FI125971B - conjugates - Google Patents

conjugates Download PDF

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Publication number
FI125971B
FI125971B FI20145552A FI20145552A FI125971B FI 125971 B FI125971 B FI 125971B FI 20145552 A FI20145552 A FI 20145552A FI 20145552 A FI20145552 A FI 20145552A FI 125971 B FI125971 B FI 125971B
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FI
Finland
Prior art keywords
dextran
conjugate
bsh
egfr1
boron
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Application number
FI20145552A
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Finnish (fi)
Swedish (sv)
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FI20145552A (en
Inventor
Anne Leppänen
Filip S Ekholm
Jari Helin
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Tenboron Oy
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Priority to FI20145552A priority Critical patent/FI125971B/en
Application filed by Tenboron Oy filed Critical Tenboron Oy
Priority to ES15734421T priority patent/ES2779974T3/en
Priority to DK15734421.9T priority patent/DK3154587T3/en
Priority to CA2952026A priority patent/CA2952026A1/en
Priority to PCT/FI2015/050422 priority patent/WO2015189477A1/en
Priority to AU2015273353A priority patent/AU2015273353B2/en
Priority to US15/318,030 priority patent/US10328149B2/en
Priority to EP15734421.9A priority patent/EP3154587B1/en
Priority to JP2015119792A priority patent/JP5924795B2/en
Priority to CN201580031651.XA priority patent/CN106456768B/en
Publication of FI20145552A publication Critical patent/FI20145552A/en
Application granted granted Critical
Publication of FI125971B publication Critical patent/FI125971B/en
Priority to US16/383,832 priority patent/US10835606B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/009—Neutron capture therapy, e.g. using uranium or non-boron material
    • A61K41/0095—Boron neutron capture therapy, i.e. BNCT, e.g. using boronated porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6865—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from skin, nerves or brain cancer cell

Description

CONJUGATES
FIELD OF THE INVENTION
The invention relates to a conjugate, a pharmaceutical composition and a method of treating or modulating the growth of EGFR1 expressing tumor cells in a human.
BACKGROUND OF THE INVENTION
Boron neutron capture therapy (BNCT) is a form of noninvasive therapy of malignant tumors such as primary brain tumors and head and neck cancer. In BNCT, a patient is injected with a drug which has the ability to localize in the tumor and which carries nonradioactive boron-10 atoms. When the drug is irradiated with low energy thermal neutrons, biologically destructive alpha particles and lithium-7 nuclei are emitted.
Drugs such as conjugates having a high content of boron-10 and capable of localizing specifically in the tumor are required for BNCT. Such conjugates should be easily produced, stable, soluble and safe. However, provision of such conjugates is complicated e.g. by that some types of chemistries do not appear to work with boron-10 containing compounds.
The purpose of the present invention is to provide conjugates that have improved properties as compared to known conjugates and that contain a high content of boron-10.
Gedda et al., Bioconjug. Chem., 1996, 7, 584-591 describes covalent coupling of EGF and sulfhydryl boron hydride to an allylated 7 0 kDa dextran chain to form a conjugate.
Wu et al., Anti-Cancer Agents in Medicinal Chemistry, 2006, 6 (2), 167-184 describes boron con- taining macromolecules and nanovehicles as delivery agents for neutron capture therapy.
Holmberg et al., Bioconjug. Chem., 1993, 4, 570-573 discloses coupling thiolated dextran to Na2Bi2HnSH.
WO 9804917 describes the preparation of a sulfhydrylborane-dextran conjugate .
WO 8705031 discloses an antibody conjugate comprising boron and an aminodextran.
WO 03068144 describes cytotoxic conjugates comprising polyethylene glycol linking groups.
SUMMARY OF THE INVENTION
The conjugate according to the present invention is characterized by what is presented in claim 1.
The pharmaceutical composition according to the present invention is characterized by what is presented in claim 18.
The conjugate or pharmaceutical composition for use as a medicament according to the present invention is characterized by what is presented in claim 19.
The conjugate or pharmaceutical composition for use in the treatment of cancer according to the present invention is characterized by what is presented in claim 20.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and together with the description help to explain the principles of the invention. In the drawings:
Figure 1. Proton-NMR spectrum of BSH-dextran. The boron linked protons resonate between 0.8-2.0 ppm, and the boron load of BSH-dextran can be estimated by comparing the integral of boron-protons to the integral of dextran protons. Unreacted allyl groups yield signals at 4.22, 5.29, 5.39 and 5.99 ppm. Sharp signal at 2.225 ppm is acetone (internal standard).
Figure 2. Gel filtration analysis of BSH-Dex-conjugates.
A. Anti-EGFRl-Fab-BSH(800B)-Dex. Conjugate elutes at 7.8 ml when analysed with Yarra SEC-3000 gel filtration column. By comparison anti-EGFRl-Fab elutes at 9.1 ml. B. Anti-EGFR1-Fab2-BSH(800B)-Dex. Conjugate elutes at 6.9 ml when analysed with Yarra SEC-3000 gel filtration column. By comparison anti-EGFRl-Fab2 elutes at 8.4 ml.
Figure 3. SDS-PAGE analysis of fluorescently labeled anti-EGFRl Fab/F(ab')2 boron conjugates with different amounts of boron in nonreducing (panel A) and reducing (panel B) conditions. Anti-EGFRl-Fab-BSH-Dex conjugates: Lane 1 (900B), lane 2 (700B) , lane 4 (560B) , lane 6 (360B) . Anti-EGFR1-F(ab')2 -BSH-Dex conjugates: Lane 3 (700B), lane 5 (560B), lane 7 (360B) . Lane 8 is Anti-EGFRl-Fab-Dex and lane 9 is a control containing a mixture of anti-EGFRl-F(ab')2 and Fc fragments (Fab fragments migrate like Fc fragments on the gel). Gel staining with Coomassie Blue.
Figure 4. Cell surface binding and internalization of fluorescently labeled anti-EGFRl-F(ab')2 (Panels A and C) and anti-EGFRl-F(ab')2 -BSH(900B)-Dex (Panels B and D) by HSC-2 cells. Incubations have been performed at +4 °C (binding to the cell surface) and at +37 °C (binding to cell surface and internalization) . Analysis has been carried out by fluorescence microscopy.
DETAILED DESCRIPTION
The present invention relates to a conjugate comprising an anti-EGFRl antibody or an EGFR1 binding fragment thereof and at least one dextran derivative, wherein the dextran derivative comprises at least one D-glucopyranosyl unit, wherein at least one carbon selected from carbon 2, 3 or 4 of the at least one D- glucopyranosyl unit is substituted by a substituent of the formula -0- (CH2)n-S-B12H112- wherein n is in the range of 3 to 10; and the dextran derivative is bound to the anti-EGFRl antibody or an EGFR1 binding fragment thereof via a bond formed by a reaction between at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative and an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
The conjugate is suitable for use in boron neutron capture therapy. "Boron neutron capture therapy" (BNCT) should be understood as referring to targeted radiotherapy, wherein nonradioactive boron-10 is irradiated with low energy thermal neutrons to yield biologically destructive alpha particles and lithium-7 nuclei. The nonradioactive boron-10 may be targeted by incorporating it in a tumor localizing drug such as a tumor localizing conjugate.
"EGFR1" herein should be understood as referring to human epidermal growth factor receptor 1 (EGFR1) having a sequence set forth in SEQ ID NO: 1.
"Anti-EGFRl antibody" should be understood as referring to an antibody that specifically binds EGFR1. The term "specifically binding" refers to the ability of the antibody to discriminate between EGFR1 and any other protein to the extent that, from a pool of a plurality of different proteins as potential binding partners, only EGFR1 is bound or significantly bound. As examples only, specific binding and/or kinetic measurements may be assayed by e.g. by utilizing surface plasmon resonance-based methods on a Biacore apparatus, by immunological methods such as ELISA or by e.g. protein microarrays.
"An EGFR1 binding fragment thereof" should be understood as referring to any fragment of an anti-EGFR1 antibody that is capable of specifically binding EGFR1.
In an embodiment, anti-EGFRl antibody is ce-tuximab, imgatuzumab, matuzumab, nimotuzumab, neci-tumumab, panitumumab, or zalutumumab.
In an embodiment, the anti-EGFRl antibody is cetuximab.
In an embodiment, cetuximab has a seguence set forth in SEQ ID NO:s 2 and 3.
In an embodiment, cetuximab comprises or consists of the sequences set forth in SEQ ID NO:s 2 and 3.
In an embodiment, the anti-EGFRl antibody is nimotuzumab.
In an embodiment, nimotuzumab has a sequence set forth in SEQ ID NO:s 4 and 5.
In an embodiment, nimotuzumab comprises or consists of the sequences set forth in SEQ ID NO:s 4 and 5 .
An anti-EGFRl antibody may be e.g. an scFv, a single domain antibody, an Fv, a VHH antibody, a di-abody, a tandem diabody, a Fab, a Fab', a F(ab')2/ a
Db, a dAb-Fc, a taFv, a scDb, a dAb2, a DVD-Ig, a
Bs (scFv)4-IgG, a taFv-Fc, a scFv-Fc-scFv, a Db-Fc, a scDb-Fc, a scDb-CH3, or a dAb-Fc-dAb. Furthermore, the anti-EGFRl antibody or an EGFR1 binding fragment thereof may be present in monovalent monospecific, multivalent monospecific, bivalent monospecific, or multivalent multispecific forms.
In an embodiment, the anti-EGFRl antibody is a human antibody or a humanized antibody. In this context, the term "human antibody", as it is commonly used in the art, is to be understood as meaning antibodies having variable regions in which both the framework and complementary determining regions (CDRs) are derived from sequences of human origin. In this context, the term "humanized antibody", as it is commonly used in the art, is to be understood as meaning antibodies wherein residues from a CDR of an antibody of human origin are replaced by residues from a CDR of a nonhuman species (such as mouse, rat or rabbit) having the desired specificity, affinity and capacity.
