapeutic
drugs.
[0018] Statements of the Invention
[0019] According to one aspect of the present invention there is
provided a pharmaceutical composition comprising a conjugate of a
cytokine and a tumor targeting moiety (TIM and a pharmaceutically
acceptable carrier, diluent or excipient, wherein the cytokine is
present in an amount which does not induce a negative feedback
mechanism. Thus, the cytokine is present in an amount which does
not induce soluble cytokine receptor shedding.
[0020] Preferably the conjugate is present in an amount to provide
a dosage in the range of 0.5 to 500 ng/kg, more preferably 1 to 50
ng/kg, and most preferably 5 to 15 ng/kg.
[0021] In one embodiment the cytokine is an inflammatory
cytokine.
[0022] In another embodiment the cytokine is a chemotherapeutic
cytokine.
[0023] Preferably the cytokine is selected from: TNF, such as
TNF.alpha., TNF.beta., IFN.alpha., IFN.beta., IFN.gamma., IL-1, 2,
4, 6, 12, 15, EMAP II, vascular endothelial growth factor (VEGF),
PDGF, PD-ECGF or a chemokine.
[0024] In a more preferred embodiment the cytokine is TNF.alpha.,
TNF-.beta. or IFN-.gamma., most preferably TNF-.alpha. or
TNF-.beta..
[0025] In one embodiment the TTM is a binding partner such as a
ligand of, or antibody to a tumor cell surface molecule such as a
receptor, marker or other extracellular component.
[0026] In another embodiment the TTM is a tumor vasculature
targeting moiety (TVTM), and may be a binding partner such as a
ligand of, or antibody to a tumor vasculature cell surface molecule
such as a receptor, marker or other extracellular component.
[0027] In one embodiment the TIM is an antibody or fragment
thereof.
[0028] In another embodiment the TTM is a ligand or fragment
thereof.
[0029] In a preferred embodiment the TTM is a peptide containing
the NGR or RGD motif, or is HIV-tat, Annexin V, Osteopontin,
Fibronectin, Collagen Type I or IV, Hyaluronate or Ephrin, or is a
binding partner, such as an antibody to oncofetal fibronectin; or
is a fragment of any of the foregoing. In a more preferred
embodiment the TTM contains the NGR motif. In a most preferred
embodiment the TTM is CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC,
linear or cyclic CNGRC.
[0030] In another preferred embodiment the TTM is a peptide
containing the RGD motif
[0031] In one embodiment the TTM is targeted to VEGFR, ICAM 1, 2 or
3, PECAM-1, CD13, VCAM-1, Selectin, Act RII, ActRIIB, ActRI,
ActRIB, CD44, aminopeptidase A, aminopeptidase N (CD13),
.alpha.v.beta.3 integrin, .alpha.v.beta.5 integrin, FGF-1, 2, 3, or
4, IL-1R, EPHR, MMP, NG2, tenascin, oncofetal fibronectin,
PD-ECGFR, TNFR, PDGFR or PSMA.
[0032] Combinations of preferred targeting moieties and cytokines
which may be used in the present invention are shown in the Table
below.
1 Cytokine Targeting Moiety TNF-.alpha. RGD-CONTAINING PEPTIDE
TNF-.beta. RGD-CONTAINING PEPTIDE IFN-.alpha. RGD-CONTAINING
PEPTIDE IFN-.beta. RGD-CONTAINING PEPTIDE IFN-.gamma.
RGD-CONTAINING PEPTIDE IL-2 RGD-CONTAINING PEPTIDE IL-12
RGD-CONTAINING PEPTIDE EMAP II RGD-CONTAINING PEPTIDE VEGF
RGD-CONTAINING PEPTIDE IL-1 RGD-CONTAINING PEPTIDE IL-6
RGD-CONTAINING PEPTIDE IL-12 RGD-CONTAINING PEPTIDE PDGF
RGD-CONTAINING PEPTIDE PD-ECGF RGD-CONTAINING PEPTIDE CXC chemokine
RGD-CONTAINING PEPTIDE CC chemokine RGD-CONTAINING PEPTIDE C
chemokine RGD-CONTAINING PEPTIDE IL-15 RGD-CONTAINING PEPTIDE
TNF-.alpha. NGR-CONTAINING PEPTIDE TNF-.beta. NGR-CONTAINING
PEPTIDE IFN-.alpha. NGR-CONTAINING PEPTIDE IFN-.beta.
NGR-CONTAINING PEPTIDE IFN-.gamma. NGR-CONTAINING PEPTIDE IL-2
NGR-CONTAINING PEPTIDE IL-12 NGR-CONTAINING PEPTIDE EMAP II
NGR-CONTAINING PEPTIDE VEGF NGR-CONTAINING PEPTIDE IL-1
NGR-CONTAINING PEPTIDE IL-6 NGR-CONTAINING PEPTIDE IL-12
NGR-CONTAINING PEPTIDE PDGF NGR-CONTAINING PEPTIDE PD-ECGF
NGR-CONTAINING PEPTIDE CXC chemokine NGR-CONTAINING PEPTIDE CC
chemokine NGR-CONTAINING PEPTIDE C chemokine NGR-CONTAINING PEPTIDE
IL-15 NGR-CONTAINING PEPTIDE TNF-.alpha. Ligand to VEGFR TNF-.beta.
Ligand to VEGFR IFN-.alpha. Ligand to VEGFR IFN-.beta. Ligand to
VEGFR IFN-.gamma. Ligand to VEGFR IL-2 Ligand to VEGFR IL-12 Ligand
to VEGFR EMAP II Ligand to VEGFR VEGF Ligand to VEGFR IL-1 Ligand
to VEGFR IL-6 Ligand to VEGFR IL-12 Ligand to VEGFR PDGF Ligand to
VEGFR PD-ECGF Ligand to VEGFR CXC chemokine Ligand to VEGFR CC
chemokine Ligand to VEGFR C chemokine Ligand to VEGFR IL-15 Ligand
to VEGFR TNF-.alpha. Ab to VEGFR TNF-.beta. Ab to VEGFR IFN-.alpha.
Ab to VEGFR IFN-.beta. Ab to VEGFR IFN-.gamma. Ab to VEGFR IL-2 Ab
to VEGFR IL-12 Ab to VEGFR EMAP II Ab to VEGFR VEGF Ab to VEGFR
IL-1 Ab to VEGFR IL-6 Ab to VEGFR IL-12 Ab to VEGFR PDGF Ab to
VEGFR PD-ECGF Ab to VEGFR CXC chemokine Ab to VEGFR CC chemokine Ab
to VEGFR C chemokine Ab to VEGFR IL-15 Ab to VEGFR TNF-.alpha.
HIV-tat TNF-.beta. HIV-tat IFN-.alpha. HIV-tat IFN-.beta. HIV-tat
IFN-.gamma. HIV-tat IL-2 HIV-tat IL-12 HIV-tat EMAP II HIV-tat VEGF
HIV-tat IL-1 HIV-tat IL-6 HIV-tat IL-12 HIV-tat PDGF HIV-tat
PD-ECGF HIV-tat CXC chemokine HIV-tat CC chemokine HIV-tat C
chemokine HIV-tat IL-15 HIV-tat TNF-.alpha. Ligand to ICAM 1, 2 or
3 TNF-.beta. Ligand to ICAM 1, 2 or 3 IFN-.alpha. Ligand to ICAM 1,
2 or 3 IFN-.beta. Ligand to ICAM 1, 2 or 3 IFN-.gamma. Ligand to
ICAM 1, 2 or 3 IL-2 Ligand to ICAM 1, 2 or 3 IL-12 Ligand to ICAM
1, 2 or 3 EMAP II Ligand to ICAM 1, 2 or 3 VEGF Ligand to ICAM 1, 2
or 3 IL-1 Ligand to ICAM 1, 2 or 3 IL-6 Ligand to ICAM 1, 2 or 3
IL-12 Ligand to ICAM 1, 2 or 3 PDGF Ligand to ICAM 1, 2 or 3
PD-ECGF Ligand to ICAM 1, 2 or 3 CXC chemokine Ligand to ICAM 1, 2
or 3 CC chemokine Ligand to ICAM 1, 2 or 3 C chemokine Ligand to
ICAM 1, 2 or 3 IL-15 Ligand to ICAM 1, 2 or 3 TNF-.alpha. Ab to
ICAM 1, 2 or 3 TNF-.beta. Ab to ICAM 1, 2 or 3 IFN-.alpha. Ab to
ICAM 1, 2 or 3 IFN-.beta. Ab to ICAM 1, 2 or 3 IFN-.gamma. Ab to
ICAM 1, 2 or 3 IL-2 Ab to ICAM 1, 2 or 3 IL-12 Ab to ICAM 1, 2 or 3
EMAP II Ab to ICAM 1, 2 or 3 VEGF Ab to ICAM 1, 2 or 3 IL-1 Ab to
ICAM 1, 2 or 3 IL-6 Ab to ICAM 1, 2 or 3 IL-12 Ab to ICAM 1, 2 or 3
PDGF Ab to ICAM 1, 2 or 3 PD-ECGF Ab to ICAM 1, 2 or 3 CXC
chemokine Ab to ICAM 1, 2 or 3 CC chemokine Ab to ICAM 1, 2 or 3 C
chemokine Ab to ICAM 1, 2 or 3 IL-15 Ab to ICAM 1, 2 or 3
TNF-.alpha. Ligand to PECAM-1/CD31 TNF-.beta. Ligand to
PECAM-1/CD31 IFN-.alpha. Ligand to PECAM-1/CD31 IFN-.beta. Ligand
to PECAM-1/CD31 IFN-.gamma. Ligand to PECAM-1/CD31 IL-2 Ligand to
PECAM-1/CD31 IL-12 Ligand to PECAM-1/CD31 EMAP II Ligand to
PECAM-1/CD31 VEGF Ligand to PECAM-1/CD31 IL-1 Ligand to
PECAM-1/CD31 IL-6 Ligand to PECAM-1/CD31 IL-12 Ligand to
PECAM-1/CD31 PDGF Ligand to PECAM-1/CD31 PD-ECGF Ligand to
PECAM-1/CD31 CXC chemokine Ligand to PECAM-1/CD31 CC chemokine
Ligand to PECAM-1/CD31 C chemokine Ligand to PECAM-1/CD31 IL-15
Ligand to PECAM-1/CD31 TNF-.alpha. Ab to PECAM-1/CD31 TNF-.beta. Ab
to PECAM-1/CD31 IFN-.alpha. Ab to PECAM-1/CD31 IFN-.beta. Ab to
PECAM-1/CD31 IFN-.gamma. Ab to PECAM-1/CD31 IL-2 Ab to PECAM-1/CD31
IL-12 Ab to PECAM-1/CD31 EMAP II Ab to PECAM-1/CD31 VEGF Ab to
PECAM-1/CD31 IL-1 Ab to PECAM-1/CD31 IL-6 Ab to PECAM-1/CD31 IL-12
Ab to PECAM-1/CD31 PDGF Ab to PECAM-1/CD31 PD-ECGF Ab to
PECAM-1/CD31 CXC chemokine Ab to PECAM-1/CD31 CC chemokine Ab to
PECAM-1/CD31 C chemokine Ab to PECAM-1/CD31 IL-15 Ab to
PECAM-1/CD31 TNF-.alpha. Ligand to VCAM-1 TNF-.beta. Ligand to
VCAM-1 IFN-.alpha. Ligand to VCAM-1 IFN-.beta. Ligand to VCAM-1
IFN-.gamma. Ligand to VCAM-1 IL-2 Ligand to VCAM-1 IL-12 Ligand to
VCAM-1 EMAP II Ligand to VCAM-1 VEGF Ligand to VCAM-1 IL-1 Ligand
to VCAM-1 IL-6 Ligand to VCAM-1 IL-12 Ligand to VCAM-1 PDGF Ligand
to VCAM-1 PD-ECGF Ligand to VCAM-1 CXC chemokine Ligand to VCAM-1
CC chemokine Ligand to VCAM-1 C chemokine Ligand to VCAM-1 IL-15
Ligand to VCAM-1 TNF-.alpha. Ab to VCAM-1 TNF-.beta. Ab to VCAM-1
IFN-.alpha. Ab to VCAM-1 IFN-.beta. Ab to VCAM-1 IFN-.gamma. Ab to
VCAM-1 IL-2 Ab to VCAM-1 IL-12 Ab to VCAM-1 EMAP II Ab to VCAM-1
VEGF Ab to VCAM-1 IL-1 Ab to VCAM-1 IL-6 Ab to VCAM-1 IL-12 Ab to
VCAM-1 PDGF Ab to VCAM-1 PD-ECGF Ab to VCAM-1 CXC chemokine Ab to
VCAM-1 CC chemokine Ab to VCAM-1 C chemokine Ab to VCAM-1 IL-15 Ab
to VCAM-1 TNF-.alpha. Ligand to Selectin TNF-.beta. Ligand to
Selectin IFN-.alpha. Ligand to Selectin IFN-.beta. Ligand to
Selectin IFN-.gamma. Ligand to Selectin IL-2 Ligand to Selectin
IL-12 Ligand to Selectin EMAP II Ligand to Selectin VEGF Ligand to
Selectin IL-1 Ligand to Selectin IL-6 Ligand to Selectin IL-12
Ligand to Selectin PDGF Ligand to Selectin PD-ECGF Ligand to
Selectin CXC chemokine Ligand to Selectin CC chemokine Ligand to
Selectin C chemokine Ligand to Selectin IL-15 Ligand to Selectin
TNF-.alpha. Ab to Selectin TNF-.beta. Ab to Selectin IFN-.alpha. Ab
to Selectin IFN-.beta. Ab to Selectin IFN-.gamma. Ab to Selectin
IL-2 Ab to Selectin IL-12 Ab to Selectin EMAP II Ab to Selectin
VEGF Ab to Selectin IL-1 Ab to Selectin IL-6 Ab to Selectin IL-12
Ab to Selectin PDGF Ab to Selectin PD-ECGF Ab to Selectin CXC
chemokine Ab to Selectin CC chemokine Ab to Selectin C chemokine Ab
to Selectin IL-15 Ab to Selectin TNF-.alpha. Ligand to ActRII,
ActRIIB, ActRI or ActRIB TNF-.beta. Ligand to ActRII, ActRIIB,
ActRI or ActRIB IFN-.alpha. Ligand to ActRII, ActRIIB, ActRI or
ActRIB IFN-.beta. Ligand to ActRII, ActRIIB, ActRI or ActRIB
IFN-.gamma. Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-2 Ligand
to ActRII, ActRIIB, ActRI or ActRIB IL-12 Ligand to ActRII,
ActRIIB, ActRI or ActRIB EMAP II Ligand to ActRII, ActRIIB, ActRI
or ActRIB VEGF Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-1
Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-6 Ligand to ActRII,
ActRIIB, ActRI or ActRIB IL-12 Ligand to ActRII, ActRIIB, ActRI or
ActRIB PDGF Ligand to ActRII, ActRIIB, ActRI or ActRIB PD-ECGF
Ligand to ActRII, ActRIIB, ActRI or ActRIB CXC chemokine Ligand to
ActRII, ActRIIB, ActRI or ActRIB CC chemokine Ligand to ActRII,
ActRIIB, ActRI or ActRIB C chemokine Ligand to ActRII, ActRIIB,
ActRI or ActRIB IL-15 Ligand to ActRII, ActRIIB, ActRI or ActRIB
TNF-.alpha. Ab to ActRII, ActRIIB, ActRI or ActRIB TNF-.beta. Ab to
ActRII, ActRIIB, ActRI or ActRIB IFN-.alpha. Ab to ActRII, ActRIIB,
ActRI or ActRIB IFN-.beta. Ab to ActRII, ActRIIB, ActRI or ActRIB
IFN-.gamma. Ab to ActRII, ActRIIB, ActRI or ActRIB IL-2 Ab to
ActRII, ActRIIB, ActRI or ActRIB IL-12 Ab to ActRII, ActRIIB, ActRI
or ActRIB EMAP II Ab to ActRII, ActRIIB, ActRI or ActRIB VEGF Ab to
ActRII, ActRIIB, ActRI or ActRIB IL-1 Ab to ActRII, ActRIIB, ActRI
or ActRIB IL-6 Ab to ActRII, ActRIIB, ActRI or ActRIB IL-12 Ab to
ActRII, ActRIIB, ActRI or ActRIB PDGF Ab to ActRII, ActRIIB, ActRI
or ActRIB PD-ECGF Ab to ActRII, ActRIIB, ActRI or ActRIB CXC
chemokine Ab to ActRII, ActRIIB, ActRI or ActRIB CC chemokine Ab to
ActRII, ActRIIB, ActRI or ActRIB C chemokine Ab to ActRII, ActRIIB,
ActRI or ActRIB IL-15 Ab to ActRII, ActRIIB, ActRI or ActRIB
TNF-.alpha. Annexin V TNF-.beta. Annexin V IFN-.alpha. Annexin V
IFN-.beta. Annexin V IFN-.gamma. Annexin V IL-2 Annexin V IL-12
Annexin V EMAP II Annexin V VEGF Annexin V IL-1 Annexin V IL-6
Annexin V IL-12 Annexin V PDGF Annexin V PD-ECGF Annexin V CXC
chemokine Annexin V CC chemokine Annexin V C chemokine Annexin V
IL-15 Annexin V TNF-.alpha. Ligand to CD44 TNF-.beta. Ligand to
CD44 IFN-.alpha. Ligand to CD44 IFN-.beta. Ligand to CD44
IFN-.gamma. Ligand to CD44 IL-2 Ligand to CD44 IL-12 Ligand to CD44
EMAP II Ligand to CD44 VEGF Ligand to CD44 IL-1 Ligand to CD44 IL-6
Ligand to CD44 IL-12 Ligand to CD44 PDGF Ligand to CD44 PD-ECGF
Ligand to CD44 CXC chemokine Ligand to CD44 CC chemokine Ligand to
CD44 C chemokine Ligand to CD44 IL-15 Ligand to CD44 TNF-.alpha. Ab
to CD44 TNF-.beta. Ab to CD44 IFN-.alpha. Ab to CD44 IFN-.beta. Ab
to CD44 IFN-.gamma. Ab to CD44 IL-2 Ab to CD44 IL-12 Ab to CD44
EMAP II Ab to CD44 VEGF Ab to CD44 IL-1 Ab to CD44 IL-6 Ab to CD44
IL-12 Ab to CD44 PDGF Ab to CD44 PD-ECGF Ab to CD44 CXC chemokine
Ab to CD44 CC chemokine Ab to CD44 C chemokine Ab to CD44 IL-15 Ab
to CD44 TNF-.alpha. Osteopontin TNF-.beta. Osteopontin IFN-.alpha.
