EA045310B1 - MONOCLONAL ANTIBODIES TO IGF-1R AND THEIR APPLICATION - Google Patents

Description

Related applications

The present invention claims priority and benefit from U.S. Provisional Application No. 62/502,288 entitled Monoclonal Antibodies to IGF-1R and Use Thereof, filed May 5, 2017, and Provisional U.S. Patent Application No. 62/545,945 entitled Monoclonal Antibodies to IGF-1Rs and their uses, filed August 15, 2017. Both of the above applications are hereby incorporated by reference in their entirety for all purposes.

State of the art

Insulin-like growth factor receptor-1 (IGF-1R) is considered as a potential therapeutic target in cancer treatment. The present invention provides an alternative method for effectively utilizing IGF-1R by delivering therapeutic radioisotopes that provide improved antitumor efficacy at substantially lower doses than the antibody alone.

Brief description of the invention

The present invention relates to monoclonal antibodies targeting the insulin-like growth factor-1 receptor and radioimmunoconjugates thereof, which exhibit improved activity and enhance excretion of a chelating moiety or a metal complex thereof when conjugated to a therapeutic moiety, targeting moiety or cross-linking group.

Accordingly, in a first aspect the invention relates to a compound having the structure: A-L'-(L ² )nB

Formula I, where A is a chelating moiety or a metal complex thereof;

L ¹ represents optionally substituted C1-C6 alkyl, substituted C1-C6 heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety targeting moiety or cross-linking group, or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L ² independently has the structure:

(-X ¹ -L ³ -Z ¹ -)

Formula II, where X ¹ represents C=O(NR1), C=S(NR1), OC=O(NR1), NR1C=O(O), NR1C=O(NR1), CH2PhC=O(NR1), -CH2Ph(NH)C=S(NR1), O, NR ¹ , and R1 is H or optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl, substituted aryl or heteroaryl; L ³ represents an optionally substituted C1-C50 alkyl or an optionally substituted C1-C50 heteroalkyl, or a C5-C20 polyethylene glycol; Z ¹ is CH2, C=O, C=S, OC=O, NR1C=O, NR ¹ , and R ¹ is hydrogen or optionally substituted C1-C6 alkyl, pyrrolidine-2,5-dione.

In some embodiments, the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-a,a',a, a'tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)1,4,7 ,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2-(10-(2,6dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid), DOTMP (1,4,6,10tetraazacyclododecane-1,4 ,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11tetraazabicyclo[6.6 .2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETRA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NENA (1,4,7,10,13,16hexaazacyclohexadecane-1,4,7,10,13,16-hexacetic acid), PEPA (1,4,7,10,13pentaazacyclopentadecane-N,N',N,N' ,N-pentaacetic acid), H ₄ Octapa (N,N'-bis(6-carboxy-2pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H ₂ Dedpa (1,2-[[6-(carboxy )-pyridin-2-yl]methylamino]ethane), H ₆ phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl1,2-diaminoethane) , TTHA (triethylenetetramine-N,N,N',N,N',N'-hexacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepenta acetic acid ), DTPA-BMA (diethylenetriaminepentaacetic acid bismethylamide), HOPO (hydroxypyridinone octadentate) or porphyrin.

One skilled in the art will understand that the use of chelating moieties in

- 1045310 The implementation of the invention is not limited to the specific constructs disclosed herein, but rather may include other known chelating moieties.

In some embodiments, the chelating moiety has the structure:

where Y ¹ represents -CH ₂ OCH ₂ (L ² )nB, C=O(L2)nB or C=S(L2)nB, and Y ² represents -CH2CO2H;

where Y ¹ represents H, Y ² represents L ¹ -(L2)nB. In some embodiments, L ¹ has the structure:

Formula III, where R ² represents optionally substituted hydrogen or -CO2H.

In some embodiments, the metal may be selected from Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, Sm, lanthanide, or actinide, for imaging or therapeutic applications. agents. Specific examples of radionuclides suitable for complexation with a compound of formula (I) include ⁴⁷ Sc, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸² Rb, ⁸⁶ Y, ⁸⁷ Y, ⁹⁰ Y, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ^117m Sn, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁹ Au, ²⁰¹ TI, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th.

In some embodiments, B is a therapeutic moiety or targeting moiety.