In an embodiment, the anti-EGFRl antibody fragment comprises a Fab fragment of cetuximab. In an embodiment, the anti-EGFRl Fab fragment has a sequence set forth in SEQ ID NO:s 6 and 3. In an embodiment, the anti-EGFRl Fab fragment comprises or consists of a sequence set forth in SEQ ID NO:s 6 and 3.
In an embodiment, the anti-EGFRl antibody comprises a F(ab')2 fragment of cetuximab. In an embodiment, the anti-EGFRl F(ab')2 fragment has a sequence set forth in SEQ ID NO:s 7 and 3. In an embodiment, the anti-EGFRl F(ab')2 fragment comprises or consists of a sequence set forth in SEQ ID NO:s 7 and 3.
"Dextran" should be understood as referring to a branched glucan composed of chains of varying lengths, wherein the straight chain consists of a a- 1,6 glycosidic linkages between D-glucopyranosyl units. Branches are bound via a-1,3 glycosidic linkages and, to a lesser extent, via a-1,2 and/or a-1,4 glycosidic linkages. A portion of a straight chain of a dextran molecule is depicted in the schematic representation below.
"D-glucopyranosyl unit" should be understood as referring to a single D-glucopyranosyl molecule. Dextran thus comprises a plurality of D-glucopyranosyl units. In dextran, each D-glucopyranosyl unit is bound to at least one other D-glucopyranosyl unit via a a- 1.6 glycosidic linkage, via a a-1,3 glycosidic linkage or via both.
Each D-glucopyranosyl unit of dextran comprises 6 carbon atoms, which are numbered 1 to 6 in the schematic representation below. The schematic representation shows a single D-glucopyranosyl unit bound to two other D-glucopyranosyl units (not shown) via a- 1.6 glycosidic linkages.
Carbons 2, 3 and 4 may contain free hydroxyl groups. In D-glucopyranosyl units bound to a second D- glucopyranosyl unit via a a-1,3 glycosidic linkage, wherein carbon 3 of the D-glucopyranosyl unit is bound via an ether bond to carbon 1 of the second D-glucopyranosyl unit, carbons 2 and 4 may be substituted by free hydroxyl groups. In D-glucopyranosyl units bound to a second D-glucopyranosyl unit via a a-1,2 or a-1,4 glycosidic linkage, wherein carbon 2 or 4 of the D-glucopyranosyl unit is bound via an ether bond to carbon 1 of the second D-glucopyranosyl unit, carbons 3 and 4 or 2 and 3, respectively, may be substituted by free hydroxyl groups.
Carbohydrate nomenclature is essentially according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur.
J. Biochem. 1998, 257, 293) .
The term "dextran derivative" should be understood as referring to dextran, wherein at least one carbon selected from carbon 2, 3 or 4 of the at least one D-glucopyranosyl unit is substituted by a substituent of the formula -O- (CH2)n-S-B12Hn2- wherein n is in the range of 3 to 10; and the dextran derivative is bound to the anti-EGFR1 antibody or an EGFR1 binding fragment thereof via a bond formed by a reaction between at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative and an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof. The dextran derivative may furher contain other modifications to the basic dextran structure, e.g. as described below.
"BSH", "Bi2Hii-SH" and "Na2Bi2HnSH" should be understood as referring to sodium borocaptate, also known as sodium mercaptododecaborate and sulfhydryl boron hydride. "Bi2Hh2”" thus refers to the boron hydride moiety of BSH.
One or more, i.e. one, two or three carbons selected from carbons 2, 3 and 4 of the at least one D-glucopyranosyl unit may be substituted by a substituent of the formula -0- (0¾) n“S-Bi2Hn2~ .
In an embodiment, n is 3, 4, 5, 6, 7, 8, 9 or 10. In an embodiment, n is in the range of 3 to 4, or in the range of 3 to 5, or in the range of 3 to 6, or in the range of 3 to 7, or in the range of 3 to 8, or in the range of 3 to 9.
D-glucopyranosyl units of dextran may be cleaved by oxidative cleavage of a bond between two adjacent carbons substituted by a hydroxyl group. The oxidative cleavage cleaves vicinal diols, i.e. D-glucopyranosyl units in which two (free) hydroxyl groups occupy vicinal positions. D-glucopyranosyl units in which carbons 2, 3 and 4 contain free hydroxyl groups may thus be oxidatively cleaved between carbons 2 and 3 or carbons 3 and 4. Thus a bond selected from the bond between carbons 2 and 3 and the bond between carbons 3 and 4 may be oxidatively cleaved. D-glucopyranosyl units of dextran may be cleaved by oxidative cleavage using an oxidizing agent such as sodium periodate, periodic acid and lead(IV) acetate, or any other oxidizing agent capable of oxidatively cleaving vicinal diols.
Oxidative cleavage of a D-glucopyranosyl unit forms two aldehyde groups, one aldehyde group at each end of the chain formed by the oxidative cleavage. In the conjugate, the aldehyde groups may in principle be free aldehyde groups. However, the presence of free aldehyde groups in the conjugate is typically undesirable. Therefore the free aldehyde groups may be capped or reacted with an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof, or e.g. with a tracking molecule.
The dextran derivative is bound to the anti-EGFRl antibody or an EGFR1 binding fragment thereof via a bond formed by a reaction between at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative and an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
The dextran derivative may also be bound to the anti-EGFRl antibody or an EGFR1 binding fragment thereof via a group formed by a reaction between at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative and an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
The aldehyde group formed by oxidative cleavage readily reacts with an amino group in solution, such as an aqueous solution. The resulting group or bond formed may, however, vary and is not always easily predicted and/or characterised. The reaction between at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative and an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof may result e.g. in the formation of a Schiff base. Thus the group via which the dextran derivative is bound to the anti-EGFRl antibody or an EGFR1 binding fragment thereof may be e.g. a Schiff base (imine) or a reduced Schiff base (secondary amine).
In an embodiment, the dextran derivative has a molecular mass in the range of about 3 to about 2000 kDa. In this context, the molecular mass of the dextran derivative should be understood as including the molecular mass of the dextran derivative containing the dextran and its substituents, but not the molecular mass of the anti-EGFRl antibody or an EGFR1 binding fragment thereof. In an embodiment, the dextran derivative has a molecular mass in the range of about 30 to about 300 kDa.
In an embodiment, the conjugate comprises about 10 to about 300 or about 20 to about 150 substituents of the formula -0- (CH2) n-S-Bi2Hn2^.
In an embodiment, the conjugate comprises about 300 boron atoms (300B), about 800 boron atoms (800B), about 900 boron atoms (900B), or about 1200 boron atoms. E.g "900B" refers to a conjugate carrying per one mole of protein one mole of dextran, that car ries ca. 900 moles of boron atoms in BSH molecules.
The anti-EGFRl antibody or an EGFR1 binding fragment thereof typically contains at least one amino group, such as an N-terminal amine group and/or the amino group of a lysine residue.
In an embodiment, the amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof is the amino group of a lysine residue of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
In an embodiment, the conjugate further comprises at least one tracking molecule bound to the dextran derivative or to the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
"Tracking molecule" refers to a detectable molecule. Such a detectable molecule may be e.g. a radioisotope, a compound comprising a radioisotope, a radionuclide, a compound comprising a radionuclide, a fluorescent label molecule (such as FITC, TRITC, the Alexa and Cy dyes, etc.), a chelator, such as DOTA (1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), or an MRI active molecule, such as gadolinium-DTPA (gadolinium-diethylenetriaminepentacetate). Procedures for accomplishing the binding of the tracking molecule to the dextran derivative or to the anti-EGFRl antibody or an EGFR1 binding fragment thereof are well known to the art. A tracking molecule may allow for locating the conjugate after it has been administered to a patient and targeted to specific cells; in this way, it is possible to direct the low energy thermal neutron irradiation to the location of the targeted conjugate.
In an embodiment, the tracking molecule is bound to the dextran derivative via a bond or a group formed by a reaction between at least one aldehyde group formed by oxidative cleavage of a D- glucopyranosyl unit of the dextran derivative and a group of the tracking molecule. A suitable group of the tracking molecule may be e.g. an amino group.
It is possible that one or more aldehyde groups formed by oxidative cleavage of a D- glucopyranosyl unit of the dextran derivative is not reacted with an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof or with a tracking molecule.
In an embodiment, the dextran derivative comprises at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative which is capped.
The at least one aldehyde group may be capped by a suitable group, such as a reduced Schiff base.
The at least one aldehyde group may also be capped by a group formed by a reaction between the at least one aldehyde group and a hydrophilic capping agent, such as ethanolamine, lysine, glycine or Tris.
The capping may be stabilized using a reducing agent, such as NaCNBH3. A capping group such as a reduced Schiff base may thus be formed.
In an embodiment, the dextran derivative comprises at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative that is not reacted with an amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof or with a tracking molecule and which is capped.
In an embodiment, essentially all aldehyde groups formed by oxidative cleavage of one or more D- glucopyranosyl units of the dextran derivative are capped.
In an embodiment, the dextran derivative comprises a plurality of aldehyde groups formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative, wherein essentially all of the aldehyde groups formed by oxidative cleavage of one or more D-glucopyranosyl units of the dextran derivative are capped.
In an embodiment, at least one carbon selected from carbon 2, 3 or 4 of at least one D- glucopyranosyl unit of the dextran derivative is substituted by a substituent of the formula -0- (CH2)mCH=CH2 wherein m is in the range of 1 to 8. While such an embodiment is typically not desirable, it may occur as a side product, when said substituent has not reacted with BSH.
In an embodiment, the conjugate is obtainable by a method comprising the steps of: a) alkenylating at least one hydroxyl group of dextran to obtain alkenylated dextran; b) reacting sodium borocaptate (BSH) with the alkenylated dextran obtainable from step a) to obtain BSH-dextran; c) oxidatively cleaving at least one D-glucopyranosyl residue of the BSH-dextran so that aldehyde groups are formed; d) reacting the oxidatively cleaved BSH-dextran obtainable from step c) with an anti-EGFRl antibody or an EGFR1 binding fragment thereof to obtain a conjugate.