Osteopontin IFN-.beta. Osteopontin IFN-.gamma. Osteopontin IL-2
Osteopontin IL-12 Osteopontin EMAP II Osteopontin VEGF Osteopontin
IL-1 Osteopontin IL-6 Osteopontin IL-12 Osteopontin PDGF
Osteopontin PD-ECGF Osteopontin CXC chemokine Osteopontin CC
chemokine Osteopontin C chemokine Osteopontin IL-15 Osteopontin
TNF-.alpha. Fibronectin TNF-.beta. Fibronectin IFN-.alpha.
Fibronectin IFN-.beta. Fibronectin IFN-.gamma. Fibronectin IL-2
Fibronectin IL-12 Fibronectin EMAP II Fibronectin VEGF Fibronectin
IL-1 Fibronectin IL-6 Fibronectin IL-12 Fibronectin PDGF
Fibronectin PD-ECGF Fibronectin CXC chemokine Fibronectin CC
chemokine Fibronectin C chemokine Fibronectin IL-15 Fibronectin
TNF-.alpha. Collagen type I or IV TNF-.beta. Collagen type I or IV
IFN-.alpha. Collagen type I or IV IFN-.beta. Collagen type I or IV
IFN-.gamma. Collagen type I or IV IL-2 Collagen type I or IV IL-12
Collagen type I or IV EMAP II Collagen type I or IV VEGF Collagen
type I or IV IL-1 Collagen type I or IV IL-6 Collagen type I or IV
IL-12 Collagen type I or IV PDGF Collagen type I or IV PD-ECGF
Collagen type I or IV CXC chemokine Collagen type I or IV CC
chemokine Collagen type I or IV C chemokine Collagen type I or IV
IL-15 Collagen type I or IV TNF-.alpha. Hyaluronate TNF-.beta.
Hyaluronate IFN-.alpha. Hyaluronate IFN-.beta. Hyaluronate
IFN-.gamma. Hyaluronate IL-2 Hyaluronate IL-12 Hyaluronate EMAP II
Hyaluronate VEGF Hyaluronate IL-1 Hyaluronate IL-6 Hyaluronate
IL-12 Hyaluronate PDGF Hyaluronate PD-ECGF Hyaluronate CXC
chemokine Hyaluronate CC chemokine Hyaluronate C chemokine
Hyaluronate IL-15 Hyaluronate TNF-.alpha. Ligand to FGF-1, 2, 3 or
4 TNF-.beta. Ligand to FGF-1, 2, 3 or 4 IFN-.alpha. Ligand to
FGF-1, 2, 3 or 4 IFN-.beta. Ligand to FGF-1, 2, 3 or 4 IFN-.gamma.
Ligand to FGF-1, 2, 3 or 4 IL-2 Ligand to FGF-1, 2, 3 or 4 IL-12
Ligand to FGF-1, 2, 3 or 4 EMAP II Ligand to FGF-1, 2, 3 or 4 VEGF
Ligand to FGF-1, 2, 3 or 4 IL-1 Ligand to FGF-1, 2, 3 or 4 IL-6
Ligand to FGF-1, 2, 3 or 4 IL-12 Ligand to FGF-1, 2, 3 or 4 PDGF
Ligand to FGF-1, 2, 3 or 4 PD-ECGF Ligand to FGF-1, 2, 3 or 4 CXC
chemokine Ligand to FGF-1, 2, 3 or 4 CC chemokine Ligand to FGF-1,
2, 3 or 4 C chemokine Ligand to FGF-1, 2, 3 or 4 IL-15 Ligand to
FGF-1, 2, 3 or 4 TNF-.alpha. Ab to FGF-1, 2, 3 or 4 TNF-.beta. Ab
to FGF-1, 2, 3 or 4 INF-.alpha. Ab to FGF-1, 2, 3 or 4 IFN-.beta.
Ab to FGF-1, 2, 3 or 4 IFN-.gamma. Ab to FGF-1, 2, 3 or 4 IL-2 Ab
to FGF-1, 2, 3 or 4 IL-12 Ab to FGF-1, 2, 3 or 4 EMAP II Ab to
FGF-1, 2, 3 or 4 VEGF Ab to FGF-1, 2, 3 or 4 IL-1 Ab to FGF-1, 2, 3
or 4 IL-6 Ab to FGF-1, 2, 3 or 4 IL-12 Ab to FGF-1, 2, 3 or 4 PDGF
Ab to FGF-1, 2, 3 or 4 PD-ECGF Ab to FGF-1, 2, 3 or 4 CXC chemokine
Ab to FGF-1, 2, 3 or 4 CC chemokine Ab to FGF-1, 2, 3 or 4 C
chemokine Ab to FGF-1, 2, 3 or 4 IL-15 Ab to FGF-1, 2, 3 or 4
TNF-.alpha. Ligand to IL-1R TNF-.beta. Ligand to IL-1R IFN-.alpha.
Ligand to IL-1R IFN-.beta. Ligand to IL-1R IFN-.gamma. Ligand to
IL-1R IL-2 Ligand to IL-1R IL-12 Ligand to IL-1R EMAP II Ligand to
IL-1R VEGF Ligand to IL-1R IL-1 Ligand to IL-1R IL-6 Ligand to
IL-1R IL-12 Ligand to IL-1R PDGF Ligand to IL-1R PD-ECGF Ligand to
IL-1R CXC chemokine Ligand to IL-1R CC chemokine Ligand to IL-1R C
chemokine Ligand to IL-1R IL-15 Ligand to IL-1R TNF-.alpha. Ab to
IL-1R TNF-.beta. Ab to IL-1R IFN-.alpha. Ab to IL-1R IFN-.beta. Ab
to IL-1R IFN-.gamma. Ab to IL-1R IL-2 Ab to IL-1R IL-12 Ab to IL-1R
EMAP II Ab to IL-1R VEGF Ab to IL-1R IL-1 Ab to IL-1R IL-6 Ab to
IL-1R IL-12 Ab to IL-1R PDGF Ab to IL-1R PD-ECGF Ab to IL-1R CXC
chemokine Ab to IL-1R CC chemokine Ab to IL-1R C chemokine Ab to
IL-1R IL-15 Ab to IL-1R TNF-.alpha. Ligand to CD31 TNF-.beta.
Ligand to CD31 IFN-.alpha. Ligand to CD31 IFN-.beta. Ligand to CD31
IFN-.gamma. Ligand to CD31 IL-2 Ligand to CD31 IL-12 Ligand to CD31
EMAP II Ligand to CD31 VEGF Ligand to CD31 IL-1 Ligand to CD31 IL-6
Ligand to CD31 IL-12 Ligand to CD31 PDGF Ligand to CD31 PD-ECGF
Ligand to CD31 CXC chemokine Ligand to CD31 CC chemokine Ligand to
CD31 C chemokine Ligand to CD31 IL-15 Ligand to CD31 TNF-.alpha. Ab
to CD31 TNF-.beta. Ab to CD31 IFN-.alpha. Ab to CD31 IFN-.beta. Ab
to CD31 IFN-.gamma. Ab to CD31 IL-2 Ab to CD31 IL-12 Ab to CD31
EMAP II Ab to CD31 VEGF Ab to CD31 IL-1 Ab to CD31 IL-6 Ab to CD31
IL-12 Ab to CD31 PDGF Ab to CD31 PD-ECGF Ab to CD31 CXC chemokine
Ab to CD31 CC chemokine Ab to CD31 C chemokine Ab to CD31 IL-15 Ab
to CD31 TNF-.alpha. Ligand
to EPHR TNF-.beta. Ligand to EPHR IFN-.alpha. Ligand to EPHR
IFN-.beta. Ligand to EPHR IFN-.gamma. Ligand to EPHR IL-2 Ligand to
EPHR IL-12 Ligand to EPHR EMAP II Ligand to EPHR VEGF Ligand to
EPHR IL-1 Ligand to EPHR IL-6 Ligand to EPHR IL-12 Ligand to EPHR
PDGF Ligand to EPHR PD-ECGF Ligand to EPHR CXC chemokine Ligand to
EPHR CC chemokine Ligand to EPHR C chemokine Ligand to EPHR IL-15
Ligand to EPHR TNF-.alpha. Ab to EPHR TNF-.beta. Ab to EPHR
IFN-.alpha. Ab to EPHR IFN-.beta. Ab to EPHR IFN-.gamma. Ab to EPHR
IL-2 Ab to EPHR IL-12 Ab to EPHR EMAP II Ab to EPHR VEGF Ab to EPHR
IL-1 Ab to EPHR IL-6 Ab to EPHR IL-12 Ab to EPHR PDGF Ab to EPHR
PD-ECGF Ab to EPHR CXC chemokine Ab to EPHR CC chemokine Ab to EPHR
C chemokine Ab to EPHR IL-15 Ab to EPHR TNF-.alpha. Ephrin
TNF-.beta. Ephrin IFN-.alpha. Ephrin IFN-.beta. Ephrin IFN-.gamma.
Ephrin IL-2 Ephrin IL-12 Ephrin EMAP II Ephrin VEGF Ephrin IL-1
Ephrin IL-6 Ephrin IL-12 Ephrin PDGF Ephrin PD-ECGF Ephrin CXC
chemokine Ephrin CC chemokine Ephrin C chemokine Ephrin IL-15
Ephrin TNF-.alpha. Ligand to MMP TNF-.beta. Ligand to MMP
IFN-.alpha. Ligand to MMP IFN-.beta. Ligand to MMP IFN-.gamma.
Ligand to MMP IL-2 Ligand to MMP IL-12 Ligand to MMP EMAP II Ligand
to MMP VEGF Ligand to MMP IL-1 Ligand to MMP IL-6 Ligand to MMP
IL-12 Ligand to MMP PDGF Ligand to MMP PD-ECGF Ligand to MMP CXC
chemokine Ligand to MMP CC chemokine Ligand to MMP C chemokine
Ligand to MMP IL-15 Ligand to MMP TNF-.alpha. Ab to MMP TNF-.beta.
Ab to MMP IFN-.alpha. Ab to MMP IFN-.beta. Ab to MMP IFN-.gamma. Ab
to MMP IL-2 Ab to MMP IL-12 Ab to MMP EMAP II Ab to MMP VEGF Ab to
MMP IL-1 Ab to MMP IL-6 Ab to MMP IL-12 Ab to MMP PDGF Ab to MMP
PD-ECGF Ab to MMP CXC chemokine Ab to MMP CC chemokine Ab to MMP C
chemokine Ab to MMP IL-15 Ab to MMP TNF-.alpha. Ligand to NG2
TNF-.beta. Ligand to NG2 IFN-.alpha. Ligand to NG2 IFN-.beta.
Ligand to NG2 IFN-.gamma. Ligand to NG2 IL-2 Ligand to NG2 IL-12
Ligand to NG2 EMAP II Ligand to NG2 VEGF Ligand to NG2 IL-1 Ligand
to NG2 IL-6 Ligand to NG2 IL-12 Ligand to NG2 PDGF Ligand to NG2
PD-ECGF Ligand to NG2 CXC chemokine Ligand to NG2 CC chemokine
Ligand to NG2 C chemokine Ligand to NG2 IL-15 Ligand to NG2
TNF-.alpha. Ab to NG2 TNF-.beta. Ab to NG2 IFN-.alpha. Ab to NG2
IFN-.beta. Ab to NG2 IFN-.gamma. Ab to NG2 IL-2 Ab to NG2 IL-12 Ab
to NG2 EMAP II Ab to NG2 VEGF Ab to NG2 IL-1 Ab to NG2 IL-6 Ab to
NG2 IL-12 Ab to NG2 PDGF Ab to NG2 PD-ECGF Ab to NG2 CXC chemokine
Ab to NG2 CC chemokine Ab to NG2 C chemokine Ab to NG2 IL-15 Ab to
NG2 TNF-.alpha. Ligand to tenascin TNF-.beta. Ligand to tenascin
IFN-.alpha. Ligand to tenascin IFN-.beta. Ligand to tenascin
IFN-.gamma. Ligand to tenascin IL-2 Ligand to tenascin IL-12 Ligand
to tenascin EMAP II Ligand to tenascin VEGF Ligand to tenascin IL-1
Ligand to tenascin IL-6 Ligand to tenascin IL-12 Ligand to tenascin
PDGF Ligand to tenascin PD-ECGF Ligand to tenascin CXC chemokine
Ligand to tenascin CC chemokine Ligand to tenascin C chemokine
Ligand to tenascin IL-15 Ligand to tenascin TNF-.alpha. Ab to
tenascin TNF-.beta. Ab to tenascin IFN-.alpha. Ab to tenascin
IFN-.beta. Ab to tenascin IFN-.gamma. Ab to tenascin IL-2 Ab to
tenascin IL-12 Ab to tenascin EMAP II Ab to tenascin VEGF Ab to
tenascin IL-1 Ab to tenascin IL-6 Ab to tenascin IL-12 Ab to
tenascin PDGF Ab to tenascin PD-ECGF Ab to tenascin CXC chemokine
Ab to tenascin CC chemokine Ab to tenascin C chemokine Ab to
tenascin IL-15 Ab to tenascin TNF-.alpha. Ligand to PD-ECGFR
TNF-.beta. Ligand to PD-ECGFR IFN-.alpha. Ligand to PD-ECGFR
IFN-.beta. Ligand to PD-ECGFR IFN-.gamma. Ligand to PD-ECGFR IL-2
Ligand to PD-ECGFR IL-12 Ligand to PD-ECGFR EMAP II Ligand to
PD-ECGFR VEGF Ligand to PD-ECGFR IL-1 Ligand to PD-ECGFR IL-6
Ligand to PD-ECGFR IL-12 Ligand to PD-ECGFR PDGF Ligand to PD-ECGFR
PD-ECGF Ligand to PD-ECGFR CXC chemokine Ligand to PD-ECGFR CC
chemokine Ligand to PD-ECGFR C chemokine Ligand to PD-ECGFR IL-15
Ligand to PD-ECGFR TNF-.alpha. Ab to PD-ECGFR TNF-.beta. Ab to
PD-ECGFR IFN-.alpha. Ab to PD-ECGFR IFN-.beta. Ab to PD-ECGFR
IFN-.gamma. Ab to PD-ECGFR IL-2 Ab to PD-ECGFR IL-12 Ab to PD-ECGFR
EMAP II Ab to PD-ECGFR VEGF Ab to PD-ECGFR IL-1 Ab to PD-ECGFR IL-6
Ab to PD-ECGFR IL-12 Ab to PD-ECGFR PDGF Ab to PD-ECGFR PD-ECGF Ab
to PD-ECGFR CXC chemokine Ab to PD-ECGFR CC chemokine Ab to
PD-ECGFR C chemokine Ab to PD-ECGFR IL-15 Ab to PD-ECGFR
TNF-.alpha. Ligand to TNFR TNF-.beta. Ligand to TNFR IFN-.alpha.
Ligand to TNFR IFN-.beta. Ligand to TNFR IFN-.gamma. Ligand to TNFR
IL-2 Ligand to TNFR IL-12 Ligand to TNFR EMAP II Ligand to TNFR
VEGF Ligand to TNFR IL-1 Ligand to TNFR IL-6 Ligand to TNFR IL-12
Ligand to TNFR PDGF Ligand to TNFR PD-ECGF Ligand to TNFR CXC
chemokine Ligand to TNFR CC chemokine Ligand to TNFR C chemokine
Ligand to TNFR IL-15 Ligand to TNFR TNF-.alpha. Ab to TNFR
TNF-.beta. Ab to TNFR IFN-.alpha. Ab to TNFR IFN-.beta. Ab to TNFR
IFN-.gamma. Ab to TNFR IL-2 Ab to TNFR IL-12 Ab to TNFR EMAP II Ab
to TNFR VEGF Ab to TNFR IL-1 Ab to TNFR IL-6 Ab to TNFR IL-12 Ab to
TNFR PDGF Ab to TNFR PD-ECGF Ab to TNFR CXC chemokine Ab to TNFR CC
chemokine Ab to TNFR C chemokine Ab to TNFR IL-15 Ab to TNFR
TNF-.alpha. Ligand to PDGFR TNF-.beta. Ligand to PDGFR IFN-.alpha.
Ligand to PDGFR IFN-.beta. Ligand to PDGFR IFN-.gamma. Ligand to
PDGFR IL-2 Ligand to PDGFR IL-12 Ligand to PDGFR EMAP II Ligand to
PDGFR VEGF Ligand to PDGFR IL-1 Ligand to PDGFR IL-6 Ligand to
PDGFR IL-12 Ligand to PDGFR PDGF Ligand to PDGFR PD-ECGF Ligand to
PDGFR CXC chemokine Ligand to PDGFR CC chemokine Ligand to PDGFR C
chemokine Ligand to PDGFR IL-15 Ligand to PDGFR TNF-.alpha. Ab to
PDGFR TNF-.beta. Ab to PDGFR IFN-.alpha. Ab to PDGFR IFN-.beta. Ab
to PDGFR IFN-.gamma. Ab to PDGFR IL-2 Ab to PDGFR IL-12 Ab to PDGFR
EMAP II Ab to PDGFR VEGF Ab to PDGFR IL-1 Ab to PDGFR IL-6 Ab to
PDGFR IL-12 Ab to PDGFR PDGF Ab to PDGFR PD-ECGF Ab to PDGFR CXC
chemokine Ab to PDGFR CC chemokine Ab to PDGFR C chemokine Ab to
PDGFR IL-15 Ab to PDGFR TNF-.alpha. Ligand to PSMA TNF-.beta.