The present invention also relates to a conjugate obtainable by a method comprising the steps of: a) alkenylating at least one hydroxyl group of dextran to obtain alkenylated dextran; b) reacting sodium borocaptate (BSH) with the alkenylated dextran obtainable from step a) to obtain BSH-dextran; c) oxidatively cleaving at least one D-glucopyranosyl residue of the BSH-dextran so that aldehyde groups are formed; d) reacting the oxidatively cleaved BSH-dextran obtainable from step c) with an anti-EGFRl antibody or an EGFR1 binding fragment thereof to obtain a conjugate.
In an embodiment, the dextran has a molecular mass in the range of about 3 to about 2000 kDa, or about 10 to about 100 kDa, or about 5 to about 200 kDa, or about 10 to about 250 kDa. The dextran having a molecular mass in said range should be understood as referring to dextran that has not been subjected to steps a)-d).
In this context, the term "alkenylation" or "alkenylating" should be understood as referring to the transfer of an alkenyl group to a D-glucopyranosyl unit of dextran to give an alkenyl ether. In other words, at least one hydroxyl group of the D-glucopyranosyl unit of dextran becomes an alkenyloxy group.
In step a) , one or more of hydroxyl groups bound to carbons 2, 3 or 4 of at least one D-glucopyranosyl unit of dextran may react in the alkenylation reaction. One or more, or a plurality of, D-glucopyranosyl units of dextran may be alkenylated.
In an embodiment, dextran is alkenylated in step a) using an alkenylating agent, wherein the alkenylating agent has a structure according to the formula X- (CH2) mCH=CH2 wherein m is in the range from 1 to 8, and X is Br, Cl, or I.
In an embodiment, m is 1, 2, 3, 4, 5, 6, 7 or 8. In an embodiment, m is in the range of 1 to 2, or in the range of 1 to 3, or in the range of 1 to 4, or in the range of 1 to 5, or in the range of 1 to 6, or in the range of 1 to 7.
In an embodiment, the alkenylating agent is allyl bromide.
In an embodiment, at least one carbon selected from carbon 2, 3 or 4 of at least one D- glucopyranosyl unit of the alkenylated dextran obtainable from step a) is substituted by a substituent of the formula -0- (CH2)mCH=CH2, wherein m is in the range of 1 to 8.
In an embodiment, m is 1, 2, 3, 4, 5, 6, 7 or 8. In an embodiment, m is in the range of 1 to 2, or in the range of 1 to 3, or in the range of 1 to 4, or in the range of 1 to 5, or in the range of 1 to 6, or in the range of 1 to 7.
In step b) , the sulfhydryl group of BSH may react with an alkenyl group of the alkenylated dextran to form BSH-dextran to give a thioether. One or more BSH molecules may react with the alkenylated dextran. Therefore, BSH-dextran obtainable from step b) may contain a plurality of BSH moieties (i.e. groups of the formula -S-Bi2Hu2~) . The the sulfhydryl groups of BSH may react with alkenyl groups of a single alkenylated D-glucopyranosyl unit containing more than one alkenyl group or with alkenyl groups of two or more alkenylated D-glucopyranosyl units.
Thus the BSH-dextran obtainable from step b) may be a dextran derivative in which at least one carbon selected from carbon 2, 3 or 4 of the at least one D-glucopyranosyl unit is substituted by a substituent of the formula -0- (CH2)n-S-B12Hn2- wherein n is in the range of 3 to 10.
In an embodiment, BSH-dextran obtainable from step b) comprises about 10 to about 100 or about 20 to 100 substituents of the formula -0- (CH2) n_S-Bi2Hn2“, wherein n is in the range of 3 to 10.
In an embodiment, BSH is reacted with the alkenylated dextran obtainable from step a) in the presence of a radical initiator in step b) . The radical initiator is capable of catalyzing the reaction between the sulfhydryl group (s) of BSH and with the alkenyl group(s) of alkenylated dextran.
In this context, "radical initiator" should be understood as referring to an agent capable of producing radical species under mild conditions and promote radical reactions. The term "radical initiator" may also refer to UV (ultraviolet) light. UV light irradiation is capable of generating radicals, e.g. in the presence of a suitable photoinitiator. Suitable radical initiators include, but are not limited to, inorganic peroxides such as ammonium persulfate or potassium persulfate, organic peroxides, and UV light.
In an embodiment, BSH is reacted with the alkenylated dextran obtainable from step a) in the presence of a radical initiator selected from the group consisting of ammonium persulfate, potassium persulfate and UV light in step b).
In step b), the weight ratio or the molar ratio of BSH to alkenylated dextran obtainable from step a) may be suitably selected in order to obtain conjugates in which the number of BSH moieties (i.e. the number of substituents of the formula -0-(CH2)n-S-Bi2Hii2”) per dextran moiety (of the dextran derivative) varies. The number of BSH moieties per dextran moiety of the BSH-dextran may be measured e.g. by nuclear magnetic resonance as described in Example 2 or by inductively coupled plasma mass spectrometry (ICP-MS) as described in Example 9.
In an embodiment, the ratio of BSH to alkenylated dextran present in step b) is in the range of 1:5 to 2:1, or in the range of 1:4 to 1:1 by weight, or in the range of 1:2 to 3:4 by weight. Typically, the higher the ratio of BSH to alkenylated dextran, the higher the number of BSH moieties per dextran moiety of the BSH-dextran.
The ratio of the radical initiator, such as ammonium persulfate or potassium persulfate, may also be varied in step b) . In an embodiment, the ratio of the radical initiator to BSH and/or to dextran present in step b) is in the range of 1:5 to 2:1, or in the range of 1:4 to 1:1 by weight, or in the range of 1:2 to 3:4 by weight.
In an embodiment, the ratio of the radical initiator to alkenylated dextran in step b) is in the range of 1:5 to 2:1, or in the range of 1:4 to 1:1 by weight, or in the range of 1:2 to 3:4 by weight.
As described above, a bond selected from the bond between carbons 2 and 3 and the bond between carbons 3 and 4 may be oxidatively cleaved in step c). In the oxidative cleavage, the D-glucopyranosyl ring is opened between vicinal diols, leaving two aldehyde groups. Aldehyde groups of the oxidatively cleaved BSH-dextran obtainable from step c) may react with an anti-EGFRl antibody or an EGFR1 binding fragment thereof to obtain a conjugate. The aldehyde groups may react with a suitable group such as an amino group.
The at least one D-glucopyranosyl residue of the BSH-dextran may, in principle, be oxidatively cleaved using any oxidizing agent capable of oxidatively cleaving the D-glucopyranosyl unit between two vicinal carbons substituted by free hydroxyl groups. The oxidizing agent may also be selected so that it essentially specifically oxidatively cleaves the at least one D-glucopyranosyl residue of the BSH-dextran.
Such an oxidizing agent may not oxidize other groups or moieties of the BSH-dextran.
In an embodiment, the at least one D-glucopyranosyl residue of the BSH-dextran is oxidatively cleaved in step c) using an oxidizing agent selected from the group consisting of sodium periodate, periodic acid and lead(IV) acetate.
In an embodiment, the at least one D-glucopyranosyl residue of the BSH-dextran is oxidatively cleaved in step c) in an aqueous solution.
In an embodiment, the method further comprises the step of reacting the oxidatively cleaved BSH-dextran obtainable from step c) or the conjugate obtainable from step d) with a tracking molecule.
In this context, the tracking molecule may be any tracking molecule described in this document.
The tracking molecule may react with at least one aldehyde group of the oxidatively cleaved BSH-dextran obtainable from step c) . A suitable group of the tracking molecule that may react with the at least one aldehyde group may be e.g. an amino group.
In an embodiment, the method further comprises the step e) of capping unreacted aldehyde groups of the oxidatively cleaved BSH-dextran obtainable from step c) or the conjugate obtainable from step d).
In an embodiment, the unreacted aldehyde groups are capped using a hydrophilic capping agent, such as ethanolamine, lysine, glycine or Tris.
In an embodiment, the hydrophilic capping agent is selected from the group consisting of ethanolamine, lysine, glycine and Tris.
In an embodiment, one or more steps selected from steps a), b), c) and d) are performed in an aqueous solution. A suitable aqueous solution may be e.g. an aqueous phosphate buffer having a pH of about 6 to 8 .
In an embodiment, all of the steps a)-d) are performed in an aqueous solution.
The anti-EGFRl antibody or an EGFR1 binding fragment thereof typically contains at least one amino group, such as the N-terminal amine group and/or the amino group of a lysine residue. In step d), the aldehyde groups of the oxidatively cleaved BSH-dextran obtainable from step c) may thus react with the at least one amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
In an embodiment, the amino group of the anti-EGFRl antibody or an EGFR1 binding fragment thereof is the amino group of a lysine residue of the anti-EGFRl antibody or an EGFR1 binding fragment thereof.
In an embodiment, the oxidatively cleaved BSH-dextran is reacted with the anti-EGFRl antibody or an EGFR1 binding fragment thereof by incubating the oxidatively cleaved BSH-dextran and the anti-EGFRl antibody or an EGFRl binding fragment thereof in room temperature in an aqueous phosphate buffer having a pH of about 6 to 8 in step d).
The conjugate may be purified e.g. by gel filtration, for instance as described in Example 4.
The present invention further relates to a pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention.
The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are well known in the art and may include e.g. phosphate buffered saline solutions, water, oil/water emulsions, wetting agents, and liposomes. Compositions comprising such carriers may be formulated by methods well known in the art. The pharmaceutical composition may further comprise other components such as vehicles, additives, preservatives, other pharmaceutical compositions administrated concurrently, and the like.
In an embodiment, the pharmaceutical composition comprises an effective amount of the conjugate according to one or more embodiments of the invention.
In an embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the conjugate according to one or more embodiments of the invention.
The term "therapeutically effective amount" or "effective amount" of the conjugate should be understood as referring to the dosage regimen for modulating the growth of cancer cells and/or treating a patient's disease when cancer cells are bombarded with neutron radiation or exposed to BNCT. The therapeutically effective amount may be selected in accordance with a variety of factors, including the age, weight, sex, diet and medical condition of the patient, the severity of the disease, and pharmacological considerations, such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular conjugate used. The therapeutically effective amount can also be determined by reference to standard medical texts, such as the Physicians Desk Reference 2004. The patient may be male or female, and may be an infant, child or adult.
The term "treatment" or "treat" is used in the conventional sense and means attending to, caring for and nursing a patient with the aim of combating, reducing, attenuating or alleviating an illness or health abnormality and improving the living conditions impaired by this illness, such as, for example, with a cancer disease.
In an embodiment, the pharmaceutical composition comprises a composition for e.g. oral, parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or for di- reet injection into tissue. Administration of the pharmaceutical composition may be effected in different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration .
The present invention further relates to the conjugate according to one or more embodiments of the present invention or the pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention for use as a medicament .
The present invention further relates to the conjugate according to one or more embodiments of the present invention or the pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention for use as a medicament for boron neutron capture therapy.
"Boron neutron capture therapy" (BNCT) should be understood as referring to targeted radiotherapy, wherein nonradioactive boron-10 is irradiated with low energy thermal neutrons to yield alpha particles and lithium-7 nuclei. The nonradioactive boron-10 may be targeted by incorporating it in a tumor localizing drug such as a tumor localizing conjugate.
The present invention further relates to the conjugate according to one or more embodiments of the present invention or the pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention for use in boron neutron capture therapy.
The present invention further relates to the conjugate according to one or more embodiments of the present invention or the pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention for use in the treatment of cancer.
In an embodiment, the cancer is a head-and-neck cancer.
In an embodiment, the cancer is selected from the group consisting of head-and-neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, multidrug resistant cancer and testicular cancer.
The present invention further relates to the conjugate according to one or more embodiments of the present invention or the pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention for use in the treatment of cancer by boron neutron capture therapy.
The present invention further relates to the use of the conjugate or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament.
The present invention further relates to the use of the conjugate or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament for boron neutron capture therapy.
The present invention further relates to the use of the conjugate or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament for the treatment of cancer.
In an embodiment, the cancer is a head-and-neck cancer.
In an embodiment, the cancer is selected from the group consisting of head-and-neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, multidrug resistant cancer and testicular cancer.
The present invention further relates to the use of the conjugate or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament for the treatment of cancer by boron neutron capture therapy.
The present invention also relates to a method of treating or modulating the growth of EGFR1 expressing tumor cells in a human, wherein the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount.
In an embodiment, the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount in boron neutron capture therapy.
In an embodiment, the concentration of boron is analysed in tumor cells after administering the conjugate or the pharmaceutical composition.
In an embodiment, the concentration of boron is analysed in blood after administering the conjugate or the pharmaceutical composition.
In an embodiment, the concentration of boron is analysed in muscle, or in other non-tumor tissue, after administering the conjugate or the pharmaceutical composition.
The concentration of boron in tumor cells, in blood or in both may be analysed or measured e.g. by inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma atomic emission spectroscopy (ICP-AES) (e.g. Example 9). These methods measure the amount (in moles) or concentration of boron atoms in the sample.
The concentration of boron in tumor cells, in blood or in both may also be analysed or measured indirectly, e.g. by using an embodiment of the conjugate comprising a tracking molecule and analysing or measuring the concentration of the tracking molecule. For instance, if the tracking molecule is fluorescent or radioactive, the fluorescence or radioactivity of the tracking molecule may be measured or visualised.
In an embodiment, the concentration of boron is analysed in tumor cells and in blood after administering the conjugate or the pharmaceutical composition, and the ratio of the concentration of boron in tumor cells to the concentration of boron in blood is higher than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1.
In an embodiment, the concentration of boron is analysed in tumor cells and in a muscle, or in other non-tumor tissue, after administering the conjugate or the pharmaceutical composition, and the ratio of the concentration of boron in tumor cells to the concentration of boron in a muscle, or other non-tumor tissue, is higher than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7 :1, or 8:1.
In an embodiment, the ratio of the concentration of boron in tumor cells to the concentration of boron in blood, in a muscle, or in other non-tumor tissue is the molar ratio of boron atoms in tumor cells to the boron atoms in blood, in a muscle, or in other non-tumor tissue.
The present invention also relates to a method for modulating the growth of a cell population expressing EGFR1 protein, wherein the method comprises the step of contacting the conjugate according to one or more embodiments of the invention or the pharmaceutical composition according to one or more embodiments of the invention with the cell population expressing EGFR1 protein.
In an embodiment, the cell population expressing EGFR1 protein is a cancer cell population or a tumor cell population.
In this context, the term "a cancer cell population" should be understood as referring to one or more cancer cell populations.
The conjugate may be contacted in vitro, in vivo and/or ex vivo to with the cell population, for example, cancer cells, including, for example, cancer of the blood, plasma, lung, breast, colon, prostate, kidney, pancreas, brain, bones, ovary, testes, and lymphatic organs; more preferably lung, colon prostrate, plasma, blood or colon cancer; "Modulating the growth of cancer cell populations" includes inhibiting the proliferation of cell populations from dividing to produce more cells; reducing the rate of increase in cell division as compared, for example, to untreated cells; killing cell populations; and/or preventing cell populations (such as cancer cells) from metastasizing. The growth of cell populations may be modulated in vitro, in vivo or ex vivo.
In an embodiment, the cancer is selected from the group consisting of head-and-neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, multidrug resistant cancer and testicular cancer.
The present invention further relates to a method of treating and/or modulating the growth of and/or prophylaxis of tumor cells in humans, wherein the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount.
In an embodiment, the effective amount is a therapeutically effective amount.
In an embodiment, the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount in boron neutron capture therapy.
In an embodiment, the tumor cells are selected from the group consisting of leukemia cells, lymphoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, smallcell lung cancer cells, head-and-neck cancer cells, multidrug resistant cancer cells, and testicular cancer cells, metastatic, advanced, drug- or hormone-resistant, multidrug resistant cancer cells, and versions thereof.
The present invention further relates to a method of treating cancer in humans, wherein the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount.
In an embodiment, the conjugate or the pharmaceutical composition according to one or more embodiments of the invention is administered to a human in an effective amount in boron neutron capture therapy.
In an embodiment, the effective amount is a therapeutically effective amount.
In an embodiment, the cancer is selected from the group consisting of head-and-neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, multidrug resistant cancer and testicular cancer.
The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodiment of the invention. A product, a use or a method to which the invention is related may comprise at least one of the embodiments of the invention described hereinbefore.
The conjugate according to one or more embodiments of the invention has a number of advantageous properties .
The conjugate according to one or more embodiments of the invention is relatively non-toxic in the absence of low energy neutron irradiation and has low antigenicity.
It contains a high number of boron-10 atoms per conjugate molecule. Further, it exhibits relatively good aqueous solubility.
The conjugate according to one or more embodiments of the invention also exhibits good pharmacokinetics. It has suitable retention in blood, high uptake in cells to which it is targeted and low uptake in cells and organs to which it is not targeted.
Its production process is relatively simple and can be performed in aqueous solutions.
The conjugate according to one or more embodiments of the invention is sufficiently stable towards chemical or biochemical degradation during manufacturing or in physiological conditions, e.g. in blood, serum, plasma or tissues.
EXAMPLES
In the following, the present invention will be described in more detail. Reference will now be made in detail to the embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The description below discloses some embodiments of the invention in such detail that a person skilled in the art is able to utilize the invention based on the disclosure. Not all steps of the embodiments are discussed in detail, as many of the steps will be obvious for the person skilled in the art based on this specification.
EXAMPLE 1. Allylation of dextran 200 mg Dextran 70 kD (Sigma) was dissolved in 2 ml of 0.6 M NaOH. 250 μΐ of allyl bromide (Sigma) was added, and the reaction was allowed to proceed for 3 h at 60°C. The reaction mixture was then neutralized with 1M acetic acid and the product was isolated by precipitation with 10 volumes of cold acetone (-20°C). Precipitate was collected by centrifugation and washed twice with acetone. The allylated dextran (Scheme 1) was subjected to 1H-NMR analysis, which showed that the level of allylation was ca. 36%.
Scheme 1. Dextran allylation by use of allyl bromide .
EXAMPLE 2. Addition of BSH to allyl dextran 50 mg allyl dextran 70 kD prepared as described in Example 1, 50 mg ammonium persulfate and 50 mg sodium borocaptate (BSH; Katchem Ltd, Czech Republic) were dissolved in 0.5 ml H2O.
The reaction was allowed to proceed for 2 h at 50°C. The reaction product, BSH-dextran (Scheme 2), was isolated with ultrafiltration using centrifugal filter (Amicon, 10K cut-off). 1H-NMR analysis showed that on average 100 BSH units were linked to allyl dextran, corresponding to 1200 boron atoms per dextran chain (Fig. 1) . With minor modifications, e.g. by use of lower allylation level in dextran, BSH dextran with ca. 900 borons or 800 borons per dextran chain were obtained.
Scheme 2. Addition of sodium borocaptate to allyl dextran in a persulfate catalyzed reaction.
By varying the amount of BSH and persulfate in the reaction described above, it was possible to prepare BSH-dextrans with a clearly lower BSH level: 1) In a reaction containing 20 mg allyl dextran, 15 mg ammonium persulfate and 15 mg BSH, the isolated BSH-dextran was found to contain ca. 700 boron atoms per dextran chain. 2) In a reaction containing 20 mg allyl dextran, 10 mg ammonium persulfate and 10 mg BSH, the isolated BSH-dextran was found to contain ca. 560 boron atoms per dextran chain. 3) In a reaction containing 20 mg allyl dextran, 5 mg ammonium persulfate and 5 mg BSH, the isolated BSH-dextran was found to contain ca. 360 boron atoms per dextran chain.