Ligand to PSMA IFN-.alpha. Ligand to PSMA IFN-.beta. Ligand to PSMA
IFN-.gamma. Ligand to PSMA IL-2 Ligand to PSMA IL-12 Ligand to PSMA
EMAP II Ligand to PSMA VEGF Ligand to PSMA IL-1 Ligand to PSMA IL-6
Ligand to PSMA IL-12 Ligand to PSMA PDGF Ligand to PSMA PD-ECGF
Ligand to PSMA CXC chemokine Ligand to PSMA CC chemokine Ligand to
PSMA C chemokine Ligand to PSMA IL-15 Ligand to PSMA TNF-.alpha. Ab
to PSMA TNF-.beta. Ab to PSMA IFN-.alpha. Ab to PSMA IFN-.beta. Ab
to PSMA IFN-.gamma. Ab to PSMA IL-2 Ab to PSMA IL-12 Ab to PSMA
EMAP II Ab to PSMA VEGF Ab to PSMA IL-1 Ab to PSMA IL-6 Ab to PSMA
IL-12 Ab to PSMA PDGF Ab to PSMA PD-ECGF Ab to PSMA CXC chemokine
Ab to PSMA CC chemokine Ab to PSMA C chemokine Ab to PSMA IL-15 Ab
to PSMA TNF-.alpha. Vitronectin TNF-.beta. Vitronectin IFN-.alpha.
Vitronectin IFN-.beta. Vitronectin IFN-.gamma. Vitronectin IL-2
Vitronectin IL-12 Vitronectin EMAP II Vitronectin VEGF Vitronectin
IL-1 Vitronectin IL-6 Vitronectin IL-12 Vitronectin PDGF
Vitronectin PD-ECGF Vitronectin CXC chemokine Vitronectin CC
chemokine Vitronectin C chemokine Vitronectin IL-15 Vitronectin
TNF-.alpha. Laminin TNF-.beta. Laminin IFN-.alpha. Laminin
IFN-.beta. Laminin IFN-.gamma. Laminin IL-2 Laminin IL-12 Laminin
EMAP II Laminin VEGF Laminin IL-1 Laminin IL-6 Laminin IL-12
Laminin PDGF Laminin PD-ECGF Laminin CXC chemokine Laminin CC
chemokine Laminin C chemokine Laminin IL-15 Laminin TNF-.alpha.
Ligand to oncofetal fibronectin TNF-.beta. Ligand to oncofetal
fibronectin IFN-.alpha. Ligand to oncofetal fibronectin IFN-.beta.
Ligand to oncofetal fibronectin IFN-.gamma. Ligand to oncofetal
fibronectin IL-2 Ligand to oncofetal fibronectin IL-12 Ligand to
oncofetal fibronectin EMAP II Ligand to oncofetal fibronectin VEGF
Ligand to oncofetal fibronectin IL-1 Ligand to oncofetal
fibronectin IL-6 Ligand to oncofetal fibronectin IL-12 Ligand to
oncofetal fibronectin PDGF Ligand to oncofetal fibronectin PD-ECGF
Ligand to oncofetal fibronectin CXC chemokine Ligand to oncofetal
fibronectin CC chemokine Ligand to oncofetal fibronectin C
chemokine Ligand to oncofetal fibronectin IL-15 Ligand to oncofetal
fibronectin TNF-.alpha. Ab to oncofetal fibronectin TNF-.beta. Ab
to oncofetal fibronectin IFN-.alpha. Ab to oncofetal fibronectin
IFN-.beta. Ab to oncofetal fibronectin IFN-.gamma. Ab to oncofetal
fibronectin IL-2 Ab to oncofetal fibronectin IL-12 Ab to oncofetal
fibronectin EMAP II Ab to oncofetal fibronectin VEGF Ab to
oncofetal fibronectin IL-1 Ab to oncofetal fibronectin IL-6 Ab to
oncofetal fibronectin IL-12 Ab to oncofetal fibronectin PDGF Ab to
oncofetal fibronectin PD-ECGF Ab to oncofetal fibronectin CXC
chemokine Ab to oncofetal fibronectin CC chemokine Ab to oncofetal
fibronectin C chemokine Ab to oncofetal fibronectin IL-15 Ab to
oncofetal fibronectin
[0033] It will be appreciated that in the above Table the term "Ab"
represents antibody, and that the antibodies and ligands include
fragments thereof.
[0034] In particularly preferred embodiments the conjugate
comprises TNF.alpha. or TNF-.beta. and an NGR-containing peptide,
or TNF-.alpha. or TNF-.beta. and an RGD-containing peptide. In a
preferred embodiment the conjugate is in the form of a fusion
protein.
[0035] In another embodiment the conjugate is in the form of
nucleic acid.
[0036] In another embodiment the composition further comprises
another antitumor agent or diagnostic tumor-imaging compound.
Preferably the further antitumor agent is doxorubicin, melphalan or
cisplatin.
[0037] According to another aspect of the present invention there
is provided use of a conjugate or a pharmaceutical composition
according to the present invention for the preparation of a
medicament for treatment or diagnosis of cancer.
[0038] In other words the present invention provides a method of
treating or diagnosing cancer comprising administering to a patient
in need of the same a conjugate or a pharmaceutical composition
according to the present invention in an effective amount, wherein
said amount does not induce a negative feedback mechanism.
[0039] Some Key Advantages of the Invention
[0040] To reach cancer cells in solid tumors, chemotherapeutic
drugs must enter the tumor blood vessels, cross the vessel wall and
finally migrate through the interstitium. Heterogeneous tumor
perfusion, vascular permeability and cell density, and increased
interstitial pressure could represent critical barriers that may
limit the penetration of drugs into neoplastic cells distant to
from tumor vessels and, consequently, the effectiveness of
chemotherapy (1). Strategies aimed at improving drug penetration in
tumors are, therefore, of great experimental and clinical
interest.
[0041] A growing body of evidence suggests that Tumor Necrosis
Factor-.alpha. (TNF), and inflammatory cytokine endowed with potent
anti-tumor activity, could be exploited for this purpose. For
example, the addition of TNF to regional isolated limb perfusion
with melphalan or doxorubicin has produced higher response rates in
patients with extremity soft-tissue sarcomas or melanomas than
those obtained with chemotherapeutic drugs alone (2-6). TNF-induced
alteration of the endothelial barrier function, reduction of tumor
interstitial pressure, increased chemotherapeutic drug penetration
and tumor vessel damage are believed to be important mechanisms of
the synergy between TNF and chemotherapy (3, 4, 7-10).
Unfortunately, systemic administration of TNF is accompanied by
prohibitive toxicity, the maximum tolerated dose (8-10 .mu.g/kg)
being 10-50 times lower than the estimated effective dose (11, 12).
For this reason, systemic administration of TNF has been abandoned
and the clinical use of this cytokine is limited to locoregional
treatmeants. Nevertheless, some features of the TNF activity, in
particular the selectivity for tumor-associated vessels and the
synergy with chemotherapeutic drugs, has continued to nourish hopes
as regards the possibility of wider therapeutic applications
(13).
[0042] The vascular effects of TNF provide the rational for
developing a "vascular targeting" strategy aimed at increasing the
local efficacy and at enabling systemic administration of
therapeutic doses. We have shown recently that targeted delivery of
TNF to tumor vessels can be achieved by coupling this protein with
the CNGRC peptide, an aminopeptidase N (CD13) ligand that targets
the tumor neovasculature (14). In the present work, we have
investigated whether vascular targeting with low doses of this
conjugate, called NGR-TNF, could enhance the penetration of
chemotherapeutic drugs in tumors and improve their efficacy. We
show that systemic administration of picogram doses of NGR-TNF (3-5
ng/kg) to mice, six orders of magnitude lower than the LD.sub.50,
is sufficient to enhance the anti-tumor activity of melphalan and
doxorubicin, with no evidence of increased toxicity. In addition,
we provide evidence that vascular targeting with NGR-TNF can reduce
drug-penetration barriers and increase the amount of doxorubicin
that reach cancer cells. Finally, we show that the delivery of
minute amounts of NGR-TNF to tumor vessels overcomes another major
problem associated with systemic administration of relatively high
doses of TNF, i.e. the induction of soluble TNF inhibitors.
DETAILED DESCRIPTION
[0043] Various preferred features and embodiments of the present
invention will now be described by way of non-limiting example.
[0044] Although in general the techniques mentioned herein are well
known in the art, reference may be made in particular to Sambrook
et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel
et al., Short Protocols in Molecular Biology (1999) 4.sup.th Ed,
John Wiley & Sons, Inc (as well as the complete version Current
Protocols in Molecular Biology).
[0045] Negative Feedback Mechanisms
[0046] Negative feedback mechanisms are known in the art and may
include: soluble receptor shedding and the induction of other
cytokines, hormones or biological agents at a systemic or local
level such that they directly or indirectly impair the activity of
the targeted cytokine of the present invention. The induction of
soluble inhibitors or decoy receptors are examples of direct
inhbitors. IL-10, TGF-.beta. or other anti-inflammatory cytokines
may indirectly prevent the cascade of events triggered by
inflammatory cytokines.
[0047] Receptor Shedding
[0048] This term relates to the ability of cells to cleave off the
extracellular domain of a cytokine receptor and to release it into
circulation as a soluble product. In many instances these cleavage
products are still capable of binding their ligands. In contrast,
however, the remaining portion of the membrane-bound receptor does
not bind ligands any longer and hence the cells become
desensibilised to the action of a particular cytokine. Such soluble
receptors have been described for a variety of cytokines, including
IL-4, 6, 6, IGF, TNF-.alpha. and IFN-.gamma..
[0049] Conjugate
[0050] The present invention relates to a conjugate which is a
molecule comprising at least one targeting protein linked to at
least cytokine formed through genetic fusion or chemical coupling.
By "linked" we mean that the first and second sequences are
associated such that the second sequence is able to be transported
by the first sequence to a target cell. Thus, conjugates include
fusion proteins in which the transport protein is linked to a
cytokine via their polypeptide backbones through genetic expression
of a DNA molecule encoding these proteins, directly synthesised
proteins and coupled proteins in which pre-formed sequences are
associated by a cross-linking agent. The term is also used herein
to include associations, such as aggregates, of the cytokine with
the targeting protein. According to one embodiment the second
sequence may comprise a polynucleotide sequence. This embodiment
may be seen as a protein/nucleic acid complex.
[0051] The second sequence may be from the same species as the
first sequence, but is present in the conjugate of the invention in
a manner different from the natural situation, or from a different
species.
[0052] The conjugates of the present invention are capable of being
directed to a cell so that an effector function corresponding to
the polypeptide sequence coupled to the transport sequence can take
place.
[0053] The peptide can be coupled directly to the cytokine or
indirectly through a spacer, which can be a single amino acid, an
amino acid sequence or an organic residue, such as
6-aminocapryl-N-hydroxysuccinimid- e.
[0054] The peptide ligand is preferably linked to the cytokine
N-terminus thus minimising any interference in the binding of the
modified cytokine to its receptor. Alternatively, the peptide can
be linked to amino acid residues which are amido- or
carboxylic-bond acceptors, which may be naturally occurring on the
molecule or artificially inserted using genetic engineering
techniques. The modified cytokine is preferably prepared by use of
a cDNA comprising a 5'-contiguous sequence encoding the
peptide.
[0055] According to a preferred embodiment, there is provided a
conjugation product between TNF and the CNGRC sequence in which the
amino-terminal of TNF is linked to the CNGRC peptide through the
spacer G (glycine).
[0056] Cytokines
[0057] Drug penetration into neoplastic cells is critical for the
effectiveness of solid-tumor chemotherapy. To reach cancer cells in
solid tumors, chemotherapeutic drugs must enter the drug blood
vessels, cross the vessel wall and finally migrate through the
interstitium. Heterogeneous tumor perfusion, vascular permeability
and cell density, and increased interstitial pressure may represent
critical barriers that may limit the penetration of drugs into
neoplastic cells and, consequently, the effectiveness of
chemotherapy. Cytokines which have the effect of affecting these
factors are therefore useful in the present invention. A
non-limiting list of cytokines which may be used in the present
invention is: TNF.alpha., TNF.beta., IFN.alpha., IFN.beta.,
IFN.gamma., IL-1, 2, 4, 6, 12, 15, EMAP II, vascular endothelial
growth factor (VEGF), PDGF, PD-ECGF or a chemokine.
[0058] TNF
[0059] TNF acts as an inflammatory cytokine and has the effect of
inducing alteration of the endothelial barrier function, reducing
of tumor interstitial pressure, and increasing chemotherapeutic
drug penetration and tumor vessel damage.
[0060] The first suggestion that a tumor necrotizing molecule
existed was made when it was observed that cancer patients
occasionally showed spontaneous regression of their tumors
following bacterial infections. Subsequent studies in the 1960s
indicated that host-associated (or endogenous) mediators,
manufactured in response to bacterial products, were likely
responsible for the observed effects. In 1975 it was shown that a
bacterially-induced circulating factor had strong anti-tumor
activity against tumors implanted in the skin in mice. This factor,
designated tumor necrosis factor (TNF), was subsequently isolated,
cloned, and found to be the prototype of a family of molecules that
are involved with immune regulation and inflammation. The receptors
for TNF and the other members of the TNF superfamily also
constitute a superfamily of related proteins.
[0061] TNF-related ligands usually share a number of common
features. These features do not include a high degree of overall
amino acid (aa) sequence homology. With the exception of nerve
growth factor (NGF) and TNF-beta, all ligands are synthesised as
type II transmembrane proteins (extracellular C-terminus) that
contain a short cytoplasmic segment (10-80 aa residues) and a
relatively long extracellular region (140-215 aa residues). NGF,
which is structurally unrelated to TNF, is included in this
superfamily only because of its ability to bind to the TNFRSF low
affinity NGF receptor (LNGFR). NGF has a classic signal sequence
peptide and is secreted. TNF-.beta., in contrast, although also
fully secreted, has a primary structure much more related to type
II transmembrane proteins. TNF-.beta. might be considered as a type
II protein with a non-functional, or inefficient, transmembrane
segment. In general, TNFSF members form trimeric structures, and
their monomers are composed of beta-strands that orient themselves
into a two sheet structure. As a consequence of the trimeric
structure of these molecules, it is suggested that the ligands and
receptors of the TNSF and TNFRSF superfamilies undergo "clustering"
during signal transduction.
[0062] TNF-.alpha.: Human TNF-.alpha. is a 233 aa residue,
nonglycosylated polypeptide that exists as either a transmembrane
or soluble protein. When expressed as a 26 kDa membrane bound
protein, TNF-.alpha. consists of a 29 aa residue cytoplasmic
domain, a 28 aa residue transmembrane segment, and a 176 aa residue
extracellular region. The soluble protein is created by a
proteolytic cleavage event via an 85 kDa TNF-alpha converting
enzyme (TACE), which generates a 17 kDa, 157 aa residue molecule
that normally circulates as a homotrimer.
[0063] TNF-.beta./LT-.alpha.: TNF-.beta., otherwise known as
lymphotoxin-.alpha. (LT-.alpha.) is a molecule whose cloning was
contemporary with that of TNF-.alpha.. Although TNF-.beta.
circulates as a 171 aa residue, 25 kDa glycosylated polypeptide, a
larger form has been found that is 194 aa residues long. The human
TNF-.beta. cDNA codes for an open reading frame of 205 aa residues
(202 in the mouse), and presumably some type of proteolytic
processing occurs during secretion. As with TNF-.alpha.,
circulating TNF-.beta. exists as a non-covalently linked trimer and
is known to bind to the same receptors as TNF-.alpha..
[0064] In one embodiment the TNF is a mutant form of TNF capable of
selectively binding to one of the INF receptors (Loetscher H et al
(1993) J Biol Chem 268:26350-7; Van Ostade X et al (1993) Nature
361:2669).
[0065] Many other inflammatory cytokines also have the property of
increasing endothelial vessel permeability, and it will be
appreciated that the invention can be applied to such cytokines,
together with agents which increase expression of such cytokines.
Inflammatory cytokines, also known as pro-inflammatory cytokines,
are a number of polypeptides and glycoproteins with molecular
weights between 5 kDa and 70 kDa. They have a stimulating effect on
the inflammatory response. The most important inflammatory
cytokines are TNF, IL-1, IL-6 and IL-8.
[0066] A Table of some cytokines classified as inflammatory
cytokines is shown below:
2 Group Individual cytokines Inflammatory Cytokines Endogenous
cytokines IL-1, TNF-.alpha., IL-6 Up-regulation IL-1, TNF-.alpha.,
IL-6, IFN-.alpha., INF-.beta., chemokines Stimulation of the IL-1,
IL-6, IL-11, TNF-.alpha., INF-.gamma., TGF-.beta., production of
acute LIF, OSM, CNTF phase reactants Chemoattractant cytokines CXC
chemokines IL-8, PF-4, PBP, NAP-2, .beta.-TG CC chemokines
MIP-1.alpha., MIP-1.beta., MCP-1, MCP-2, MCP- 3, RANTES C
chemokines Lymphotactin Stimulation of IL-12 inflammatory cytokines
TGF-.beta.: transforming growth factor, LIF: leukemia inhibitory
factor; OSM: oncostatin M; CNTF: ciliary neurotrophic factor; PF-4:
platelet factor 4; PBP: platelet basic protein; NAP-2: neutrophil
activating protein 2; .beta.-TG: .beta.-thromboglobulin; MIP:
macrophage inflammatory protein; MCP: monocyte chemoattractant
protein.
[0067] The up-regulation of inflammatory response is also performed
by IL-11, IFN-.alpha., IFN-.beta., and especially by the members of
the chemokine superfamily. TGF-.beta. in some situations has a
number of inflammatory activities including chemoattractant effects
on neutrophils, T lymphocytes and inactivated monocytes.
[0068] IL2
[0069] Because of the central role of the IL-2/IL-2R system in
mediation of the immune and inflammatory responses, it is obvious
that monitoring and manipulation of this system has important
diagnostic and therapeutic implications. IL-2 has shown promise as
an anti-cancer drug by virtue of its ability to stimulate the
proliferation and activities of tumor-attacking LAK and TIL
(tumor-infiltrating lymphocytes) cells. However, problems with IL-2
toxicity are still of concern and merit investigation. The present
invention addresses this problem.