EXAMPLE 3. Oxidation of BSH-dextran 50 mg of BSH-dextran prepared as described in Example 2 was dissolved in 3 ml of 25 mM NaI04 in 0.1 M sodium acetate, pH 5.5. The reaction tube was covered with aluminium foil and incubated at RT overnight. The reaction product, oxidized BSH-dextran (Scheme 3), was isolated with ultrafiltration using a centrifugal filter (Amicon, 10K cut-off).
Scheme 3. Oxidation of BSH-dextran by use of sodium periodate.
EXAMPLE 4. Conjugation of oxidized BSH-dextran to anti -EGFR1 Fab/F(ab')2 2 mg (40 nmol) of anti-EGFRl Fab in 2 ml of phosphate buffered saline (PBS) was mixed with 5.1 mg (60 nmol) of oxidized BSH-dextran (Example 3) in 1.6 ml of PBS. Reaction was allowed to proceed overnight at RT. 400 μΐ of 0.5 M NaCNBH3 was added to the reaction to stabilize the aldehyde-lysine linkages and the reaction was incubated for 2 hours at RT. 800 μΐ of 0.2 M ethanolamine-HCl pH 8 was added and the reaction was incubated for 1 hour at RT. 400 μΐ of 0.5 M NaCNBH3 was added to stabilize ethanolamine capping and the reaction was incubated for 2 hours at RT. Low molecular weight reagents were removed by a Amicon centrifugal filter unit (MWCO 30K) according to the manufacturer's instructions using PBS as the washing eluent.
2 mg (40 nmol) of anti-EGFRl F(ab')2 in 2 ml of phosphate buffered saline (PBS) was mixed with 2.56 mg (30 nmol) of oxidized BSH-dextran (Example 3) in 1.6 ml of PBS. Conjugate was stabilized, capped and purified by ultrafiltration as above.
Both conjugates were analyzed by Äkta purifier (GE Healthcare) with a Yarra 3 pm SEC-3000 gel filtration column (300 x 7.8 mm; Phenomenex) using 10 % acetonitrile (ACN)-50 mM Tris-HCl, pH 7.5 as the elution buffer (Fig. 2).
EXAMPLE 5. Generation of Anti-EGFR1-Fab and -F(ab')2 , and control-Fab and -F(ab')2 fragments
Fab and F(ab')2 fragments were generated either from commercial cetuximab (Erbitux, Roche) or ce-tuximab produced in CHO cells (Freedom CHO-S kit, Invitrogen). Freedom CHO-S Kit (Life Technologies) was used for the development of stable cell lines producing cetuximab. The work was done according to manufacturer's instructions. Optimized nucleotide sequences encoding the heavy and light chain sequences were purchased from GeneArt (Life Technologies) and cloned separately into pCEP4 expression vectors (Life Technologies) . For stable expression, the Freestyle™ CHO-S cells were transfected with linearized 1:1 light chain and heavy chain vectors. Transfectants were selected with puromycin and methotrexate after which clone isolation was done by limited dilution cloning. Cloned cell lines were scaled up and assessed for productivity.
Control-Fab and -F(ab')2 fragments were generated from commercial omalizumab (anti-IgE) (Xolair, Novartis).
Anti-EGFRl Fab fragments were prepared by digesting antibody with immobilized papain (Pierce) according to manufacturer's instructions with minor modifications. The used ratio of enzyme to substrate was 1:60 (w/w) and incubation time was 7 h. Fab fragments were separated from undigested IgG and Fc fragments with a column of immobilized protein A (Thermo
Scientific) according to the manufacturer's instructions .
Anti-EGFRl F(ab')2 fragments were prepared by digesting the antibody with either FragIT MaxiSpin (Genovis) according to manufacturer's instructions or with Fabricator enzyme (Genovis) according to the manufacturer's instructions with minor modifications. Fabricator enzyme digestion was performed with 120 Units of enzyme per mg of antibody in 50 mM sodium phosphate buffer pH 6.6 and incubation time was 1 h at +37°C. F(ab')2 fragments were purified with an immo bilized HiTrap protein L column (GE Healthcare) according to the manufacturer's instructions. Reaction buffer was changed to PBS with Amicon Ultra concentrator (Millipore) (10 kDa cutoff).
The generated fragments were identified with SDS-PAGE and the protein concentration of each fragment was determined by measuring UV absorbance at 280 nm.
EXAMPLE 6. SDS-PAGE analysis of boron conjugates
Boron conjugates of anti-EGFRl Fab and F(ab')2 fragments were analyzed using SDS-PAGE in order to verify that the conjugations have been successful and that unconjugated Fab or F(ab')2 fragments are not present after conjugation. Figure 3 shows an SDS-PAGE analysis of anti-EGFRl Fab/F(ab')2 boron conjugates with different amounts of boron in a gradient gel (Bio-Rad, 4-15 %) under nonreducing (panel A) and reducing (panel B) conditions. The results of panel A show that conjugation has been complete (or near complete) because unconjugated Fab or F(ab')2 fragments were not visible. BSH is a negatively charged molecule and when conjugated to a protein the migration velocity of a conjugate is faster on a gel than expected based on its theoretical molecular weight. The example of Figure 3 (Panel A) indicates that con- jugates with high amount of boron migrate faster on a nonreducing gel than conjugates with lower amount of boron (e.g. compare lanes 1, 2, 4 and 6). The results of Figure 3 (Panel A) also indicate that most of the conjugates are separated into two bands on a nonreducing gel implying that the samples contain a mixture of two different kinds of conjugates. SDS-PAGE analysis of boron conjugates in reducing conditions (Figure 3, panel B) show that all Fab conjugates with different amounts of boron migrate similarly on the gel under reducing conditions (Lanes 1, 2, 4, 6) . Likewise, re duced F(ab')2 conjugates with different amounts of boron migrate identically (Lanes 3, 5, 7). In general, reduced boron conjugates migrate faster on the gel than nonreduced conjugates.
EXAMPLE 7. In vitro internalization assays of boron conjugates
AlexaFluor488 labeling of boron conjugates 5 pg AlexaFluor488 carboxylic acid, succin-imidyl ester label (Invitrogen) was incubated with 100 pg of boron conjugates (anti-EGFRl-Fab, anti-EGFRl-F(ab')2, anti-EGFRl-mAb, control-Fab, control-F(ab')2, control-mAb) or corresponding nonconjugated compounds for 15 min at room temperature in a buffer containing 10 pi 1 M NaHC03, pH 9 in 100 pi PBS. After incubation excess label was removed by changing the buffer to PBS with Amicon Ultra concentrator (Millipore) (10 kDa cutoff). Protein concentration of each compound was determined by measuring UV absorbance at 280 nm and the degree of labeling was calculated according to the manufacturer's instructions (Invitrogen).
Tritium labeling of boron conjugates
After removal of toluene solvent by evaporation, 100 pCi tritium labeled N-succinimidyl propio- nate (Perkin Elmer) was incubated with 100 pg of anti-EGFRl-Fab-BSH(800B)-Dex, anti-EGFRl-F(ab')2 BSH(800B)-Dex, anti-EGFRl-mAb and control-mAb in a buffer containing 20 μΐ 1 M Na-borate buffer, pH 8.8 in 100 μΐ PBS. Reaction was allowed to proceed overnight at room temperature and then excess label was removed by changing the buffer to PBS with an Amicon Ultra concentrator (10 kDa cutoff). The amount of radioactivity was measured with a scintillation counter in the presence of a scintillation fluid cocktail (Ultima Gold, Perkin Elmer). The amount of tritium label in compounds was calculated as cpm/pg protein.
Cell culture HSC-2 cells (human squamous cell carcinoma of mouth, JCRP Cellbank, Japan) and FaDu cells (human squamous cell carcinoma of pharynx, ATCC) were cultured in T75 flasks in Eagle's minimal essential medium with 2 % glutamine, 10 % fetal bovine serum and 1 % penicillin/streptomycin. HEK (Human Embryonic Kidney, ATCC) cells were cultured in T75 flasks in Dulbecco’s Modified Eagle Medium with 2 % glutamine, 10 % fetal bovine serum and 1 % penicillin/streptomycin.
Internalization assay visualized in fluorescence microscopy HSC-2 cells (5xl04) were seeded on a chamber slide and allowed to grow for 24 h. Then the cells were incubated for 3h at + 37°C or at +4°C in 100μ1 media containing 10 pg/ml AlexaFluor488 labeled BSH-conjugates. After incubation cells were washed two times with PBS and fixed with 4 % paraformaldehyde for 20 min. Mounting media (Prolong Gold antifade reagent with DAPI) was added and the cells were covered with microscopy cover slips. Cells were photographed with fluorescence microscopy (Zeiss Axio Scope A1; ProgRes C5, JENOPTIK AG).
Internalization of anti-EGFRl-F(ab')2 BSH(900B)-Dex and nonconjugated anti-EGFRl-F(ab')2 by HSC-2 tumor cell line was analyzed by fluorescence microscopy (Figure 4). The experiment was carried out at +4°C (compounds bind to the cell surface but cannot be internalized) and at +37°C (cells are able to internalize the surface-bound compounds). Both nonconjugated anti-EGFRl-F(ab' )2 and boron conjugate bound to the cell surface at +4°C (Panels A and B) and were internalized at +37°C (Panels C and D). In fact, boron conjugate was internalized more efficiently than nonconjugated anti-EGFRl-F(ab')2. Internalization assay with anti-EGFRl-Fab-BSH(900B)-Dex and EGFRl-mAb-BSH(90OB)-Dex and corresponding nonconjugated anti-EGFRl-Fab and anti-EGFRl-mAb gave very similar results to the data presented in Figure 4 (not shown). The effect of boron load for internalization was examined using boron conjugates (anti-EGFRl-Fab-BSH-Dex and anti-EGFRl-F(ab')2-BSH-Dex) with different amounts of boron. The results indicated that conjugates with more boron were internalized more efficiently by HSC-2 cells than conjugates with low boron load at +37°C (not shown). Con-trol-F(ab')2 -BSH(900B)-Dex was internalized only very weakly (not shown).
Internalization assay (FACS) HSC-2, FaDu and HEK cells (2xl05) were seeded on a 24 well plate and allowed to grow for 24 h. Then the cells were incubated for 3 h at +37°C in 300 μΐ media containing 5 pg/ml AlexaFluor488 labeled compounds. After incubation the cells were washed two times with PBS and detached by incubating with 100 μΐ Trypsin-EDTA for 10 min at +37°C. Cells were neutralized by adding 300 μΐ of media and resuspended in PBS and analyzed using a flow cytometer (FACS LRS II). The mean fluorescence intensity of each sample was calculated using FACS Diva software. The data presented in
Tables 1-3 is expressed as "Normalized mean fluorescence intensity" where the fluorescence intensity has been normalized to the degree of labeling for each compound.
Assays with FACS
Internalization of fluorescently labeled boron conjugates (900 boron atoms) and nonconjugated Ab fragments by human HNC cancer cell line HSC-2 was evaluated using FACS. The results represent internalized plus cell surface bound compounds that occurs when cells have been incubated at +37°C (Table 1). An-ti-EGFRl-Fab-BSH-Dex was internalized more efficiently than other boron conjugates or nonconjugated anti-EGFRl-Fab. Other anti-EGFRl boron conjugates (anti-EGFR1-F(ab')2-BSH-Dex and anti-EGFRl-mAb-BSH-Dex) were internalized equally well to nonconjugated anti-EGFRl-Fab and anti-EGFRl-F(ab')2 . Boron conjugates of con-trol-F(ab')2 and -mAb were internalized very weakly.
Table 1. Cell surface binding and internalization of fluorescently labeled boron conjugates and nonconjugated compounds by HSC-2 cells. Analysis has been carried out by FACS and fluorescence intensity has been normalized to the degree of labeling for each compound.
Boron conjugates with different amounts of boron (360-900 boron atoms) were synthesized from an-ti-EGFRl F(ab')2 and -Fab to study the effect of boron load in the internalization process. Example shows internalization assay with fluorescently labeled conjugates using human HNC cancer cell line HSC-2 and a control human cell line HER. The results from flow cytometric analysis represent internalized plus cell surface bound compounds that occurs when cells have been incubated at +37°C (Table 2). Internalization of all boron conjugates of anti-EGFRl Ab fragments was very similar as analyzed by flow cytometry. However, experiments with microscopy revealed that conjugates with more boron were internalized more efficiently than conjugates with low boron load (not shown).
Table 2. Cell surface binding and internalization of fluorescently labeled boron conjugates with different amounts of boron by HSC-2 and HER cells. Analysis has been carried out by flow cytometry and fluorescence intensity has been normalized to the degree of labeling for each compound.
Internalization of fluorescently labeled boron conjugates (1200 or 800 boron atoms) and nonconju-gated Ab fragments by human HNC cancer cell lines (HSC-2 and FaDu) and a control cell line HEK was evaluated using flow cytometry. The results represent internalized plus cell surface bound compounds that occurs when cells have been incubated at + 37°C (Table 3). Anti-EGFRl-Fab-BSH(1200B)-Dex and nonconjugated anti-EGFRl-Fab showed strongest internalization by HSC-2 and FaDu cells. Internalization by FaDu cells has been consistently weaker than by HSC-2 cells, likely due to the smaller amount of EGFR1 receptors at the cell surface. Control boron conjugates (control-Fab-BSH(800B)-Dex and control-F(ab')2 -BSH(800B)-Dex) and corresponding nonconjugated compounds were internalized very weakly. Control cell line HEK internalized the boron conjugates and nonconjugated compounds only very weakly.
Table 3. Cell surface binding and internalization of fluorescently labeled boron conjugates (1200B or 800B) and nonconjugated compounds by HSC-2, FaDu and HEK cells. Analysis has been carried out by flow cytometry and fluorescence intensity has been normalized to the degree of labeling for each compound.
HSC-2 FaDu HEK
Internalization assay with radiolabeled samples HSC-2, FaDu and HER cells (2xl05) were seeded on a 24 well plate and allowed to grow for 24 h. Then the cells were incubated for 3 h at +37°C in 300 μΐ media containing 5 pg/ml tritium labeled compounds. After incubation media was removed and cells were washed three times with PBS and lysed by adding 300 μΐ 1 M NaOH. The amount of radioactivity in media and cell lysates was measured with scintillation counter in the presence of scintillation fluid cocktail (Ultima Gold) . The amount of internalized compounds was calculated from the total amount of radioactivity per well and normalized to 100 000 cells.
Boron conjugates (800 boron atoms) of anti-EGFRl-Fab and -F(ab')2 as well as nonconjugated anti-EGFRl-mAb were labeled with tritium to the lysine residues of a protein part. Internalization assay with radiolabeled compounds was carried out using human HNC cancer cell lines, HSC-2 and FaDu, as well as a control cell line HER. The results represent internalized plus cell surface bound compounds that occur when cells have been incubated at +37°C. The results (Table 4) indicate that boron conjugates of anti-EGFRl-Fab and -F(ab')2 were internalized as efficiently as non-conjugated anti-EGFRl-mAb by HSC-2 and FaDu cells. Internalization by HSC-2 cells was 100 times stronger than by FaDu cells likely due to the higher amount of EGFR1 receptors at the cell surface in HSC-2 cells. Control cell line HER showed only very weak internalization .
Table 4. Internalization of radiolabeled boron conjugates by HSC-2, FaDu and HER cells. The amount of internalized compounds has been calculated from the total amount of radioactivity per well and normalized to 100 000 cells. The results are an average of three determinations +/- S.D.
EXAMPLE 8. In vivo experiments with tritium labeled conjugates
Preparation of mouse tissues and blood samples for liquid scintillation counting
Weighted mouse organs were dissolved to 1 ml of tissue solubilizer (Solvable™, Perkin Elmer) per 0.2 g tissue. Samples were incubated overnight at +60°C. Then 150 μΐ of H2O2 was added per 300 μΐ of dis solved organ and samples were incubated for one hour at +60°C. Bones were treated first with 1 M HCl overnight at +60°C and then with Solvable and H2O2. The amount of radioactivity in the organs was measured with scintillation counter in a presence of scintillation fluid cocktail (Ultima Gold™, Perkin Elmer). Data is presented as percent of total injected dose in g of tissue. The results are an average of three mice + /-SEM. Since each of the mice had two tumors, the results in tumors are an average of six determinations + /- SEM.
Blood samples in clearance tests were collected in Eppendorf tubes and the volumes were measured after adding 100 μΐ of Solvable and overnight incubation at +60°C. Then 100 μΐ of H2O2 was added and samples were incubated for one hour at +60°C. The amount of radioactivity in the blood samples was measured with scintillation counter in the presence of scintillation fluid cocktail (Ultima Gold, Perkin Elmer) . Data is presented as a percent of total injected dose. The results are an average of two mice.
Blood clearance of boron conjugates in non-tumor mice
Female adult mice of the same age (Harlan HSD:Athymic nude Foxnlnu) were used. Radiolabeled (3H) boron conjugates of anti-EGFRl-Fab and -F(ab')2 with 800B and 300B boron load were injected i.v. via tail vein in 100 μΐ PBS. Injected dose was 30 pg = 1.3-2 x 106 cpm per mouse and two mice per sample were used. Blood samples of approximately 10 μΐ were collected before and after injection at different time points and counted for radioactivity. At the end of the experiment (48 h) mice were sacrificed and organs were collected and counted for radioactivity for determination of tissue biodistribution of the conjugates.
Blood clearance study in non-tumor mice was carried out using 3H-labeled boron conjugates of anti- EGFRl-Fab and -F(ab')2 with 800B and 300B boron load. Two different boron loads were used to see whether the boron load has an effect on the clearance rate of the conjugate from blood circulation. The results indicate that blood clearance of boron conjugates was rapid and independent on the boron load (Table 5) . Clearance rate was comparable to the clearance of corresponding non-conjugated F(ab')2 and Fab fragments (not shown). Tissue distribution study indicated that the boron conjugates were not accumulated into any organs at 48 h (not shown).
Table 5. Blood clearance of boron conjugates in non-tumor mice. The results are an average of two determinations. Time is time after administration (min) and values % of total injected dose.
Biodistribution of boron conjugates in HSC-2 tumor mice
Female adult mice of the same age (Harlan HSD:Athymic nude Foxnlnu) were used. Two and half to three million HSC-2 cells (JCRP Cellbank, Japan) in 150 μΐ in EME-media and 50 % Matrigel were inoculated to both flanks of nude mice. The dosing was given when at least one tumor per mouse has grown to at least 6 mm diameter in size (6-10 mm) corresponding roughly to tumor volume of 100-500 mm3. Radiolabeled (3H) boron conjugates (800B) of anti-EGFRl-Fab/F(abf)2 and control-Fab/F (ab' ) 2 were injected i.v. via tail vein in 100 μΐ PBS. Injected dose was 50 pg = 1.3-2.6 x 106 cpm per mouse and three mice per sample were used. Mice were sacrificed at different time points (24 h, 48 h and 72 h) and organs were collected and counted for radioactivity for determination of tissue biodistribution of the conjugates.
Tissue distribution of boron conjugates (Table 6) show that boron conjugates of anti-EGFRl-Fab and -F(ab')2 accumulated into tumors but not in any other organs, whereas control boron conjugates did not significantly accumulate into tumors. Tumor accumulation of boron conjugates of anti-EGFRl-Fab and F(ab')2 was highest at 24 h and slowly decreased at later time points (48 h and 72 h).
Table 6. Biodistribution of boron conjugates in HSC-2 tumor mice. The results represent an average of three determinations + /- SEM except for tumors that are an average of six determinations + /- SEM. Values are % of total injected dose/g organ.