[0070] IL-15
[0071] Interleukin 15 (IL-15) is a novel cytokine that shares many
biological properties with, but lacks amino acid sequence homology
to, IL-2. IL-15 was originally identified in media conditioned by a
monkey kidney epithelial cell line (CVI/EBNA) based on its
mitogenic activity on the murine T cell line, CTLL-2. IL-15 was
also independently discovered as a cytokine produced by a human
adult T cell leukemia cell line (HuT-102) that stimulated T cell
proliferation and was designated ST. By virtue of its activity as a
stimulator of T cells, NK cells, LAK cells, and TILs, IL-2 is
currently in clinical trials testing its potential use in
treatments for cancer and for viral infections. Because of its
similar biological activities, IL-15 should have similar
therapeutic potential.
[0072] Chemokines
[0073] Chemokines are a superfamily of mostly small, secreted
proteins that function in leukocyte trafficking, recruiting, and
recirculation. They also play a critical role in many
pathophysiological processes such as allergic responses, infectious
and autoimmune diseases, angiogenesis, inflammation, tumor growth,
and hematopoietic development. Approximately 80 percent of these
proteins have from 66 to 78 amino acids (aa) in their mature form.
The remainder are larger with additional aa occurring upstream of
the protein core or as part of an extended C-terminal segment. All
chemokines signal through seven transmembrane domain G-protein
coupled receptors. There are at least seventeen known chemokine
receptors, and many of these receptors exhibit promiscuous binding
properties whereby several different chemokines can signal through
the same receptor.
[0074] Chemokines are divided into subfamilies based on conserved
aa sequence motifs. Most family members have at least four
conserved cysteine residues that form two intramolecular disulfide
bonds. The subfamilies are defined by the position of the first two
cysteine residues:
[0075] The alpha subfamily, also called the CXC chemokines, have
one aa separating the first two cysteine residues. This group can
be further subdivided based on the presence or absence of a
glu-leu-arg (ELR) aa motif immediately preceding the first cysteine
residue. There are currently five CXC-specific receptors and they
are designated CXCR1 to CXCR5. The ELR.sup.+ chemokines bind to
CXCR2 and generally act as neutrophil chemoattractants and
activators. The ELR-chemokines bind CXCR3 to -5 and act primarily
on lymphocytes. At the time of this writing, 14 different human
genes encoding CXC chemokines have been reported in the scientific
literature with some additional diversity contributed by
alternative splicing.
[0076] In the beta subfamily, also called the CC chemokines, the
first two cysteines are adjacent to one another with no intervening
aa. There are currently 24 distinct human beta subfamily members.
The receptors for this group are designated CCR1 to CCR11. Target
cells for different CC family members include most types of
leukocytes.
[0077] There are two known proteins with chemokine homology that
fall outside of the alpha and beta subfamilies. Lymphotactin is the
lone member of the gamma class (C chemokine) which has lost the
fist and third cysteines. The lymphotactin receptor is designated
XCR1. Fractalkine, the only known member of the delta class
(CX.sub.3C chemokine), has three intervening aa between the first
two cysteine residues. This molecule is unique among chemokines in
that it is a transmembrane protein with the N-terminal chemokine
domain fused to a long mucin-like stalk The fractalkine receptor is
known as CX.sub.3CR1.
[0078] VEGF
[0079] The present invention is also applicable to Vasculature
Endothelial Growth Factor (VEGF). Angiogenesis is a process of new
blood vessel development from pre-existing vasculature. It plays an
essential role in embryonic development, normal growth of tissues,
wound healing, the female reproductive cycle (i.e., ovulation,
menstruation and placental development), as well as a major role in
many diseases. Particular interest has focused on cancer, since
tumors cannot grow beyond a few millimeters in size without
developing a new blood supply. Angiogenesis is also necessary for
the spread and growth of tumor cell metastases.
[0080] One of the most important growth and survival factors for
endothelium is VEGF. VEGF induces angiogenesis and endothelial cell
proliferation and it plays an important role in regulating
vasculogenesis. VEGF is a heparin-binding glycoprotein that is
secreted as a homodimer of 45 kDa. Most types of cells, but usually
not endothelial cells themselves, secrete VEGF. Since the initially
discovered VEGF, VEGF-A, increases vascular permeability, it was
known as vascular permeability factor. In addition, VEGF causes
vasodilatation, partly through stimulation of nitric oxide synthase
in endothelial cells. VEGF can also stimulate cell migration and
inhibit apoptosis. There are several splice variants of VEGF-A. The
major ones include: 121, 165, 189 and 206 amino acids (aa), each
one comprising a specific exon addition.
[0081] EMAP II
[0082] Endothelial-Monocyte Activating Polypeptide-II (EMAP-II) is
a cytokine that is an antiangiogenic factor in tumor vascular
development, and strongly inhibits tumor growth. Recombinant human
EMAP-II is an 18.3 kDa protein containing 166 amino acid residues.
EMAP II has also bee found to increase endothelial vessel
permeability.
[0083] PDGF
[0084] It has also been proposed that platelet-derived growth
factor (PDGF) antagonists might increase drug-uptake and
therapeutic effects of a broad range of anti-tumor agents in common
solid tumors. PDGF is a cytokine of 30 kDA and is released by
platelets on wounding and stimulates nearby cells to grow and
repair the wound.
[0085] PD-ECGF
[0086] As its name suggests, platelet-derived endothelial cell
growth factor (PD-ECGF) was originally isolated from platelets
based on its ability to induce mitosis in endothelial cells. Its
related protein is gliostatin.
[0087] Targeting Moiety
[0088] We have found that the therapeutic index of cytokines can be
increased by homing of targeting the cytokine to tumor vessels. In
addition, since it is known that tumor cells can form part of the
lining of tumor vasculature, the present invention encompasses
targeting to tumor cells directly as well as to its vasculature.
Any convenient tumor or tumor vasculature, particular endothelial
cell, targeting moiety may be used in the conjugate of the present
invention. Many such targeting moieties are known and these and any
which subsequently become available are encompassed within the
scope of the present invention. In one embodiment, the targeting
moiety is a binding partner, such as a ligand, of a receptor
expressed by a tumor cell, or a binding partner, such as an
antibody, to a marker or a component of the extracellular matrix
associated with tumor cells. More particularly the targeting moiety
is binding partner, such as a ligand of, a receptor expressed by
tumor-associated vessels, or a binding partner, such as an
antibody, to an endothelial marker or a component of the
extracellular matrix associated with angiogenic vessels. The term
binding partner is used here in its broadest sense and includes
both natural and synthetic binding domains, including ligand and
antibodies or binding fragments thereof. Thus, said binding partner
can be an antibody or a fragment thereof such as Fab, Fv,
single-chain Fv, a peptide or a peptido-mimetic, namely a
peptido-like molecule capable of binding to the receptor, marker of
extracellular component of the cell.
[0089] The following represent a non-limiting examples of suitable
targeting domains and receptors/markers to which the conjugate may
be targeted:
[0090] CD13
[0091] It has surprisingly been found that the therapeutic index of
certain cytokines can be remarkably improved and their
immunotherapeutic properties can be enhanced by coupling with a
ligand of aminopeptidase-N receptor (CD13). CD13 is a
trans-membrane glycoprotein of 150 kDa highly conserved in various
species. It is expressed on normal cells as well as in myeloid
tumor lines, in the angiogenic endothelium and is some epithelia
CD13 receptor is usually identified as "NGR" receptor, in that its
peptide ligands share the amino acidic "NGR" motif. The ligand is
preferably a straight or cyclic peptide comprising the NGR motif,
such as CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC or cycloCNGRC,
or more preferably the peptide. CNGRC. Further details can be found
in our WO01/61017 which is incorporated herein by reference.
[0092] TNF Receptor
[0093] As with members of the TNF Superfamily, members of the TNF
Receptor Superfamily (TNFRSF) also share a number of common
features. In particular, molecules in the TNFRSF are all type I
(N-terminus extracellular) transmembrane glycoproteins that contain
one to six ligand-binding, 40 aa residue cysteine-rich motifs in
their extracellular domain. In addition, functional TNFRSF members
are usually trimeric or multimeric complexes that are stabilised by
intracysteine disulfide bonds. Unlike most members of the TNFSF,
INFRSF members exist in both membrane-bound and soluble forms.
Finally, although aa sequence homology in the cytoplasmic domains
of the superfamily members does not exceed 25%, a number of
receptors are able to transduce apoptotic signals in a variety of
cells, suggesting a common function.
[0094] CD40: CD40 is a 50 kDa, 277 aa residue transmembrane
glycoprotein most often associated with B cell proliferation and
differentiation. Expressed on a variety of cell types, human CD40
cDNA encodes a 20 aa residue signal sequence, a 173 aa residue
extracellular region, a 22 aa residue transmembrane segment, and a
62 aa residue cytoplasmic domain. There are four cysteine-rich
motifs in the extracellular region that are accompanied by a
juxtamembrane sequence rich in serines and threonines. Cells known
to express CD40 include endothelial cells.
[0095] TNFRI/p55/CD120a: TNFRI is a 55 kDa, 455 aa residue
transmembrane glycoprotein that is apparently expressed by
virtually all nucleated mammalian cells. The molecule has a 190 aa
residue extracellular region, a 25 aa residue transmembrane
segment, and a 220 aa residue cytoplasmic domain. Both
TNF-.alpha.and TNF-.beta. bind to TNFRI. Among the numerous cells
known to express TNFRI are endothelial cells.
[0096] TNFRII/p75/CD120b: Human TNFRII is a 75 kDa, 461 aa residue
transmembrane glycoprotein originally isolated from a human lung
fibroblast library. This receptor consists of a 240 aa residue
extracellular region, a 27 aa residue transmembrane segment and a
173 aa residue cytoplasmic domain. Soluble forms of TNFRII have
been identified, resulting apparently from proteolytic cleavage by
a metalloproteinase termed TRRE (TNF-Receptor Releasing Enzyme).
The shedding process appears to be independent of that for soluble
TNFRI. Among the multitude of cells known to express TNFRII are
endothelial cells.
[0097] CD134L/OX40L: OX40, the receptor for OX40L, is a T cell
activation marker with limited expression that seems to promote the
survival (and perhaps prolong the immune response) of CD4.sup.+ T
cells at sites of inflammation. OX40L also shows limited
expression. Currently only activated CD4.sup.+, CD8.sup.+ T cells,
B cells, and vascular endothelial cells have been reported to
express this factor. The human ligand is a 32 kDa, 183 aa residue
glycosylated polypeptide that consists of a 21 aa residue
cytoplasmic domain, a 23 aa residue transmembrane segment, and a
139 aa residue extracellular region.
[0098] VEGF Receptor Family
[0099] There are three receptors in the VEGF receptor family. They
have the common properties of multiple IgG-like extracellular
domains and tyrosine kinase activity. The enzyme domains of VEGF
receptor 1 (VEGF R1, also known as Flt-1), VEGF R2 (also known as
KDR or Flk-1), and VEGF R3 (also known as Flt-4) are divided by an
inserted sequence. Endothelial cells also express additional VEGF
receptors, Neuropilin-1 and Neuropilin-2. VEGF-A binds to VEGF R1
and VEGF R2 and to Neuropilin-1 and Neuropilin-2. PIGF and VEGF-B
bind VEGF R1 and Neuropilin-1. VEGF-C and -D bind VEGF R3 and VEGF
R2. HIV-tat and peptides derived therefrom have also been found to
target the VEGFR.
[0100] PDGF Receptors
[0101] PDGF receptors are expressed in the stromal compartment in
most common solid tumors. Inhibition of stromally expressed PDGF
receptors in a rat colon carcinoma model reduces the tumor
interstitial fluid pressure and increases tumor transcapillary
transport.
[0102] PSMA
[0103] Prostate specific membrane antigen (PSMA) is also an
excellent tumor endothelial marker, and PSMA antibodies can be
generated.
[0104] Cell Adhesion Molecules (CAMs)
[0105] Cell adhesion molecules (CAMs) are cell surface proteins
involved in the binding of cells, usually leukocytes, to each
other, to endothelial cells, or to extracellular matrix. Specific
signals produced in response to wounding and infection control the
expression and activation of certain of these adhesion molecules.
The interactions and responses then initiated by binding of these
CAMs to their receptors/ligands play important roles in the
mediation of the inflammatory and immune reactions that constitute
one line of the body's defence against these insults. Most of the
CAMs characterised so far fall into three general families of
proteins: the immunoglobulin (Ig) superfamily, the integrin family,
or the selectin family.
[0106] A member of the Selectin family of cell surface molecules,
L-Selectin consists of an NH2-terminal lectin type C domain, an
EGF-like domain, two complement control domains, a 15 amino acid
residue spacer, a transmembrane sequence and a short cytoplasmic
domain.
[0107] Three ligands for L-Selectin on endothelial cells have been
identified, all containing O-glycosylated mucin or mucin-like
domains. The first ligand, GlyCAM-1, is expressed almost
exclusively in peripheral and mesenteric lymph node high
endothelial venules. The second L-Selectin ligand, originally
called sgp90, has now been shown to be CD34. This sialomucin-like
glycoprotein, often used as a surface marker for the purification
of pluripotent stem cells, shows vascular expression in a wide
variety of nonlymphoid tissues, as well as on the capillaries of
peripheral lymph nodes. The third ligand for L-Selectin is MadCAM
1, a mucin-like glycoprotein found on mucosal lymph node high
endothelial venules.
[0108] P-Selectin, a member of the Selectin family of cell surface
molecules, consists of an NH2-terminal lectin type C domain, an
EGF-like domain, nine complement control domains, a transmembrane
domain, and a short cytoplasmic domain.
[0109] The tetrasaccharide sialyl Lewisx (sLex) has been identified
as a ligand for both P-- and E-Selectin, but P- E- and L-Selectin
can all bind sLex and sLea under appropriate conditions. P-Selectin
also reportedly binds selectively to a 160 kDa glycoprotein present
on murine myeloid cells and to a glycoprotein on myeloid cells,
blood neutrophils, monocytes, and lymphocytes termed P-Selectin
glycoprotein ligand-1 (PSGL-1), a ligand that also can bind
E-Selectin. P-Selectin-mediated rolling of leukocytes can be
completely inhibited by a monoclonal antibody specific for PSLG-1,
suggesting that even though P-Selectin can bind to a variety of
glycoproteins under in vitro conditions, it is likely that
physiologically important binding is more limited. A variety of
evidence indicates that P-Selectin is involved in the adhesion of
myeloid cells, as well as B and a subset of T cells, to activated
endothelium.
[0110] Ig Superfamily CAMs
[0111] The Ig superfamily CAMs are calcium-independent
transmembrane glycoproteins. Members of the Ig superfamily include
the intercellular adhesion molecules (ICAMs), vascular-cell
adhesion molecule (VCAM-1), platelet-endothelial-cell adhesion
molecule (PECAM-1), and neural-ceu adhesion molecule (NCAM). Each
Ig superfamily CAM has an extracellular domain, which contains
several Ig-like intrachain disulfide-bonded loops with conserved
cysteine residues, a transmembrane domain, and an intracellular
domain that interacts with the cytoskeleton. Typically, they bind
integrins or other Ig superfamily CAMs. The neuronal CAMs have been
implicated in neuronal patterning. Endothelial CAMs play an
important role in immune response and inflammation.
[0112] In more detail, vascular cell adhesion molecule (VCAM-1,
CD106, or INCAM-110), platelet endothelial cell adhesion molecule
(PECAM-1/CD31) and intercellular adhesion molecules 1, 2 &3
(ICAM-1, 2 & 3) are five functionally related CAM/IgSF
molecules that are critically involved in leukocyte-connective
tissue/endothelial cell interactions. Expressed principally on
endothelial cells, these molecules in general regulate leukocyte
migration across blood vessel walls and provide attachment points
for developing endothelium during angiogenesis and are all suitable
for targeting in the present invention.
[0113] Human CD31 is a 130 kDa, type I (extracellular N-terminus)
transmembrane glycoprotein that belongs to the cell adhesion
molecule (CAM) or C2-like subgroup of the IgSFl. The mature
molecule is 711 amino acid (aa) residues in length and contains a
574 aa residue extracellular region, a 19 aa residue transmembrane
segment, and a 118 aa residue cytoplasmic tail. In the
extracellular region, there are nine potential N-linked
glycosylation sites, and, with a predicted molecular weight of 80
kDa, it appears many of these sites are occupied. The most striking
feature of the extracellular region is the presence of six
Ig-homology units that resemble the C2 domains of the IgSF.
Although they vary in number, the presence of these modules is a
common feature of all IgSF adhesion molecules (ICAM-1, 2, 3 &
VCAM-1).
[0114] Integrins
[0115] Integrins are non-covalently linked heterodimers of .alpha.
and .beta. subunits. To date, 16 .alpha. subunits and 8 .beta.
subunits have been identified. These can combine in various ways to
form different types of integrin receptors. The ligands for several
of the integrins are adhesive extracellular matrix (ECM) proteins
such as fibronectin, vitronectin, collagens and laminin Many
integrins recognise the amino acid sequence RGD
(arginine-glycine-aspartic acid) which is present in fibronectin or
the other adhesive proteins to which they bind. Peptides and
protein fragments containing the RGD sequence can be used to
modulate the activities of the RGD-recognising integrins. Thus the
present invention may employ as the targeting moiety peptides
recognised by integrins. These peptides are conventionally known as
"RGD-containing peptides". These peptides may include peptides
motifs which have been identified as binding to integrins. These
motifs include the amino acid sequences: DGR, NGR and CRGDC. The
peptide motifs may be linear or cyclic. Such motifs are described
in more detail in the following patents which are herein
incorporated by reference in relation to their description of an
RGD peptides: U.S. Pat. No. 5,536,814 which describes cyclasized
CRGDCL, CRGDCA and GACRGDCLGA. U.S. Pat. No. 4,578,079 relates to
synthetic peptides of formula X-RGD-T/C-Y where X and Y are amino
acids. U.S. Pat. No. 5,547,936 describes a peptide counting the
sequence X-RGD-XX where X may be an amino acid. U.S. Pat. No.
4,988,621 describes a number of RGD-counting peptides. U.S. Pat.
No. 4,879,237 describes a general peptide of the formula RGD-Y
where Y is an amino acid, and the peptide G-RGD-AP. U.S. Pat. No.