Tumor vs. blood distribution of boron conjugates in HSC-2 xenograft mice was calculated at different time points (24 h, 48 h and 72 h) (Table 7) . Tumor/blood ratio was 4-5 for anti-EGFRl-Fab conjugate and 2-6 for anti-EGFRl- F(ab')2 conjugate. Anti-EGFRl-Fab-BSH-Dex reached the maximum ratio earlier (24 h) than anti-EGFRl-F(ab')2-BSH-Dex (48 h). Tumor/blood ratio of control conjugates remained at a constant level throghout the study (approximately 1-2).
Table 7. Tumor/blood distribution of boron conjugates in HSC-2 tumor mice. The results are based on an average of three determinations for blood samples and an average of six determinations for tumors (2 tumors per mouse) +/- S.D.
Biodistribution of boron conjugates in FaDu tumor mice Female adult mice of the same age (Charles River CrlrAthymic nude Foxnlnu) were used. Three million FaDu cells (ATCC) in 150 μΐ in EME-media and 50 % Matrigel were inoculated to both flanks of nude mice. The dosing was given when at least one tumor per mouse has grown to at least 6 mm diameter in size (6-10 mm) corresponding roughly to tumor volume of 100-500 mm3. Radiolabeled (3H) boron conjugates (800B or 1200B) of anti-EGFRl-Fab/F(ab')2 and control-Fab/F(ab')2 were injected i.v. via tail vein in 100 μΐ PBS. Injected dose was 50 μg = 2.3-2.7 x 106 cpm per mouse and three mice per sample were used. Mice were sacrificed at two different time points (24 h and 48 h) and organs were collected and counted for radioactivity for determination of tissue biodistribution of the conjugates.
Biodistribution study in FaDu xenograft tumor mice was carried out using anti-EGFRl-F(ab')2-BSH(8 0 OB)-Dex and anti-EGFRl-Fab(800B or 1200B)-BSH-Dex and boron conjugates (800B) of control-F(ab')2 and -Fab. The conjugates were radiolabeled (3H) to lysine residues of a protein. Radioactivity in tissue samples, including tumors and blood, were counted at two different time points (24h and 48h) . Tissue distribution of boron conjugates (Table 8) show that boron conjugates of anti-EGFRl-Fab and -F(ab')2 accumulated into tumors but not significantly in any other organs, whereas control boron conjugates did not significantly accumulate into tumors. Control-F(ab')2-BSH(800B)-Dex can be still be found in blood circulation and in all organs at 24 h, but is cleared from circulation at 48 h. Tumor accumulation of boron conjugates of anti-EGFRl-Fab and -F(ab')2 was highest at 24 h and decreased at 48 h.
Table 8. Biodistribution of boron conjugates in FaDu tumor mice. The results represent an average of three determinations + /- SEM except for tumors that are an average of six determinations + /- SEM. values are % of total injected dose/g organ.
Tumor vs. blood distribution of boron conjugates in FaDu xenograft mice was calculated at 24 h and 48 h (Table 9). Tumor/blood ratio was approximately 7 for anti-EGFRl-Fab and -F(ab')2 conjugates with 1200 borons at 24 h, and the ratio decreased to 3-4 at 48 h suggesting that the labeled protein is degraded and is secreted out of the cells. Tumor/blood ratio of anti-EGFRl-Fab conjugate with 800 borons was approximately 4-5 at both time points. The ratio of control conjugates remained at a constant level (approximately 1-2) .
Table 9. Tumor /blood distribution of boron conjugates in FaDu tumor mice. The results are based on an average of three determinations for blood samples and an average of six determinations for tumors (2 tumors per mouse) +/- S.D.
EXAMPLE 9. Quantitation of boron in BSH-Dextran by inductively coupled plasma mass spectrometry (ICP-MS) (mol boron per mol BSH-Dextran)
The boron load of BSH-dextran was estimated from proton-NMR spectrum of BSH-dextran (Figure 1) and ICP-MS was used to quantitate the amount of boron in the samples. The BSH-Dextran sample analyzed in this example was estimated to contain about 1200 borons based on NMR analysis. Approximately 2.1 pg (0.0228 nmol) of BSH-Dextran (average MW 92 kDa) was liquefied with microwave-assisted wet ashing and analyzed by ICP-MS essentially as described in Laakso et al., 2001, Clinical Chemistry 47, 1796-1803. Different dilutions of the sample were analyzed by ICP-MS and the background boron was subtracted from the samples. The results representing an average of 7 determinations indicate that the sample contains approximately 0.341 pg (31.5 nmol) of boron atoms, or one mole of the BSH-Dextran contain 1381 moles of boron atoms.
EXAMPLE 10. In vivo experiments and boron quantitation
Female adult mice of the same age (Charles River Crl:Athymic nude Foxnlnu) were used. 2.3 million HSC-2 or 5 million FaDu cells in 150 μΐ in EME-media and 50 % Matrigel were inoculated to the right flank of nude mice. The dosing was given when the tumor was grown to at least 6 mm diameter in size (6 - 10 mm) corresponding roughly to tumor volume of 100-500 mm3. Anti-EGFR-Fab-BSH(1200)-dex or anti-EGFR-F(ab')2-BSH(1200)-dex (both non-labeled) conjugates were injected i.v. via tail vein in 100 μΐ PBS. Injected dose was 50 pg or 250 pg per mouse and three mice per sample were used. Mice were sacrificed at 24 h and 48 h and organs were collected for boron determination.
Tissue samples (including blood) were digested in closed teflon vessels in a microwave oven (Milestone, ETHOS 1200). The digestion temperature was 200 C and duration of the digestion was 50 min. Acid used in the digestions was HNO3 (6,0 ml, E. Merck, Supra-pur). After cooling the resultant solution was diluted to 25 ml with Milli-Q water. The digested samples were diluted further (1:10 or 1:50) with 1 % HNO3 for ICP-MS analysis. The internal standard beryllium was added to the sample to gain the final concentration, 10 ppb of Be, in the samples. Standard solutions with concentrations of 1, 5, 10 and 20 pg/L for analyses were diluted from Spectrascan's single element standard solution (1000 ug/ml boron as H3BO3 in H20). Control sample for analysis was prepared from multielemental standard solution by SPEX (CLMS-4). Analyses were performed with the high resolution sector field inductively coupled plasma mass spectrometer (HR-ICP-MS, Element2, Thermo Scientific). The concentration of boron in diluted samples was defined from the peaks of lOB and 11B with both low resolution (R % 300) and medium resolution (R » 4000) mode. Between the samples the samples introduction system was washed first with 5 % HNO3 and then with 1 % HN03 to exclude the memory effect typical for boron.
Initial boron analysis of two HSC-2 tumor mice at 24h indicated that boron tumor per muscle ratios were 5.3 and 6.3.
The muscle was used as a control tissue instead of blood because initial boron measurements from blood were inconclusive or beyond detection limit.
It is obvious to a person skilled in the art that with the advancement of technology, the basic idea of the invention may be implemented in various ways. The invention and its embodiments are thus not limited to the examples described above, instead they may vary within the scope of the claims.