5,169,930 describes the peptide RGDSPK which binds to .alpha.v
.beta.1 integrin. U.S. Pat. Nos. 5,498,694 and 5,700,908 relate to
the cytoplasmic domain of the .beta.3 integrin sub-unit that
strictly speaking is not an RGD-containing peptide; although it
does contain the sequence RDG. WO97/08203 describes cyclic peptides
that are structural mimics or RGD-binding sites. U.S. Pat. No.
5,612,311 describes 15 RGD-containing peptides that are capable of
being cyclized either by C-C linkage or through other groups such
as penicillamine or mecapto propionic acid analogs. U.S. Pat. No.
5,672,585 describes a general formula encompassing RGD-containing
peptides. A preferred group of peptides are those where the
aspartic acid residue of RGD is derivatised into an O-methoxy
tyrosine derivative. U.S. Pat. No. 5,120,829 describes an RGD cell
attachment promoting binding site and a hydrophobic attachment
domain. The D form is described in U.S. Pat. No. 5,587,456. U.S.
Pat. No. 5,648,330 describes a cyclic RGD-containing peptide that
has high affinity for GP Iib/IIIa.
[0116] In a preferred embodiment of the present invention the
targeting moiety is a ligand for .alpha.v .beta.3 or .alpha.v
.beta.5 integrin.
[0117] Activin
[0118] Cells known to express ActRII include endothelial cells.
ActRIIB expression parallels that for ActRII, and is again found in
endothelial cells. Cells known to express ActRI include vascular
endothelial cells. ActRIB has also been identified in endothelial
cells.
[0119] Angiogenin
[0120] Angiogenin (ANG) is a 14 kDa, non-glycosylated polypeptide
so named for its ability to induce new blood vessel growth.
[0121] Annexin V
[0122] Annexin V is a member of a calcium and phospholipid binding
family of proteins with vascular anticoagulant activity. Various
synomyms for Annexin V exist: placental protein 4 (PP4), placental
anticoagulant protein I (PAP I), calphobindin I (CPB-I), calcium
dependent phospholipid binding protein 33 (CaBP33), vascular
anticoagulant protein alpha (VACa), anchorin CII, lipocortin-V,
endonexin II, and thromboplastin inhibitor. The number of binding
sites for Annexin V has been reported as 6-24.times.106/cell in
tumor cells and 8.8.times.106/cell for endothelial cells.
[0123] CD44
[0124] Another molecule apparently involved in white cell adhesive
events is CD44, a molecule ubiquitously expressed on both
hematopoietic and non-hematopoietic cells. CD44 is remarkable for
its ability to generate alternatively spliced forms, many of which
differ in their activities. This remarkable flexibility has led to
speculation that CD44, via its changing nature, plays a role in
some of the methods that tumor cells use to progress successfully
through growth and metastasis. CD44 is a 80-250 kDa type I
(extracellular N-terminus) transmembrane glycoprotein. Cells known
to express CD44H include vascular endothelial cells.
[0125] There are multiple ligands for CD44, including osteopontin,
fibronectin, collagen types I and IV and hyaluronate. Binding to
fibronectin is reported to be limited to CD44 variants expressing
chrondroitin sulfate, with the chrondroitin sulfate attachment site
localised to exons v8-v11. Hyaluronate binding is suggested to be
possible for virtually all CD44 isoforms. One of the principal
binding sites is proposed to be centred in exon 2 and to involve
lysine and arginine residues. Factors other than the simple
expression of a known hyaluronate-binding motif also appear to be
necessary for hyaluronate binding. Successful hyaluronate binding
is facilitated by the combination of exons expressed, a distinctive
cytoplasmic tail, glycosylation patterns, and the activity state of
the cell. Thus, in terms of its hyaluronate-binding function, a
great deal of "potential" flexibility exists within each
CD44-expressing cell.
[0126] Fibroblast Growth Factor (FGF)
[0127] The name "fibroblast growth factor" (FGF) is a limiting
description for this family of cytokines. The function of FGFs is
not restricted to cell growth. Although some of the FGFs do,
indeed, induce fibroblast proliferation, the original FGF molecule
(FGF-2 or FGF basic) is now known to also induce proliferation of
endothelial cells, chondrocytes, smooth muscle cells, melanocytes,
as well as other cells. It can also promote adipocyte
differentiation, induce macrophage and fibroblast IL-6 production,
stimulate astrocyte migration, and prolong neuronal survival. To
date, the FGF superfamily consists of 23 members, all of which
contain a conserved 120 amino acid (aa) core region that contains
six identical, interspersed amino acids.
[0128] FGF-1: Human FGF-1 (also known as FGF acidic, FGFa, ECGF and
HBGF-1) is a 17-18 kDa non-glycosylated polypeptide that is
expressed by a variety of cells from all three germ layers. The
binding molecule may be either an FGF receptor. Cells known to
express FGF-1 include endothelial cells.
[0129] FGF-2: Human FGF-2, otherwise known as FGF basic, HBGF-2,
and EDGF, is an 18 kDa, non-glycosylated polypeptide that shows
both intracellular and extracellular activity. Following secretion,
FGF-2 is sequestered on either cell surface HS or matrix
glycosaminoglycans. Although FGF-2 is secreted as a monomer, cell
surface HS seems to dimerize monomeric FGF-2 in a non-covalent
side-to-side configuration that is subsequently capable of
dimerizing and activating FGF receptors. Cells known to express
FGF-2 include endothelial cells.
[0130] FGF-3: Human FGF-3 is the product of the int-2 gene [i.e.,
derived from integration region-2, a region on mouse chromosome 7
that contains a gene (int-2/FGF-3) accidentally activated following
retroviral insertion]. The molecule is synthesised as a 28-32 kDa,
222 aa glycoprotein that contains a number of peptide motifs. Cells
reported to express FGF-3 are limited to developmental cells and
tumors. Tumors known to express FGF-3 include breast carcinomas and
colon cancer cell lines.
[0131] FGF4: Human FGF-4 is a 22 kDa, 176 aa glycoprotein that is
the product of a developmentally-regulated gene. The molecule is
synthesised as a 206 aa precursor that contains a large,
ill-defined 30 aa signal sequence plus two heparin-binding motifs
(at aa 51-55 and 140-143). The heparin-binding sites directly
relate to FGF4 activity; heparin/heparan regulate the ability of
FGF-4 to activate FGFR1 and FGFR2. Cells known to express FGF-4
include both tumor cells and embryonic cells. Its identification in
human stomach cancer gives rise to one alternative designation
(/hst-1/hst), while its isolation in Kaposi's sarcoma provides
grounds for another (K-FGF).
[0132] IL-1R
[0133] IL-1 exerts its effects by binding to specific receptors.
Two distinct IL-1 receptor binding proteins, plus a non-binding
signalling accessory protein have been identified. Each have three
extracellular immunoglobulin-like (Ig-like) domains, qualifying
them for membership in the type IV cytokine receptor family. The
two receptor binding proteins are termed type I IL-1 receptor (IL-1
RI) and type II IL-1 receptor (IL-1 RII) respectively. Human IL-1
RI is a 552 aa, 80 kDa transmembrane glycoprotein that has been
isolated from endothelium cells.
[0134] RTK
[0135] The new family of receptor tyrosine kinase (RTK), the Eph
receptors and their ligands ephrins, have been found to be involved
in vascular assembly, angiogenesis, tumorigenesis, and metastasis.
It has also been that class A Eph receptors and their ligands are
elevated in tumor and associated vasculature.
[0136] MMP
[0137] Matrix metalloproteinases (MMPs) have been implicated in
tumor growth, angiogenesis, invasion, and metastasis. They have
also been suggested for use as tumor markers.
[0138] NG2
[0139] NG2 is a large, integral membrane, chondroitin sulfate
proteoglycan that was first identified as a cell surface molecule
expressed by immature neural cells. Subsequently NG2 was found to
be expressed by a wide variety of immature cells as well as several
types of tumors with high malignancy. NG2 has been suggested as a
target molecule in the tumor vasculature. In particular,
collagenase-1 (C1) is the predominant matrix metalloproteinase
present in newly formed microvessels and serves as a marker of
neovascularization.
[0140] Oncofetal Fibronectin
[0141] The expression of the oncofetal fragment of fibronectin
(Fn-f) has also been found to be increased during angiogenesis and
has been suggested as a marker of tumor angiogenesis. In one
embodiment the TTM is an antibody or fragment thereof to the
oncofetal ED-B domain of fibronectin. The preparation of such an
antibody and its conjugation with EL-12 is described in Halin et al
(2002) Nature Biotechnology 20:264-269.
[0142] Tenascin
[0143] Tenascin is a matrix glycoprotein seen in malignant tumors
including brain and breast cancers and melanoma Its expression is
malignant but not well differentiated tumors and association with
the blood vessels of tumors makes it an important target for both
understanding the biology of malignant tumors and angiogenesis, but
is a therapeutic cancer target and marker as well.
[0144] The targeting moiety is preferably a polypeptide which is
capable of binding to a tumor cell or tumor vasculature surface
molecule. As well as those mentioned above other such surface
molecules which are known or become available may also be targeted
by the first sequence.
[0145] It will be appreciated that one can apply conventional
protein binding assays to identify molecules which bind to surface
molecules. It will also be appreciated that one can apply
structural-based drug design to develop sequences which bind to
surface molecules.
[0146] High throughput screening, as described above for synthetic
compounds, can also be used for identifying targeting
molecules.
[0147] This invention also contemplates the use of competitive drug
screening assays in which neutralising antibodies capable of
binding a target specifically compete with a test compound for
binding to a target.
[0148] Binding Partner (BP)
[0149] The targeting moiety generally take the form of a binding
partner (BP) to a surface molecule comprising or consisting of one
or more binding domains.
[0150] Ligand
[0151] The targeting moiety of the present invention may take the
form of a ligand. The ligands may be natural or synthetic. The term
"ligand" also refers to a chemically modified ligand. The one or
more binding domains of the BP may consist of, for example, a
natural ligand for a receptor, which natural ligand may be an
adhesion molecule or a growth-factor receptor ligand (e.g.
epidermal growth factor), or a fragment of a natural ligand which
retains binding affinity for the receptor.
[0152] Synthetic ligands include the designer ligands. As used
herein, the term means "designer ligands" refers to agents which
are likely to bind to the receptor based on their three dimensional
shape compared to that of the receptor.
[0153] Antibodies
[0154] Alternatively, the binding domains may be derived from heavy
and light chain sequences from an immunoglobulin (Ig) variable
region. Such a variable region may be derived from a natural human
antibody or an antibody from another species such as a rodent
antibody. Alternatively the variable region may be derived from an
engineered antibody such as a humanised antibody or from a phage
display library from an immunised or a non-immunised animal or a
mutagenised phage-display library. As a second alternative, the
variable region may be derived from a single-chain variable
fragment (scFv). The BP may contain other sequences to achieve
multimerisation or to act as spacers between the binding domains or
which result from the insertion of restriction sites in the genes
encoding the BP, including Ig hinge sequences or novel spacers and
engineered linker sequences.
[0155] The BP may comprise, in addition to one or more
immunoglobulin variable regions, all or part of an Ig heavy chain
constant region and so may comprise a natural whole Ig, an
engineered Ig, an engineered Ig-like molecule, a single-chain Ig or
a single-chain Ig-like molecule. Alternatively, or in addition, the
BP may contain one or more domains from another protein such as a
toxin.
[0156] As used herein, an "antibody" refers to a protein consisting
of one or more polypeptides substantially encoded by immunoglobulin
genes or fragments of immunoglobulin genes. Antibodies may exist as
intact immunoglobulins or as a number of fragments, including those
well-characterised fragments produced by digestion with various
peptidases. While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that antibody fragments may be synthesised de novo
either chemically or by utilising recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody
fragments either produced by the modification of whole antibodies
or synthesised de novo using recombinant DNA methodologies.
Antibody fragments encompassed by the use of the term "antibodies"
include, but are not limited to, Fab, Fab', F (ab') 2, scFv, Fv,
dsFv diabody, and Fd fragments.
[0157] The invention also provides monoclonal or polyclonal
antibodies to the surface proteins. Thus, the present invention
further provides a process for the production of monoclonal or
polyclonal antibodies to polypeptides of the invention
[0158] If polyclonal antibodies are desired, a selected mammal
(e.g., mouse, rabbit, goat, horse, etc.) is immunised with an
immunogenic polypeptide bearing an epitope(s). Serum from the
immunised animal is collected and treated according to known
procedures. If serum containing polyclonal antibodies to an epitope
contains antibodies to other antigens, the polyclonal antibodies
can be purified by immunoaffinity chromatography. Techniques for
producing and processing polyclonal antisera are known in the art.
In order that such antibodies may be made, the invention also
provides polypeptides of the invention or fragments thereof
haptenised to another polypeptide for use as immunogens in animals
or humans.
[0159] Monoclonal antibodies directed against binding cell surface
epitopes in the polypeptides can also be readily produced by one
skilled in the art. The general methodology for making monoclonal
antibodies by hybridomas is well known. Immortal antibody-producing
cell lines can be created by cell fusion, and also by other
techniques such as direct transformation of B lymphocytes with
oncogenic DNA, or transfection with Epstein-Barr virus. Panels of
monoclonal antibodies produced against epitopes can be screened for
various properties; i.e., for isotype and epitope affinity.
[0160] An alternative technique involves screening phage display
libraries where, for example the phage express scFv fragments on
the surface of their coat with a large variety of complementarity
determining regions (CDRs). This technique is well known in the
art.
[0161] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes fragments of whole
antibodies which retain their binding activity for a target
antigen. As mentioned above such fragments include Fv, F(ab) and
F(ab').sub.2 fragments, as the use of the term "antibodies"
include, but are not limited to, Fab, Fab', F (ab') 2, scFv, Fv,
dsFv diabody, and Fd fragments.
[0162] The invention also provides monoclonal or polyclonal
antibodies to the surface proteins. Thus, the present invention
further provides a process for the production of monoclonal or
polyclonal antibodies to polypeptides of the invention.
[0163] If polyclonal antibodies are desired, a selected mammal
(e.g., mouse, rabbit, goat, horse, etc.) is immunised with an
immunogenic polypeptide bearing an epitope(s). Serum from the
immunised animal is collected and treated according to known
procedures. If serum containing polyclonal antibodies to an epitope
contains antibodies to other antigens, the polyclonal antibodies
can be purified by immunoaffinity chromatography. Techniques for
producing and processing polyclonal antisera are known in the art.
In order that such antibodies may be made, the invention also
provides polypeptides of the invention or fragments thereof
haptenised to another polypeptide for use as immunogens in animals
or humans.
[0164] Monoclonal antibodies directed against binding cell surface
epitopes in the polypeptides can also be readily produced by one
skilled in the art. The general methodology for making monoclonal
antibodies by hybridomas is well known. Immortal antibody-producing
cell lines can be created by cell fusion, and also by other
techniques such as direct transformation of B lymphocytes with
oncogenic DNA, or transfection with Epstein-Barr virus. Panels of
monoclonal antibodies produced against epitopes can be screened for
various properties; i.e., for isotype and epitope affinity.
[0165] An alternative technique involves screening phage display
libraries where, for example the phage express scFv fragments on
the surface of their coat with a large variety of complementarity
determining regions (CDRs). This technique is well known in the
art.
[0166] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes fragments of whole
antibodies which retain their binding activity for a target
antigen. As mentioned above such fragments include Fv, F(ab') and
F(ab').sub.2 fragments, as well as single chain antibodies (scFv).
Furthermore, the antibodies and fragments thereof may be humanised
antibodies, for example as described in EP-A-239400.
[0167] Screens
[0168] In one aspect, the invention relates to a method of
screening for an agent capable of binding to a tumor or tumor
vasculature cell surface molecule, the method comprising contacting
the cell surface molecule with an agent and determining if said
agent binds to said cell surface molecule.
[0169] As used herein, the term "agent" includes, but is not
limited to, a compound, such as a test compound, which may be
obtainable from or produced by any suitable source, whether natural
or not. The agent may be designed or obtained from a library of
compounds which may comprise peptides, as well as other compounds,
such as small organic molecules and particularly new lead
compounds. By way of example, the agent may be a natural substance,
a biological macromolecule, or an extract made from biological
materials such as bacteria, fungi, or animal (particularly
mammalian) cells or tissues, an organic or an inorganic molecule, a
synthetic test compound, a semi-synthetic test compound, a
structural or functional mimetic, a peptide, a peptidomimetics, a
derivatised test compound, a peptide cleaved from a whole protein,
or a peptides synthesised synthetically (such as, by way of
example, either using a peptide synthesizer) or by recombinant
techniques or combinations thereof, a recombinant test compound, a
natural or a non-natural test compound, a fusion protein or
equivalent thereof and mutants, derivatives or combinations
thereof.
[0170] The agent can be an amino acid sequence or a chemical
derivative thereof. The substance may even be an organic compound
or other chemical. The agent may even be a nucleotide
sequence--which may be a sense sequence or an anti-sense
sequence.
[0171] Protein
[0172] The term "protein" includes single-chain polypeptide
molecules as well as multiple-polypeptide complexes where
individual constituent polypeptides are linked by covalent or
non-covalent means. The term "polypeptide" includes peptides of two
or more amino acids in length, typically having more than 5, 10 or
20 amino acids.
[0173] Polypeptide Homologues
[0174] It will be understood that polypeptide sequences for use in
the invention are not limited to the particular sequences or
fragments thereof but also include homologous sequences obtained
from any source, for example related viral/bacterial proteins,
cellular homologues and synthetic peptides, as well as variants or
derivatives thereof. Polypeptide sequences of the present invention
also include polypeptides encoded by polynucleotides of the present
invention.
[0175] Polypeptide Variants, Derivatives and Fragments
[0176] The terms "variant" or "derivative" in relation to the amino
acid sequences of the present invention includes any substitution
of, variation of, modification of, replacement of, deletion of or
addition of one (or more) amino acids from or to the sequence
providing the resultant amino acid sequence preferably has
targeting activity, preferably having at least 25 to 50% of the
activity as the polypeptides presented in the sequence listings,
more preferably at least substantially the same activity.
[0177] Thus, sequences may be modified for use in the present
invention. Typically, modifications are made that maintain the
activity of the sequence. Thus, in one embodiment, amino acid
substitutions maybe made, for example from 1, 2 or 3 to 10, 20 or
30 substitutions provided that the modified sequence retains at
least about 25 to 50% of, or substantially the same activity.
However, in an alternative embodiment, modifications to the amino
acid sequences of a polypeptide of the invention may be made
intentionally to reduce the biological activity of the polypeptide.
For example truncated polypeptides that remain capable of binding
to target molecule but lack functional effector domains may be
useful.
[0178] In general, preferably less than 20%, 10% or 5% of the amino
acid residues of a variant or derivative are altered as compared
with the corresponding region depicted in the sequence
listings.
[0179] Amino acid substitutions may include the use of
non-naturally occurring analogues, for example to increase blood
plasma half-life of a therapeutically administered polypeptide (see
below for further details on the production of peptide derivatives
for use in therapy).
[0180] Conservative substitutions may be made, for example
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
3 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q
Polar - charged D E K R AROMATIC H F W Y
[0181] Polypeptides of the invention also include fragments of the
above mentioned polypeptides and variants thereof, including
fragments of the sequences. Preferred fragments include those which
include an epitope. Suitable fragments will be at least about 5,
e.g. 10, 12, 15 or 20 amino acids in length. They may also be less
than 200, 100 or 50 amino acids in length Polypeptide fragments of
the proteins and allelic and species variants thereof may contain
one or more (e.g. 2, 3, 5, or 10) substitutions, deletions or
insertions, including conserved substitutions. Where substitutions,
deletion and/or insertions have been made, for example by means of
recombinant technology, preferably less than 20%, 10% or 5% of the
amino acid residues depicted in the sequence listings are
altered.
[0182] Proteins of the invention are typically made by recombinant
means, for example as described below. However they may also be
made by synthetic means using techniques well known to skilled
persons such as solid phase synthesis. Various techniques for
chemical synthesising peptides are reviewed by Borgia and Fields,
2000, TibTech 18: 243-251 and described in detail in the references
contained therein.
[0183] Therapeutic Peptides
[0184] Peptides of the present invention may be administered
therapeutically to patients. It is preferred to use peptides that
do not consisting solely of naturally-occurring amino acids but
which have been modified, for example to reduce immunogenicity, to
increase circulatory half-life in the body of the patient, to
enhance bioavailability and/or to enhance efficacy and/or
specificity.
[0185] A number of approaches have been used to modify peptides for
therapeutic application. One approach is to link the peptides or
proteins to a variety of polymers, such as polyethylene glycol
(PEG) and polypropylene glycol (PPG)--see for example U.S. Pat.
Nos. 5,091,176, 5,214,131 and U.S. Pat. No. 5,264,209.
[0186] Replacement of naturally-occurring amino acids with a
variety of uncoded or modified amino acids such as D-amino acids
and N-methyl amino acids may also be used to modify peptides
[0187] Another approach is to use bifunctional crosslinkers, such
as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl
6-[3-(2 pyridyldithio) propionamido] hexanoate, and
sulfosuccinimidyl 6-[3-(2 pyridyldithio) propionamido]hexanoate
(see U.S. Pat. No. 5,580,853).
[0188] It may be desirable to use derivatives of the peptides of
the invention which are conformationally constrained.
Conformational constraint refers to the stability and preferred
conformation of the three-dimensional shape assumed by a peptide.
Conformational constraints include local constraints, involving
restricting the conformational mobility of a single residue in a
peptide; regional constraints, involving restricting the
conformational mobility of a group of residues, which residues may
form some secondary structural unit; and global constraints,
involving the entire peptide structure.
[0189] The active conformation of the peptide may be stabilised by
a covalent modification, such as cyclization or by incorporation of
gamma-lactam or other types of bridges. For example, side chains
can be cyclized to the backbone so as create a L-gamma-lactam
moiety on each side of the interaction site. See, generally, Hruby
et al., "Applications of Synthetic Peptides," in Synthetic
Peptides: A User's Guide: 259-345 (W. H. Freeman & Co. 1992).
Cyclization also can be achieved, for example, by formation of
cysteine bridges, coupling of amino and carboxy terminal groups of
respective terminal amino acids, or coupling of the amino group of
a Lys residue or a related homolog with a carboxy group of Asp, Glu
or a related homolog. Coupling of the alpha-amino group of a
polypeptide with the epsilon-amino group of a lysine residue, using
iodoacetic anhydride, can be also undertaken See Wood and Wetzel,
1992, Int'l J. Peptide Protein Res. 39: 533-39.
[0190] Another approach described in U.S. Pat. No. 5,891,418 is to
include a metal-ion complexing backbone in the peptide structure.
Typically, the preferred metal-peptide backbone is based on the
requisite number of particular coordinating groups required by the
coordination sphere of a given complexing metal ion. In general,
most of the metal ions that may prove useful have a coordination
number of four to six. The nature of the coordinating groups in the
peptide chain includes nitrogen atoms with amine, amide, imidazole,
or guanidino functionalities; sulfur atoms of thiols or disulfides;
and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl
functionalities. In addition, the peptide chain or individual amino
acids can be chemically altered to include a coordinating group,
such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano,
pyridino, piperidino, or morpholino. The peptide construct can be
either linear or cyclic, however a linear construct is typically
preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly
which has four nitrogens (an N.sub.4 complexation system) in the
back bone that can complex to a metal ion with a coordination
number of four.
[0191] A further technique for improving the properties of
therapeutic peptides is to use non-peptide peptidomimetics. A wide
variety of useful techniques may be used to elucidating the precise
structure of a peptide. These techniques include amino acid
sequencing, x-ray crystallography, mass spectroscopy, nuclear
magnetic resonance spectroscopy, computer-assisted molecular
modelling, peptide mapping, and combinations thereof. Structural
analysis of a peptide generally provides a large body of data which
comprise the amino acid sequence of the peptide as well as the
three-dimensional positioning of its atomic components. From this
information, non-peptide peptidomimetics may be designed that have
the required chemical functionalities for therapeutic activity but
are more stable, for example less susceptible to biological
degradation. An example of this approach is provided in U.S. Pat.
No. 5,811,512.
[0192] Techniques for chemically synthesising therapeutic peptides
of the invention are described in the above references and also
reviewed by Borgia and Fields, 2000, TibTech 18: 243-251 and
described in detail in the references contained therein.
[0193] Bifunctional Derivatives
[0194] A further embodiment of the invention is provided by
bifunctional derivatives in which the cytokines modified with a TTM
are conjugated with antibodies, or their fragments, against tumoral
antigens or other tumor angiogenic markers, e.g. .alpha.v
integrins, metalloproteases or the vascular growth factor, or
antibodies or fragments thereof directed against components of the
extracellular matrix, such as anti-tenascin antibodies or
anti-fibronectin EDB domain. The preparation of a fusion product
between TNF and the hinge region of a mAb against the
tumor-associated TAG72 antigen expressed by gastric and ovarian
adenocarcinoma has recently been reported.
[0195] A further embodiment of the invention is provided by the
tumoral pre-targeting with the biotin/avidin system. According to
this approach, a ternary complex is obtained on the tumoral
antigenic site, at different stages, which is formed by 1)
biotinylated mAb, 2) avidin (or streptavidin) and 3) bivalent
cytokine modified with the TTM and biotin. A number of papers
proved that the pre-targeting approach, compared with conventional
targeting with immunoconjugates, can actually increase the ratio of
active molecule homed at the target to free active molecule, thus
reducing the treatment toxicity. This approach produced favorable
results with biotinylated TNF, which was capable of inducing
cytotoxicity in vitro and decreasing the tumor cells growth under
conditions in which normal TNF was inactive. The pre-targeting
approach can also be carried out with a two-phase procedure by
using a bispecific antibody which at the same time binds the
tumoral antigen and the modified cytokine. The use of a bispecific
antibody directed against a carcinoembryonic antigen and TNF has
recently been described as a means for TNF turmoral
pre-targeting.
[0196] According to a further embodiment, the invention comprises a
cytokine conjugated to both a TTM and an antibody, or a fragment
thereof (directly or indirectly via a boitin-avidin bridge), on
different TNF subunits, where the antibody or its fragments are
directed against an antigen expressed on tumor cells or other
components of the tumor stroma, e.g. tenacin and fibronectin EDB
domain. This results in a further improvement of the tumor homing
properties of the modified cytokine and in the slow release of the
latter in the tumor microenvironment through trimer-monomer-trimer
transitions. The modified subunits of e.g. TNF conjugates can
disassociate from the targeting complexes and reassociate so as to
form unmodified trimeric TNF molecules, which then diffuse in the
tumor microenvironment. The release of bioactive TNF has been shown
to occur within 24-48 hours after targeting.
[0197] The preparation of cytokines in the form of liposomes can
improve the biological activity thereof. It has, in fact, been
observed that acylation of the TNF amino groups induces an increase
in its hydrophobicity without loss of biological activity in vitro.
Furthermore, it has been reported that TNF bound to lipids has
unaffected cytotoxicity in vitro, immunomodulating effects and
reduced toxicity in vivo.
[0198] Polynucleotides
[0199] Polynucleotides for use in the invention comprise nucleic
acid sequences encoding the polypeptide conjugate of the invention.
It will be understood by a skilled person that numerous different
polynucleotides can encode the same polypeptide as a result of the
degeneracy of the genetic code. In addition, it is to be understood
that skilled persons may, using routine techniques, make nucleotide
substitutions that do not affect the polypeptide sequence encoded
by the polynucleotides of the invention to reflect the codon usage
of any particular host organism in which the polypeptides of the
invention are to be expressed.
[0200] Polynucleotides of the invention may comprise DNA or RNA.
They may be single-stranded or double-stranded. They may also be
polynucleotides which include within them synthetic or modified
nucleotides. A number of different types of modification to
oligonucleotides are known in the art. These include
methylphosphonate and phosphorothioate backbones, addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present invention, it is to be
understood that the polynucleotides described herein may be
modified by any method available in the art. Such modifications may
be carried out in order to enhance the in vivo activity or life
span of polynucleotides of the invention.
[0201] Nucleotide Vectors
[0202] Polynucleotides of the invention can be incorporated into a
recombinant replicable vector. The vector may be used to replicate
the nucleic acid in a compatible host cell. Thus in a further
embodiment, the invention provides a method of making
polynucleotides of the invention by introducing a polynucleotide of
the invention into a replicable vector, introducing the vector into
a compatible host cell, and growing the host cell under conditions
which bring about replication of the vector. The vector may be
recovered from the host cell. Suitable host cells include bacteria
such as E. coli, yeast, mammalian cell lines and other eukaryotic
cell lines, for example insect Sf9 cells.
[0203] Preferably, a polynucleotide of the invention in a vector is
operably linked to a control sequence that is capable of providing
for the expression of the coding sequence by the host cell, i.e.
the vector is an expression vector. The term "operably linked"
means that the components described are in a relationship
permitting them to function in their intended manner. A regulatory
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
condition compatible with the control sequences.
[0204] The control sequences may be modified, for example by the
addition of further transcriptional regulatory elements to make the
level of transcription directed by the control sequences more
responsive to transcriptional modulators.
[0205] Vectors of the invention may be transformed or transfected
into a suitable host cell as described below to provide for
expression of a protein of the invention. This process may comprise
culturing a host cell transformed with an expression vector as
described above under conditions to provide for expression by the
vector of a coding sequence encoding the protein, and optionally
recovering the expressed protein.
[0206] The vectors may be for example, plasmid or virus vectors
provided with an origin of replication, optionally a promoter for
the expression of the said polynucleotide and optionally a
regulator of the promoter. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene
in the case of a bacterial plasmid or a neomycin resistance gene
for a mammalian vector. Vectors may be used, for example, to
transfect or transform a host cell.
[0207] Control sequences operably linked to sequences encoding the
protein of the invention include promoters/enhancers and other
expression regulation signals. These control sequences may be
selected to be compatible with the host cell for which the
expression vector is designed to be used in. The term "promoter" is
well-known in the art and encompasses nucleic acid regions ranging
in size and complexity from minimal promoters to promoters
including upstream elements and enhancers.
[0208] The promoter is typically selected from promoters which are
functional in mammalian cells, although prokaryotic promoters and
promoters functional in other eukaryotic cells may be used. The
promoter is typically derived from promoter sequences of viral or
eukaryotic genes. For example, it may be a promoter derived from
the genome of a cell in which expression is to occur. With respect
to eukaryotic promoters, they may be promoters that function in a
ubiquitous manner (such as promoters of a-actin, b-actin, tubulin)
or, alternatively, a tissue-specific manner (such as promoters of
the genes for pyruvate kinase). Tissue-specific promoters specific
for certain cells may also be used. They may also be promoters that
respond to specific stimuli, for example promoters that bind
steroid hormone receptors. Viral promoters may also be used, for
example the Moloney murine leukaemia virus long terminal repeat
(MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or
the human cytomegalovirus (CMV) EI promoter.
[0209] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the heterologous gene
can be regulated during the life-time of the cell. Inducible means
that the levels of expression obtained using the promoter can be
regulated.
[0210] In addition, any of these promoters may be modified by the
addition of further regulatory sequences, for example enhancer
sequences. Chimeric promoters may also be used comprising sequence
elements from two or more different promoters described above.
[0211] Host Cells
[0212] Vectors and polynucleotides of the invention may be
introduced into host cells for the purpose of replicating the
vectors/polynucleotides and/or expressing the proteins of the
invention encoded by the polynucleotides of the invention. Although
the proteins of the invention may be produced using prokaryotic
cells as host cells, it is preferred to use eukaryotic cells, for
example yeast, insect or mammalian cells, in particular mammalian
cells.
[0213] Vectors/polynucleotides of the invention may introduced into
suitable host cells using a variety of techniques known in the art,
such as transfection, transformation and electroporation. Where
vectors/polynucleotides of the invention are to be administered to
animals, several techniques are known in the art, for example
infection with recombinant viral vectors such as retroviruses,
herpes simplex viruses and adenoviruses, direct injection of
nucleic acids and biolistic transformation.
[0214] Protein Expression and Purification
[0215] Host cells comprising polynucleotides of the invention may
be used to express proteins of the invention. Host cells may be
cultured under suitable conditions which allow expression of the
proteins of the invention. Expression of the proteins of the
invention may be constitutive such that they are continually
produced, or inducible, requiring a stimulus to initiate
expression. In the case of inducible expression, protein production
can be initiated when required by, for example, addition of an
inducer substance to the culture medium, for example dexamethasone
or IPTG.
[0216] Proteins of the invention can be extracted from host cells
by a variety of techniques known in the art, including enzymatic,
chemical and/or osmotic lysis and physical disruption.
[0217] Administration
[0218] Proteins of the invention may preferably be combined with
various components to produce compositions of the invention.
Preferably the compositions are combined with a pharmaceutically
acceptable carrier, diluent or excipient to produce a
pharmaceutical composition (which may be for human or animal use).
Suitable carriers and diluents include isotonic saline solutions,
for example phosphate-buffered saline. Details of excipients may be
found in The Handbook of Pharmaceutical Excipients, 2nd Edn, Eds
Wade & Weller, American Pharmaceutical Association. The
composition of the invention may be administered by direct
injection. The composition may be formulated for parenteral,
intramuscular, intravenous, subcutaneous, intraocular, oral or
transdermal administration. Typically, each protein may be
administered at a dose of from in the range of 0.5 to 500 ng/kg,
preferably in the range of 1 to 50 ng/kg, more preferably in the
range of 5 to 15 ng/kg.
[0219] In an alternative aspect the present invention provides a
pharmaceutical composition comprising a conjugate, wherein the
conjugate is present in an amount such that the conjugate or a
metabolite thereof is provided to the blood plasma of the subject
to be treated in an amount of no greater than about 35,000 ng/day,
preferably about 3,500 ng/day, more preferably about 1,000
ng/day.
[0220] The above dosage relate to a dosage for a 70 kg subject. A
person skilled in the art would readily be able to modify the
recited dosage for a subject having as mass other than 70 kg.
[0221] The dosage per day is calculated by dividing the dose to be
administered to the subject by the anticipated dosage period in
days. The anticipated dosage period will typically be the period
until the next administration, the period over which the dose is to
have effect or the period over which the dose is required to have
effect.
[0222] The composition may be formulated such that administration
daily, weekly or monthly will provide the desired daily dosage. It
will be appreciated that the composition may be conveniently
formulated for administrated less frequently, such as every 2, 4,
6, 8, 10 or 12 hours.
[0223] Polynucleotides/vectors encoding polypeptide components may
be administered directly as a naked nucleic acid construct,
preferably further comprising flanking sequences homologous to the
host cell genome.
[0224] Uptake of naked nucleic acid constructs by mammalian cells
is enhanced by several known transfection techniques for example
those including the use of transfection agents. Example of these
agents include cationic agents (for example calcium phosphate and
DEAE-dextran) and lipofectants (for example lipofectam.TM. and
transfectam.TM.). Typically, nucleic acid constructs are mixed with
the transfection agent to produce a composition.
[0225] Preferably the polynucleotide or vector of the invention is
combined with a pharmaceutically acceptable carrier or diluent to
produce a pharmaceutical composition. Suitable carriers and
diluents include isotonic saline solutions, for example
phosphate-buffered saline. The composition may be formulated for
parenteral, intramuscular, intravenous, subcutaneous, intraocular
or transdermal administration.
[0226] The routes of administration and dosage regimens described
are intended only as a guide since a skilled practitioner will be
able to determine readily the optimum route of administration and
dosage regimens for any particular patient and condition.
[0227] Viral Vectors
[0228] In a preferred embodiment the conjugate is administered
using a viral vector, more preferably a retroviral vector.
[0229] Retroviruses
[0230] The retroviral vector for use the present invention may be
derived from or may be derivable from any suitable retrovirus. A
large number of different retroviruses have been identified.
Examples include: murine leukemia virus (MLV), human
immunodeficiency virus (HIV), simian immunodeficiency virus, human
T-cell leukemia virus (HTLV). equine infectious anaemia virus
(EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV),
Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus
(Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine
sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV),
Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis
virus (AEV). A detailed list of retroviruses may be found in Coffin
et al., 1997, "retroviruses", Cold Spring Harbour Laboratory Press
Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763.