Claims (21)

1. Konjugaatti, joka käsittää anti-EGFRl-vasta-aineen tai sen EGFRl:tä sitovan fragmentin ja vähintään yhden dekstraanijohdannaisen, jossa dekstraanijohdannainen käsittää vähintään yhden D-glukopyranosyyliyksikön, jossa vähintään yhden D-glukopyranosyyliyksikön vähintään yksi hiili, joka on valittu hiilestä 2, 3 tai 4, on substituoitu sub- stituentilla, jolla on kaava -0- (CHzJn-S-BizHu2- jossa n on 3 - 10/ ja dekstraanijohdannainen on sitoutunut anti-EGFRl-vasta-aineeseen tai sen EGFR1:tä sitovaan fragmenttiin sidoksella, joka on muodostettu dekstraani j ohdannaisen D-glukopyranosyyliyksikön oksidatii-visella lohkaisulla muodostetun vähintään yhden alde-hydiryhmän ja anti-EGFRl-vasta-aineen tai sen EGFRlrtä sitovan fragmentin aminoryhmän välisellä reaktiolla.A conjugate comprising an anti-EGFR1 antibody or an EGFR1 binding fragment thereof and at least one dextran derivative, wherein the dextran derivative comprises at least one D-glucopyranosyl unit, wherein the at least one D-glucopyranosyl unit is at least one carbon selected from carbon 2, 3 or 4, is substituted with a substituent of the formula -O- (CH2Jn-S-BizHu2- wherein n is 3 to 10) and the dextran derivative is bound to the anti-EGFR1 antibody or its EGFR1-binding fragment by a bond, formed by the reaction of at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of a dextran with an amino group of an anti-EGFR1 antibody or an EGFR1 binding fragment thereof. 2. Patenttivaatimuksen 1 mukainen konjugaatti, jossa dekstraanijohdannaisen molekyylimassa on noin 3 - noin 2000 kDa tai noin 30 - noin 300 kDa.The conjugate of claim 1, wherein the dextran derivative has a molecular weight of about 3 to about 2000 kDa or about 30 to about 300 kDa. 3. Patenttivaatimuksen 1 tai 2 mukainen konjugaatti, jossa konjugaatti käsittää noin 10 - noin 300 substituenttia tai noin 20 - noin 150 substituent-tia, joilla on kaava -0-(CH2) n-S-Bi2Hn2”.The conjugate of claim 1 or 2, wherein the conjugate comprises about 10 to about 300 substituents or about 20 to about 150 substituents of the formula -O- (CH 2) n -S-Bi 2 Hn 2 '. 4. Jonkin patenttivaatimuksista 1-3 mukainen konjugaatti, jossa anti-EGFRl-vasta-aineen tai sen EGFRl:tä sitovan fragmentin aminoryhmä on anti-EGFRl-vasta-aineen tai sen EGFRlrtä sitovan fragmentin ly-siinitähteen aminoryhmä.The conjugate of any one of claims 1-3, wherein the amino group of the anti-EGFR1 antibody or its EGFR1 binding fragment is the amino group of the lysine residue of the anti-EGFR1 antibody or its EGFR1 binding fragment. 5. Jonkin patenttivaatimuksista 1-4 mukainen konjugaatti, jossa konjugaatti käsittää edelleen dekstraanijohdannaiseen tai anti-EGFRl-vasta-aineeseen tai sen EGFRlrtä sitovaan fragmenttiin sitoutuneen vähintään yhden detektoitavissa olevan molekyylin.The conjugate of any one of claims 1-4, wherein the conjugate further comprises at least one detectable molecule bound to a dextran derivative or anti-EGFR1 antibody or an EGFR1 binding fragment thereof. 6. Jonkin patenttivaatimuksista 1-5 mukainen konjugaatti, jossa dekstraanijohdannainen käsittää vähintään yhden aldehydiryhmän, joka on muodostettu dekstraanijohdannaisen D-glukopyranosyyliyksikön oksi-datiivisella lohkaisulla ja joka on suojattu.The conjugate according to any one of claims 1 to 5, wherein the dextran derivative comprises at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative and protected. 7. Patenttivaatimuksen 6 mukainen konjugaatti, jossa dekstraanijohdannainen käsittää joukon alde-hydiryhmiä, jotka on muodostettu dekstraanijohdannaisen D-glukopyranosyyliyksikön oksidatiivisella lohkaisulla, ja olennaisesti kaikki aldehydiryhmät, jotka on muodostettu dekstraanijohdannaisen yhden tai useamman D-glukopyranosyyliyksikön oksidatiivisella lohkaisulla, ovat suojattuja.The conjugate of claim 6, wherein the dextran derivative comprises a plurality of aldehyde groups formed by oxidative cleavage of the D-glucopyranosyl moiety of the dextran derivative, and substantially all of the aldehyde groups formed by one or more D-glucohydroxy moieties of the dextran derivative. 8. Jonkin patenttivaatimuksista 1-7 mukainen konjugaatti, joka on saatavissa menetelmällä, joka käsittää vaiheet, joissa: a) alkenyloidaan dekstraanin vähintään yksi hydroksyyliryhmä alkenyloidun dekstraanin saamiseksi; b) annetaan natriumboorikaptaatin (BSH) reagoida vaiheesta a) saatavan alkenyloidun dekstraanin kanssa BSH-dekstraanin saamiseksi; c) lohkaistaan oksidatiivisesti BSH-dekstraanin vähintään yksi D-glukopyranosyylitähde, niin että muodostuu aldehydiryhmiä; d) annetaan vaiheesta c) saatavan oksidatiivisesti lohkaistun BSH-dekstraanin reagoida anti-EGFRl-vasta-aineen tai sen EGFRl:tä sitovan fragmentin kanssa konjugaatin saamiseksi.The conjugate of any one of claims 1 to 7 obtainable by a process comprising the steps of: a) alkenylating at least one hydroxyl group of dextran to obtain an alkenylated dextran; b) reacting sodium borate captate (BSH) with alkenylated dextran from step a) to obtain BSH dextran; c) oxidatively cleaving at least one D-glucopyranosyl residue of BSH-dextran to form aldehyde groups; d) reacting the oxidatively cleaved BSH dextran from step c) with an anti-EGFR1 antibody or an EGFR1 binding fragment thereof to obtain a conjugate. 9. Patenttivaatimuksen 8 mukainen konjugaatti, jossa dekstraani alkenyloidaan vaiheessa a) käyttäen alkenyloivaa ainetta, jossa alkenyloivalla aineella on seuraavan kaavan mukainen rakenne X- (CH2) mCH=CH2 jossa m on 1 - 8 ja X on Br, Cl tai I.The conjugate of claim 8, wherein the dextran is alkenylated in step a) using an alkenylating agent wherein the alkenylating agent has the structure X- (CH 2) m CH = CH 2 wherein m is 1-8 and X is Br, Cl or I. 10. Patenttivaatimuksen 8 tai 9 mukainen konjugaatti, jossa vaiheesta a) saatavan alkenyloidun dekstraanin vähintään yhden D-glukopyranosyyliyksikön vähintään yksi hiili, joka on valittu hiilestä 2, 3 tai 4,on substituoitu substituentilla, jolla on kaava -o- (CH2)mCH=CH2, jossa m on 1 - 8.The conjugate of claim 8 or 9, wherein at least one carbon of at least one D-glucopyranosyl unit of the alkenylated dextran from step a) selected from carbon 2, 3 or 4 is substituted with a substituent of the formula -O- (CH 2) mCH =. CH 2 where m is 1 to 8. 11. Jonkin patenttivaatimuksista 8-10 mukainen konjugaatti, jossa BSH:n annetaan reagoida vaiheesta a) saatavan alkenyloidun dekstraanin kanssa ra-dikaali-initiaattorin läsnä ollessa, jossa radikaali-initiaattori valitaan ryhmästä, joka koostuu ammonium-persulfaatista, kaliumpersulfaatista ja UV-valosta, vaiheessa b).The conjugate according to any one of claims 8 to 10, wherein the BSH is reacted with the alkenylated dextran from step a) in the presence of a radical initiator, wherein the radical initiator is selected from the group consisting of ammonium persulfate, potassium persulfate and UV light, in step b). 12. Jonkin patenttivaatimuksista 8-11 mukainen konjugaatti, jossa BSH-dekstraanin vähintään yksi D-glukopyranosyylitähde lohkaistaan oksidatiivi-sesti vaiheessa c) käyttäen hapettavaa ainetta, joka valitaan ryhmästä, joka koostuu natriumperjodaatista, perjodihaposta ja lyijy(IV)asetaatista.The conjugate of any one of claims 8 to 11, wherein at least one D-glucopyranosyl residue of BSH-dextran is oxidatively cleaved in step c) using an oxidizing agent selected from the group consisting of sodium periodate, periodic acid and lead (IV) acetate. 13. Jonkin patenttivaatimuksista 8-12 mukainen konjugaatti, jossa menetelmä käsittää edelleen vaiheen, jossa annetaan vaiheesta c) saatavan oksida-tiivisesti lohkaistun BSH-dekstraanin tai vaiheesta d) saatavan konjugaatin reagoida detektoitavissa olevan molekyylin kanssa.The conjugate of any one of claims 8 to 12, wherein the method further comprises the step of reacting the oxidatively cleaved BSH dextran from step c) or the conjugate from step d) with a detectable molecule. 14. Jonkin patenttivaatimuksista 8-13 mukainen konjugaatti, jossa menetelmä käsittää edelleen vaiheen e), jossa suojataan vaiheesta c) saatavan ok-sidatiivisesti lohkaistun BSH-dekstraanin tai vaiheesta d) saatavan konjugaatin reagoimattomat aldehydiryh-mät.The conjugate according to any one of claims 8 to 13, wherein the process further comprises the step of e) protecting the unreacted aldehyde groups of the oxidatively cleaved BSH dextran from step c) or the conjugate of step d). 15. Patenttivaatimuksen 14 mukainen konjugaatti, jossa reagoimattomat aldehydiryhmät suojataan käyttäen hydrofiilistä suojaavaa ainetta, kuten etano-liamiini, lysiini, glysiini tai tris.The conjugate of claim 14, wherein the unreacted aldehyde groups are protected using a hydrophilic protecting agent such as ethanolamine, lysine, glycine or tris. 16. Jonkin patenttivaatimuksista 8-15 mukainen konjugaatti, jossa dekstraanin molekyylimassa on noin 3 - noin 2000 kDa tai noin 10 - noin 100 kDa tai noin 5 - noin 200 kDa tai noin 10 - noin 250 kDa.The conjugate according to any one of claims 8 to 15, wherein the dextran has a molecular weight of about 3 to about 2000 kDa or about 10 to about 100 kDa or about 5 to about 200 kDa or about 10 to about 250 kDa. 17. Jonkin patenttivaatimuksista 8-16 mukainen konjugaatti, jossa oksidatiivisesti lohkaistun BSH-dekstraanin annetaan reagoida anti-EGFRl-vasta-aineen tai sen EGFRlrtä sitovan fragmentin kanssa in-kuboimalla oksidatiivisesti lohkaistua BSH-dekstraania ja anti-EGFRl-vasta-ainetta tai sen EGFRlrtä sitovaa fragmenttia huoneenlämpötilassa vesipitoisessa fosfaattipuskurissa, jonka pH on noin 6-8, vaiheessa d) .The conjugate of any one of claims 8 to 16, wherein the oxidatively cleaved BSH-dextran is reacted with an anti-EGFR1 antibody or an EGFR1-binding fragment thereof by incubating an oxidatively cleaved BSH-dextran and an anti-EGFR1 antibody or its EGFR1. binding fragment at room temperature in aqueous phosphate buffer at pH 6-8 in step d). 18. Farmaseuttinen koostumus, joka käsittää jonkin patenttivaatimuksista 1-17 mukaisen konjugaa-tin.A pharmaceutical composition comprising the conjugate of any one of claims 1-17. 19. Jonkin patenttivaatimuksista 1-17 mukainen konjugaatti tai patenttivaatimuksen 18 mukainen farmaseuttinen koostumus käytettäväksi lääkkeenä.The conjugate of any one of claims 1-17 or the pharmaceutical composition of claim 18 for use as a medicament. 20. Jonkin patenttivaatimuksista 1-17 mukainen konjugaatti tai patenttivaatimuksen 18 mukainen farmaseuttinen koostumus käytettäväksi syövän hoidossa .The conjugate of any one of claims 1-17 or the pharmaceutical composition of claim 18 for use in the treatment of cancer. 21. Konjugaatti tai farmaseuttinen koostumus käytettäväksi patenttivaatimuksen 20 mukaisesti, jossa syöpä on pään ja kaulan alueen syöpä.The conjugate or pharmaceutical composition for use according to claim 20, wherein the cancer is head and neck cancer.
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