[0231] Details on the genomic structure of some retroviruses may be
found in the art. By way of example, details on HIV and Mo-MLV may
be found from the NCBI Genbank (Genome Accession Nos. AF033819 and
AF033811, respectively).
[0232] Retroviruses may be broadly divided into two categories:
namely, "simple" and "complex". Retroviruses may even be further
divided into seven groups. Five of these groups represent
retroviruses with oncogenic potential. The remaining two groups are
the lentiviruses and the spumaviruses. A review of these
retroviruses is presented in Coffin et al., 1997 (ibid).
[0233] The lentivirus group can be split even further into
"primate" and "on-primate". Examples of primate lentiviruses
include human immunodeficiency virus (HIV), the causative agent of
human auto-immunodeficiency syndrome (AIDS), and simian
immunodeficiency virus (SIV). The non-primate lentiviral group
includes the prototype "slow virus" visna/maedi virus (VMV), as
well as the related caprine arthritis-encephalitis virus (CAEV),
equine infectious anaemia virus (EIAV) and the more recently
described feline immunodeficiency virus (FIV) and bovine
immunodeficiency virus (BIV).
[0234] This invention also relates to the use of vectors for the
delivery of a conjugate in the form of a nucleotide sequence to a
haematopoietic stem cell (HSC).
[0235] Gene transfer involves the delivery to target cells, such as
HSCs, of an expression cassette made up of one or more nucleotide
sequences and the sequences controlling their expression. This can
be carried out ex vivo in a procedure in which the cassette is
transferred to cells in the laboratory and the modified cells are
then administered to a recipient. Alternatively, gene transfer can
be carried out in vivo in a procedure in which the expression
cassette is transferred directly to cells within an individual. In
both strategies, the transfer process is usually aided by a vector
that helps deliver the cassette to the appropriate intracellular
site.
[0236] Bone marrow has been the traditional source of HSCs for
transduction, more recent studies have suggested that peripheral
blood stem cells or cord blood cells may be equally good or better
target cells (Cassel et al 1993 Exp Hematol 21: 585-591; Bregni et
al 1992 Blood 80: 1418-1422; Lu et al 1993 J Exp Med 178:
2089-2096).
[0237] Further Anticancer Agents
[0238] The conjugate of the present invention may be used in
combination with one or more other active agents, such as one or
more cytotoxic drugs. Thus, in one aspect of the present invention
the method further comprises administering another active
pharmaceutical ingredient, such as a cytotoxic drug, either in
combined dosage form with the conjugate or in a separate dosage
form. Such separate cytotoxic drug dosage form may include solid
oral, oral solution, syrup, elixir, injectable, transdermal,
transmucosal, or other dosage form. The conjugate and the other
active pharmaceutical ingredient can be combined in one dosage form
or supplied in separate dosage forms that are usable together or
sequentially.
[0239] Examples of cytotoxic drugs which may be used in the present
invention include: the alkylating drugs, such as cyclophosphamide,
ifospfamide, chlorambucil, melphalan, busulfan, lomustine,
carmustine, chlormethhine (mustine), estramustine, treosulfan,
thiotepa, mitobronitol; cytotoxic antibiotics, such as doxorubicin,
epirubicin, aclarubicin, idarubicin, daunorubicin, mitoxantrone
(mitozantrone), bleomycin, dactinomycin and mitomycin;
antimetabolites, such as methotrexate, capecitabine, cytarabine,
fludarabine, cladribine, gemcitabine, fluorouracil, raltitrexed,
mercaptopurine, tegafur and tioguanine; vinca alkaloids, such as
vinblastine, vincristine, vindesine and vinorelbine, and etoposide;
other neoplastic drugs, such as amsacrine, altretamine,
crisantaspase, dacarbazine and temozolomide, hydroxycarbamide
(hydroxyurea), pentostatin, platinum compounds including:
carboplatin, cisplatin and oxaliplatin, porfimer sodium,
procarbazine, razoxane, taxanes including: docetaxel and
paclitaxel, topoisomerase I inhibitors including: irinotecan and
topotecan, trastuzumab, and tretinoin.
[0240] In a preferred embodiment the further cytotoxic drug is
doxorubicin, melphalan or cisplatin.
[0241] The conjugate of the present invention can also be used to
use the permeability of tumor cells and vessels to compounds for
diagnostic purposes. For instance, the conjugate can be used to
increase the tumor uptake of radiolabelled antibodies or hormones
(tumor-imaging compounds) in radioimmunoscintigraphy or
radiotherapy of tumors.
FIGURES AND EXAMPLES
[0242] The present invention will further be described by reference
to the following non-limiting Examples and Figures in which:
[0243] FIG. 1 Effect of mTNF and NGR-mTNF on tumor growth and body
weight of animals bearing RMA-T lymphomas.
[0244] Animals bearing RMA-T tumors (5 mice/group) were treated
i.p. with NGR-mTNF or mTNF at day 12 after tumor implantation (A)
or at day 10, 11 and 12 (B), in two separate experiments (Exp. 1
and Exp. 2). Tumor volumes in Exp. 1 (A) and Exp. 2 (B) and animal
body weight in Exp. 1 (C) 1-4 days after treatment are shown. The
arrowheads in panel C indicate the time of treatment.
[0245] FIG. 2 Circulating levels of sTNF-R2 and their role in
regulating the activity of NGR-mTNF and NGR-hTNF.
[0246] Panel A: serum levels of sTNF-R1 and sTNF-R2 in B16F1 tumor
bearing mice 1 hour after treatment with various doses of NGR-mTNF
or mTNF. Animals (3 mice/group) were treated at day 6.
[0247] Panel B: effect of the anti-sTNF-R2 mAb 6G1 on the
anti-tumor activity of NGR-mTNF. MAb 6G1 (100 .mu.g) was
administered to animals bearing B16F1 tumors at day 5 and 8. One
hour later each animal was treated with NGR-mTNF at the indicated
doses, and 2 hours later with melphalan (90 .mu.g, 5
mice/group).
[0248] Panel C: effect NGR-hTNF and hTNF on the growth of RMA-T
tumors. Mice were treated with various doses of each cytokine at
day 11. N.S.: not significant (t-test).
[0249] FIG. 3 Effect of melphalan, alone (A) or in combination with
NGR-mTNF (C) or mTNF (D), on the tumor growth (A-D) and body weight
(E-F) of mice bearing B16F1 melanoma.
[0250] The animals were treated (i.p.) with the drugs and the doses
indicated in each panel (5 animals/group) at day 4, 7 and 9 after
tumor implantation (indicated by arrows).
[0251] FIG. 4 Effect of various doses of doxorubicin, alone (white
bars) or in combination with NGR-mTNF (black bars) on the tumor
growth (A, B) body weight (C. D) and survival (E) of mice bearing
B16F1 melanomas.
[0252] The drugs were administered to the animals (5 mice/group
i.p.) 5 days after tumor implantation.
[0253] FIG. 5 Role of TNF receptors in the synergistic activity of
NGR-mTNF and melphalan.
[0254] Panel A: effect of mAb V1q (an anti-mTNF neutralizing
antibody) on the anti-tumor activity of melphalan in combination
with NGR-mTNF in the B16F1 model. The drugs were administered at
day 5. Mab V1q and NGR-TNF were pre-mixed and incubated for 1 hour
before injection into animals.
[0255] Panel B: effect of melphalan in combination with NGR-hTNF at
the indicated doses.
[0256] FIG. 6 Effect of NGR-mTNF on the penetration of doxorubicin
in B16F1 and RMA-T tumors.
[0257] Panel A: bright field (upper panels) and fluorescence (lower
panels) microscopy of B16F1 cells incubated in vitro with 100
.mu.g/ml doxorubicin (30 min, 37.degree. C.). Inset: merge of
bright field and fluorescence images.
[0258] Panel B: stability of the B16F1 fluorescence signal after in
vitro treatment with doxorubicin. B16F1 cells were incubated with
various doses of doxorubicin in culture medium (30 min, 37.degree.
C.), washed with 0.9% sodium chloride, and fixed with 4%
formaldehyde. The cells were then incubated for 0 hours or 24 hours
in culture medium at 4.degree. C., washed again and analyzed by
FACS.
[0259] Panel C, F: representative FACS analysis of cells recovered
from B16F1 (C) or RMA-T (i) tumors 2 hours after in vitro
administration of doxorubicin alone (320 .mu.g) or in combination
with NGR-mTNF (0.1 ng). Dashed lines indicate the fluorescence
interval considered positive.
[0260] Panel D, G: mean.+-.SE fluorescence of B16F1 (D) or RMA-T
(G) cells recovered from tumors.
[0261] Panel E, H: mean.+-.SE of positive cells recovered from
B16F1 (E) RMA-T (H) tumors.
[0262] Statistical analysis by two-tailed t-test, P<0.05
(*).
[0263] FIG. 7 Schematic representation of the hypothetical
interations of low (A), moderate (B) and high (C) doses of NGR-TNF
with soluble and membrane receptors in normal vessels
(CD13-negative) and in tumor associated vessels
(CD13-positive).
[0264] Black arrows indicate TNF receptor signalling or
extracellular domain shedding.
[0265] FIG. 8 Effect of RGD-TNF on the tumor growth (FIG. 8A) and
body weight (FIG. 8B) of an animal bearing RMA-T.
[0266] FIG. 9 Effect of a single treatement (arrow) with
IFN.gamma.-NGR on the growth of RMA lymphoma tumors in C57B6 mice
(FIG. 9A & FIG. 9C) and on animal weight (FIG. 9B).
[0267] FIG. 10 Effect of NGR-TNF and cisplatinum on RMA tumors in
C57/BL6 mice.
EXAMPLE 1
Tumor Cell Lines and Reagents
[0268] Mouse B16F1 melanoma and RM lymphoma cells were cultured as
described previously (14, 15). Mb 6G1 (rat anti-p75 mTNF receptor
antagonist) was produced and characterized as described previously
(16, 17). MAb V1q (rat anti-mTNF) (was kindly supplied by Dr. D.
Mannel (University of Regensburg, Germany). Melphalan (Alkeran) was
obtained from Glaxo-Wellcome (London, Great Britain). Doxorubicin
(Adriblastina) was purchased from Pharmacia-Upjohn (Milan,
Italy).
EXAMPLE 2
Preparation of Human and Murine TNF and NGR-TNF
[0269] Human and murine TNF and NGR-TNF (consisting of TNF fused
with the C-terminus of CNGRCG) were prepared by recombinant DNA
technology and purified from E. coli cell extracts, as described
(14). All solutions used in the chromatographic steps were prepared
with sterile and endotoxin-free water (Salf, Bergamo, Italy).
Protein concentration was measured with a commercial protein
quantification assay kit (Pierce, Rockford, EL). The in vitro
cytolytic activity of human TNF (hTNF), estimated from a standard
cytolytic assay with LM mouse fibroblasts (18), was
5.4.times.10.sup.7 Units/mg, whereas that of purified NGR-hTNF was
1.4.times.10.sup.8 Units/mg. The cytolytic activity of murine TNF
(mTNF), was 7.6.times.10.sup.7 Units/mg, whereas that of NGR-mTNF
was 9.1.times.10.sup.7 Units/mg. The hydrodynamic volumes of
NGR-mTNF, NGR-hTNF and mTNF were similar to those of hTNF, a
homotrimeric protein (19), by gel filtration chromatography on a
Superdex 75 HR column (Pharmacia, Sweden). Electrospray mass
spectrometry of each product determined the following molecular
masses: NGR-hTNF, 17937.6.+-.1.9 Da (expected for
CNGRCG-hTNF.sub.1-157 Monomers, 17939.4 Da); hTINF, 17349.+-.1.3
(expected for hTNF.sub.1-157, 17350.7); NGR-mTNF, 17841.16.+-.2.5
(expected for CNGRCG-mTNF.sub.1-156, 17844.2), mTNF, 17384.9.+-.2
(expected for Met-mTNF.sub.1-156, 17386.7). The endotoxin content
of each product, measured using the quantitative chromogenic
limulus amoebocyte lysate (LAL) test (BioWhittaker), was: NGR-hTNF,
0.079 Units/.mu.g; hTNF, 0.117 Units/.mu.g; NGR-mTNF, 0.082
Units/.mu.g; mTNF, 1.61 Units/.mu.g.
EXAMPLE 3
In Vivo Studies
[0270] Studies on animal models were approved by the Ethical
Committee of the San Raffaele H Scientific Institute and performed
according to the prescribed guidelines. C57BL/6 mice (Charles River
Laboratories, Calco, Italy) weighing 16-18 g were challenged with
subcutaneous injection in the left flank of 5.times.10.sup.4 RMA-T
or B16F1 living cells; 4-12 days later, the mice were treated with
TNF or NGR-TNF solutions (100 .mu.l) followed 2 hours later by
administration of melphalan or doxorubicin solution (100 .mu.l).
Unless specified, all drugs were administered intraperitoneally
(i.p.). All drugs were diluted with 0.9% sodium chloride,
containing 100 fig/ml endotoxin-free human serum albumin
(Farma-Biagini, Lucca, Italy), except for doxorubicin, which was
diluted with 0.9% sodium chloride alone. Tumor growth was monitored
daily by measuring the tumors with calipers as previously described
(20). Animals were sacrificed before the tumors reached 1.0-1.5 cm
in diameter. Tumor sizes are shown as mean.+-.SE (5
animals/group).
EXAMPLE 4
Soluble TNF Receptor Assays
[0271] Soluble p55-TNF receptor (sTNF-R1) and soluble p75-TNF
receptor (sTNF-R2) in animal sera were measured using the
Quantikine M kit (R & D Systems, Minneapolis, Minn. 55413).
EXAMPLE 5
Detection of Doxorubicin in Tumors
[0272] C57/BL6 mice bearing B16F1 or RMA-T tumors (diameter, 0.5-1
cm) were treated with or without NGR-m TNF (0.1 ng, i.p.), followed
2 hours later by doxorubicin (320 .mu.g, i.p.). After 2 hours the
animals were sacrificed and the tumors were excised. Each tumor was
weighed, disaggregated, resuspended in cold phosphate-buffered
saline (PBS) and filtered through 70 .mu.m filters. The cells were
resuspended with cold PBS (50 ml), centrifuged (1500 rpm, 10 min,
4.degree. C.), resuspended in cold PBS (2.5 ml/g of tumor tissue)
and mixed with freshly prepared PBS containing 8% formaldehyde (2.5
ml/g of tissue). The cells were stored in the dark at 4.degree. C.
overnight, and then analyzed by FACS. The FACScan
(Becton-Dickinson) was calibrated with cells recovered from
untreated tumors. Each sample was then analyzed using the FL-3
filter and Cell Quest software.
EXAMPLE 6
Dose-Response Curves of NGR-mTNF and mTNF in Murine Lymphoma and
Melanoma Models
[0273] The anti-tumor activity of NGR-mTNF and mTNF was first
characterized in the absence of chemotherapeutic drugs. To compare
the dose-response curves of NGR-mTNF and mTNF we performed several
experiments based on single or repeated administration (i.p.) of
various doses of NGR-mTNF and mTNF (from 0.01 to 10000 ng) to RMA-T
lymphoma- or B16F1 melanoma-bearing mice. Murine TNF delayed tumor
growth when adminstered at high doses (10000 ng) (FIG. 1A); no
effects were induced by doses lower than 100 ng, either with single
(FIG. 1A) or repeated administrations (FIG. 1B). NGR-mTNF was
markedly more potent. In this case we observed anti-tumor effects
even with doses as low as 0.01 ng (FIG. 1A, B). However, the
dose-response curve was more complex. For instance, the effect of
10 ng was surprisingly lower than that of 0.01-0.1 ng and
1000-10000 ng. A bell-shaped dose-response curve was observed in
several other experiments conducted in the RMA-T model as well as
in the B16F1 melanoma model (not shown). These results suggest that
1) the efficacy of low doses of NGR-mTNF is markedly higher than
that of mTNF, and 2) doses of NGR-mTNF greater than 1-10 ng
activate negative feed-back mechanisms that inhibit its potential
anti-tumor activity.
EXAMPLE 7
Nanogram Doses of NGR-TNF, But Not Picograms, Induce Soluble TNF
Receptor Shedding
[0274] The protective mechanisms responsible for the bell-shaped
dose-response curve of NGR-mTNF were then investigated. Since
exogenously administered TNF can induce soluble TNF receptor
(sTNF-Rs) shedding in vivo (21), we hypothesized that the lower
efficacy of 10 ng of NGR-TNF was related to induction of sTNF-R1
and/or sTNF-R2 and, consequently, to neutralization of its
interation with membrane receptors.
[0275] To test this hypothesis, we measured the levels of sTNF-R1
and sTNF-R2 in the serum of tumor bearing mice collected 1 hour
after administration of various doses of mTNF and NGR-mTNF. As
expected, both products induced sTNF-R2 shedding, but not sTNF-R1
shedding, at doses greater than 4 ng (FIG. 2A).
[0276] To assess whether sTNF-R2 shedding regulates the activity of
NGR-mTNF, we coadminstered this cytokine with mAb 6G1, an
antagonist anti-sTNF-R2 antibody that prevents the binding of mTNF
to soluble and membrane murine TNF-R2 (16). The anti-tumor activity
of 10 ng of NGR-mTNF was potentiated by mAb 6G1 (FIG. 2B), in line
with the hypothesis that sTNF-R2 plays a role in inhibiting the
anti-tumor effects of NGR-mTNF.
[0277] To further support this hypothesis we compared the in vivo
dose-response curve of NGR-mTNF with that of NGR-hTNF, taking
advantage of the fact that the human cytokine cannot bind murine
sTNF-R2 (22). We found that the dose-response curve of NGR-hTNF was
not bell-shaped, and that 10 ng of NGR-hTNF is as active as 1 ng
(FIG. 2C). It is also remarkable that 1 ng was sufficient to induce
the maximum anti-tumor effect. This may suggest that receptor
binding on vessels can be achieved with very low blood levels of
NGR-hTNF.
[0278] Taken together, the results of these experiments strongly
suggest that NGR-mTNF and mTNF, at doses greater than 4 ng, induce
shedding of sTNF-R2 in amounts sufficient to inhibit their
anti-tumor activity.
EXAMPLE 8
Picogram Doses of NGR-mTNF are Sufficient to Enhance the
Therapeutic Effect of Melphalan and Doxorubicin
[0279] We then investigated whether targeted delivery of low doses
of NGR-mTNF to tumor vessles could enhance the anti-tumor activity
of chemotherapeutic drugs. These experiments were conducted in the
B16F1 model, a spontaneous mouse melanoma characterized by scarce
immunogenicity and low sensitivity to melphalan. Melphalan (90
.mu.g) was unable to effect the growth of tumors when injected
alone (FIG. 3A). Similarly, mTNF (0.1 ng alone, i.p.) was virtually
inactive, while the same dose of NGR-mTNF modestly delayed the
tumor growth (FIG. 3B, upper panels). The combination of melphalan
with 0.1 ng of NGR-mTNF induced stronger anti-tumor effects than
the single agents, indicating a synergistic effect (FIG. 3C).
Remarkably, the combination of melphalan with 0.1 ng of NGR-mTNF
was more effective than the combination with 5000 ng of mTNF (FIG.
3C-D). We observed this synergisim even when NGR-mTNF (0.1 ng) was
injected i.v. (not shown).
[0280] Two similar experiments were conducted with doxorubicin in
the B16F1 model. Animals were treated five days after tumor
implantation, with or without NGR-mTNR and, 2 hours later, with
various doses of doxorubicin (20-320 .mu.g, i.p.). In both
experiments, the effect of doxorubicin plus NGR-mTNF was stronger
than that of doxorubicin alone (FIG. 4A, B, E), indicating that
NGR-mTNF markedly improves the efficacy of this drug. For example,
the effect of doxorubicin (40 .mu.g) plus NGR-mTNF (0.1 ng) was
stronger than that of 320 .mu.g of doxorubicin alone (FIG. 4B),
while, the effect of doxorubicin (20 .mu.g) plus NGR-mTNF was lower
(FIG. 4A). From these results we estimate that the activity of
doxorubicin is potentiated 8-10 fold by NGR-mTNF.
[0281] In conclusion, these results suggest that picogram doses of
NGR-TNF are sufficient to improve the response of tumors to both
melphalan and doxorubicin.
EXAMPLE 9
Low Doses of NGR-mTNF Are Not Toxic and Do Not Increase the
Toxicity of Melphalan
[0282] To estimate the efficacy/toxicity ratio of each treatment,
we monitored the animal body weight daily and animal survival after
treatment. While therapeutic doses of mTNF (10000 ng) induced
marked loss of body weight in RMA-T bearing animals (FIG. 1C,
left), therapeutic doses of NGR-mTNF (0.01-1 ng) did not cause loss
of body weight (FIG. 1C, right). Moreover, neither NGR-mTNF nor
mTNF (1 ng each) increased the lethality of 200 .mu.g of melphalan
in mice bearing the RNA-T tumor (Table 1).
4TABLE 1 Effect of mTNF and NGR-mTNF on the toxicity of Melphalan
(high dose) in tumor-bearing mice.sup.a No. of mice alive Treatment
3 days after treatment None 5/5 (100%) Melphalan 7/10 (70%)
Melphalan + mTNF 8/10 (80%) Melphalan + NGR-mTNF 8/10 (80%)
.sup.aC57BL6 mice, bearing 11-day old tumors, were treated (i.p.)
with 1 ng of NGR-mTNF or mTNF and, 2 hours later, with 200 .mu.g of
melphalan.
[0283] In the B16F1 model, therapeutic doses of NGR-mTNF (0.1 ng)
did not cause loss of body weight, even when combined with
melphalan (FIG. 3E). In contrast, melphalan combined with
therapeutic doses of MTNF (5 .mu.g) induced marked loss of body
weight (FIG. 3F). In addition, NGR-mTNF (0.1 ng) did not increase
the loss of body weight causes by high doses of doxorubicin (FIG.
4C-D).
[0284] These results suggest that picogram doses of NGR-mTNF
increase the response of tumors to melphalan and doxorubicin with
no evidence of increased toxicity.
EXAMPLE 10
TNF-R1 Activation is Necessary and Sufficient For the Synergism
Between NGR-TNF and Chemotherapeutic Drugs
[0285] The mechanisms of the synergism between low doses of
NGR-mTNF and chemotherapy were then investigated.
[0286] To assess whether these mechanisms rely on TNF-Rs activation
we tested the effect of mAbV1q, a neutralizing anti-mTNF antibody,
on the anti-tumor activity of NGR-mTNF (0.1 ng) in combination with
melphalan (90 .mu.g). MAb V1q inhibited, at least partially, the
anti-tumor activity of these drugs in the B16F1 model (FIG. 5A).
This suggests that the interaction between the TNF moity and TNF-Rs
is critical for the activity of the conjugate.
[0287] The role of TNF-R1 and TNF-R2 was then studied. To this end,
we evaluated the effect of melphalan in combination with 0.01 ng or
0.1 ng of NGR-hTNF, a TNF-R1 specific agonist (22). The effect of
melphalan in the B16F1 model was potentiated by NGR-hTNF (FIG. 5B)
suggesting that TNF-R1 activation is sufficient for the
synergism.
EXAMPLE 11
The Synergy Between NGR-TNF and Chemotherapy is Not Dependent on
Tumor Cell Cytotoxicity
[0288] To assess whether the synergism depends directly on
cytotoxicity against tumor cells we measured the effect of each
compound, alone or in combination, on cultured B16F1 cells. Neither
melphalan or NGR-mTNF, alone or in combination, killed these cells
in a 48 hour in vitro assay (not shown). Similarly, NGR-mTNF did
not enhance the cytotoxic activity of doxorubicin in vitro (not
shown). These results suggest that the synergism observed in vivo
is not directly dependent on cytotoxic effects against tumor cells
and points to an indirect role of a component of the tumor stroma,
e.g. the endothelial lining of tumor vessels.
EXAMPLE 12
NGR-TNF Increases the Penetration of Doxorubicin in Murine
Melanomas and Lymphomas
[0289] We then investigated whether NGR-mTNF could increase the
penetration of chemotherapeutic drugs in tumors. To this aim we
measured the amount of doxorubicin that had penetrated B16F1 and
RMA-T tumors, 2 hours after administration, taking advantage from
the fluorescent properties of this drug (23). Preliminary
experiments showed that the nuclei of B16F1 cells become
fluorescent after these cells are exposed to doxorubicin in vitro
(FIG. 6A). The fluorescence signal is dose-dependent and stable for
at least 24 hours, when the cells are fixed with formaldehyde and
kept at 4.degree. C., as measured by FACS (FIG. 6B). Thus, the
fluorescence intensity of tumor cells recovered from animals after
treatment is an indication of the amount of doxorubicin that has
penetrated tumors. We observed that 0.1 ng of NGR-mTNF,
administered 2 hours before doxorubicin, increased the fluorescence
intensity and the percentage of positive cells recovered from both
B16F1 and RMA-T tumors, 2 hours after treatment (2-5-fold, FIG.
6C-H). This suggests that NGR-mTNF increased the number of cells
that were reached by doxorubicin as well as the intracellular
amount of drug.
EXAMPLE 13
Effect of RGD-TNF on Tumors
[0290] C57/BL6 mice bearing RMA-T tumors (5 mice/group) were
treated intraperitoneally with melphalan alone, or in combination
with RGD-TNF or NGR-TNF. The effect on tumor volume is shown in
FIG. 8A and the effect on animal weight is shown in FIG. 8B. These
results demonstrate that RGD-TNF is also active in the picogram
range.
EXAMPLE 14
Effect of IFN.gamma.-NGR on Tumors
[0291] C57/BL6 mice bearing RMA lymphoma tumors were treated with
or without IFN.gamma. (3 ng) or IFN.gamma.-NGR (3 ng). After 21
days the animals were sacrificed. The results of tumor volume and
of animal weight loss can be seen in FIGS. 9A and 9B. In addition,
C57/BL6 mice bearing RMA lymphoma tumors were treated with or or
without IFN.gamma. (3 ng), IFN.gamma.-NGR (3 ng), IFN.gamma.-NGR (3
ng) plus anti-CD13 R3-63 mAb (25 .mu.g) or FNy-NGR (3 ng) plus mAB
19E12 (50 .mu.g). The mAB 19E12 is an irrelevant IgG used as a
negative control in the experiment. The results demonstrate that
IFN.gamma.-NGR is active in the picogram range.
EXAMPLE 15
Picogram Doses of NGR-TNF Are Sufficient to Enhance the Therapeutic
Effect of Cisplatinum
[0292] We investigated whether targeted delivery of low doses of
NGR-TNF to tumor vessels could enhance the anti-tumor activity of
the anti-cancer drug, cisplatinum. These experiments were carried
out in C57/BL6/N mice bearing RMA-T tumors of initial age 8 weeks.
The mice were treated with or without cisplatinum or NGR-mTNF with
treatment at day 10. The results are shown in FIG. 10. In this
figure, Cys=Cisplatino Teva solution 0.5 mg/ml; NGR=NGR-mTNF. The
diluent was HAS 100 mg/ml in NaCl 0.9%. The results suggest that
pciogram doses of NGR-TNF are sufficient to enhance the therapeutic
effect of cisplatinum.
[0293] Summary of Advantages
[0294] Alteration of vascular permeability and interstitial
pressure, endothelial cell damage and fibrin deposition are
important mechanisms for the anti-tumor activity of TNF, either
alone or in combination with chemotherapeutic drugs. After,
infusion in animals or patients TNF can also induce negative
feedback mechanisms that neutralize most of these effects. For
example, TNF, even at moderate doses, can induce the release of
soluble p55 and p75 TNF receptors that may prevent its interation
with membrane receptos (21, 24). Although these soluble inhibitors
may protect the body from the harmful effects of this cytokine,
they may also prevent its anti-tumor activity and could explain, in
part, the need of high doses of TNF for effective therapy. In this
work we postulated that homing low doses of TNF to tumor vessels
represents a new strategy to avoid toxic reactions as well as
negative feedback mechanisms, while preserving its synergism with
chemotherapy. To verify this hypothesis, we have investigated the
anti-tumor activity of high and low doses of NGR-mTNF and mTNF,
ranging from picogram to microgram quantities, in two murine models
based on subcutaneous RMA-T lymphoma and B16F1 melanoma tumors. The
study was carried out using these cytokines alone or in combination
with melphalan or doxorubicin. While mTNF was virtually inactive in
these models at lower doses than 100-1000 ng, we found that
NGR-mTNF, even alone, could induce anti-tumor effects with doses as
low as 0.01-0.1 ng. Since the LD.sub.50 values of mTNF and NGR-mTNF
are similar and correspond to about 50,000 ng in RMA-T
tumor-bearing mice (14), these results indicate that the
efficacy/toxicity ratio of NGR-mTNF is 10.sup.4-10.sup.5 times
greater than that of mTNF.
[0295] Administration of minute amounts of NGR-mTNF (0.1 ng) to
tumor-bearing animals potentiated the anti-tumor activity of
melphalan and doxorubicin with no evidence of increased toxicity,
as judged by tumor mass reduction, animal survival and weight loss
after treatment. This suggests that NGR-mTNF improves the
therapeutic index of these drugs. It is noteworthy that
5.times.10.sup.4-fold greater doses of mTNF were necessary to
enhance the effect of melphalan to comparable levels, causing
marked loss of body weight.
[0296] The fact that both melphalan and doxorubicin at doses
virtually inactive in the B16F1 model reduced tumor growth when
combined with NGR-mTNF indicates that these drugs act
synergistically. Studies on the mechanism of action showed that the
synergism relies on the interaction of NGR-mTNF with TNF-R1 on
stromal cells, most likely endothelial cells, and much less on
tumor cells. In addition, we found that vascular targeting with
NGR-mTNF improves cytotoxic drug penetration in tumors. Noteworthy,
NGR-mTNF increased both the percentage of cancer cells that can be
reached by doxorubicin in 2 hours as well as the intracellular
amount of drug, suggesting that NGR-TNF can alter drug-penetration
barriers. Previous studies showed that TNF can rapidly increase
endothelial permeability (25, 26), and can decrease interstitial
fluid pressure (8) that are believed to be important barriers for
drug penetration in tumors (1). Possibly, these mechanisms increase
convective transport of drugs through the tumor vessel wall and
interstitium, finally resulting in increased drug uptake by tumor
cells. The timing of administration is likely critical for these
mechanisms, as TNF can also induce intravascular coagulation (27)
leading to vessel occlusion and reduction of tumor perfusion.
According to this view, we observed that the effect of melphalan
was higher when this drug was administered 2 hours after NGR-TNF
than after 6 hours (data not shown).
[0297] The hypothesis that vascular targeting could avoid negative
feedback mechanisms, usually associated with TNF therapy, is
supported by the observation that picogram doses of NGR-mTNF do not
induce soluble receptor shedding, while both NGR-mTNF and mTNF
rapidly induce the release of sTNF-R2 into the circulation at doses
greater than 410 ng. These levels of sTNF-R2 inhibited most of the
anti-tumor activity of 10 ng of NGR-mTNF and may explain the
paradoxical observation that 10 ng is less active than 0.1 ng.
Likely, a large proportion of injected molecules were rapidly
complexed by sTNF-Rs and their activity was blocked.
[0298] The molecular mechanisms underlying the selective
interaction of low doses of NGR-mTNF with tumor blood vessels has
been partially elucidated. We have shown recently that different
CD13 isoforms are expressed in tumor-associated vessels, in
epithelia and in myeloid cells, and that the NGR domain of NGR-TNF
selectively recognizes a CD13 isoform associated with tumor vessels
(28). We hypothesize, therefore, that low blood levels of NGR-mTNF
can rapidly interact with CD13-positive endothelial cells because
of high-avidity multivalent binding involving both CD13 and TNF-Rs,
and little or not at all with CD13-negative endothelial cells of
normal vessels, because of lower avidity. A schematic
representation of these concepts and of the hypothetical
interactions of NGR-TNF with soluble and membrane receptors is
shown in FIG. 7.
[0299] We have also found that RGD-TNF and IFN.gamma.-NGR are
active in the picogram range. We have also shown that NGR-TNF
increases the effect of cisplatinum.
[0300] In conclusion, we have found that targeted delivery of
picogram doses of cytokines to tumor vessels enhances the
anti-tumor activity of chemotherapeutic drugs in mice without
inducing soluble receptor shedding. Given that the CNGRC motif is
expected to target murine as well as human tumor associated vessels
(29), our results suggest that the combination of low doses of
targeted cytokines with anti-cancer drugs could increase their
therapeutic index in human patients.
[0301] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are apparent to those skilled in molecular biology or related
fields are intended to be within the scope of the following
claims.
REFERENCES
[0302] 1. Jain, R. K. 1994. Sci Am. 271:58-65.
[0303] 2. Lienard, D., et al. 1992 J. Clin. Oncol. 10:52-60.
[0304] 3. Eggermont, A. M., et al. 1996. J. Clin. Oncol.
14:2653-2665.
[0305] 4. Lejeune, F. J. 1995. Eur. J. Cancer. 31A:1009-1016.
[0306] 5. Fraker, D. L., et al. 1996. J. Clin. Oncol.
14:479-489.
[0307] 6. Rossi, C. R., et al. 1999. Cancer. 86:1742-1749.
[0308] 7. van der Veen, A. H., et al. 2000. Br. J. Cancer.
82:973-980.
[0309] 8. Kristensen, C. A., et al. 1996. Br. J. Cancer.
74:533-536.
[0310] 9. Suzul, S., et al. 1990. Int. J. Cancer. 46:1095-1100.
[0311] 10. de Wilt, J. H., et al. 2000. Br. J. Cancer.
82:1000-1003.
[0312] 11. Fiers, W. 1995. Biologic therapy with TNF: preclinical
studies. In Biologic therapy of cancer: principles and practice. V.
De Vita, S. Hellman, and S. Rosenberg, editors. J.B. Lippincott
Company, Philadelphia. 295-327.
[0313] 12. Fraker, D. L., H. I Alexander, and H. I. Pass. 1995.
Biologic therapy with TNF: systemic administration and
isolation-perfusion. In Biologic therapy of cancer: principles and
practice. V. De Vita, S. Hellman and S. Rosenberg, editors. J.B.
Lippincott Company, Philadelphia 329-345.
[0314] 13. Corti, A., and F. Marcucci. 1998. J. Drug Targ.
5:403-413.
[0315] 14. Cumis, F., Aet al. 2000. Nat. Biotechnol.
18:1185-1190.
[0316] 15. Moro, M., et al. 1997. Cancer. Res. 57:1922-1928.
[0317] 16. Corti, A., et al. 1999. Infect Immun. 67:5762-5767.
[0318] 17. Pelagi, M., et al. 2000. Eur. Cytokine Net.
11:580-588.
[0319] 18. Corti, A., et al. 1994. J. Immunol. Meth.
177:191-198.
[0320] 19. Smith, R. A., and C. Baglioni. 1987. J. Biol. Chem.
262:6951-6954.
[0321] 20. Gasparri, A., et al. 1999. Cancer Res. 59:2917-2923.
[0322] 21. Aderka, D., et al. 1998. J. Clin. Invest.
101:650-659.
[0323] 22. Tartaglia, L. A., et al. 1991. Proc. Natl. Acad. Sci.
USA. 88:9292-9296.
[0324] 23. Luk, C. K, and I. F. Tannock. 1989. J. Natl. Cancer
Inst. 81:55-59.
[0325] 24. Sella, A., et al. 1995. Cancer Biother. 10:225-235.
[0326] 25. Brett, J., et al. 1989. J. Exp. Med. 169:1977-1991.
[0327] 26. Goldblum, S. E., and W. L. Sun. 1990. Am. J. Physiol.
258:L57-67.
[0328] 27. Clauss, M., et al. 1992. Modulation of endothelial cell
hemostatic properties by TNF: insights into the role of endothelium
in the host response to inflammatory stimuli. In Tumour necrosis
factors: the molecules and their emerging roles in medicine. B.
Beutler, editor, Raven Press, New York. 4963.
[0329] 28. Cumis, F., et al. 2002. Cancer Res.: 62:867-874.
[0330] 29. Arap, W., et al. 1998. Science. 279:377-380.
* * * * *