In some embodiments, the therapeutic fragment or targeting fragment is an antibody, an antigen binding fragment thereof, or other targeting protein such as nanobodies, affibodies, and fibronectin type III domain consensus sequences.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds the insulin-like growth factor-1 receptor (IGF-1R), such as figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. In some embodiments, the antibody or antigen binding fragment thereof is AVE1642.

In some embodiments, the antibody or antibody-binding fragment thereof includes a light chain variable domain including at least one, two, or all three complementarity determining regions (CDRs) selected from:

(a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1;

(b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the antibody or antibody-binding fragment thereof includes a heavy chain variable domain including at least one, two, or all three CDRs selected from:

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5;

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7.

In certain embodiments, the antibody or antibody-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain comprising at least one, two, three, four, five, or all six CDRs selected from:

(a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1;

(b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2;

(c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3;

(d) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5;

(e) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6; and (f) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7.

In other embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the cross-linking group is an amino-reactive cross-linking group, a methionine-reactive cross-linking group, a thiol-reactive cross-linking group

- 2 045310 or sortase-mediated binding sequence.

In some embodiments, the amino-reactive, methionine-reactive, or thiol-reactive cross-linking group comprises an activated ester, such as a hydroxysuccinimide ester, N-hydroxysulfosuccinimide, a 2,3,5,6-tetrafluorophenol ester, a 4-nitrophenol ester, or an imidate, anhydride, thiol, disulfide , maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate or oxaziridine.

In some embodiments, the sortase recognition sequence may consist of a terminal amino acid sequence of glycine-glycine-glycine (GGG) and/or LPTXG, where X is any amino acid.

One skilled in the art will appreciate that the use of crosslinking groups in the practice of the invention is not limited to the specific designs disclosed herein, but may instead include other known crosslinking groups.

In some embodiments, the crosslinking group is selected from the group consisting of:

In some embodiments, Y ¹ is H. In some embodiments, X ¹ is C=O(NR1), and R ¹ is H.

In some embodiments, Z ¹ is -CH ₂ .

In some embodiments, L ² has a value of n equal to 1.

In some embodiments, the compound is selected from the group consisting of:

or

In some embodiments, the metal is a radionuclide. In some embodiments, the radionuclide is ¹¹¹ In. In some embodiments, the radionuclide is ⁶⁸ Ga. In some embodiments, the radionuclide is ⁸⁶ Y. In some embodiments, the metal is a beta-emitting radionuclide.

In some embodiments, the radionuclides are ⁶⁷ Cu, ¹⁷⁷ Lu, or ⁹⁰ Y.

In some embodiments, the metal is a radionuclide that emits alpha radiation.

In some embodiments, the radionuclide is ²²⁵ Ac, ²¹² Pb, ²²⁷ Th, or their decay products (daughter isotopes).

Another aspect of the invention relates to a pharmaceutical composition comprising any of the above compounds and a pharmaceutically acceptable excipient.

Another aspect of the invention relates to a method of planning radiation therapy and/or radiation therapy, comprising administering to a subject in need thereof any of the foregoing compounds or pharmaceutical compositions.

Another aspect of the invention relates to a method of detecting and/or treating cancer, comprising administering to a subject in need thereof a first dose of any of the foregoing compounds or pharmaceutical compositions in an amount effective for radiation therapy planning, followed by administration of additional doses of any of the foregoing compounds or pharmaceutical compositions in a therapeutically effective amount.

In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose or subsequent doses are the same.

In some embodiments, the compound or composition administered in the first dose, and with

- 3 045310 the unit or composition administered in the second dose, or subsequent doses, is different.

In some embodiments, the cancer is a solid tumor or hematologic cancer (hemoblastosis).

In some embodiments, the solid tumor is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing's sarcoma, multiple myeloma, or acute myeloid leukemia.

In some embodiments, the above methods further comprise administering an antiproliferative agent, a radiosensitizer, or an immunoregulatory or immunomodulatory agent.

In some embodiments, any of the foregoing compounds or compositions containing them, and the antiproliferative agent or radiosensitizer are administered at 28 day intervals (e.g., at 14, 7, 6, 5, 4, 3, 2, or 1 day(s) interval) .

In some embodiments, any of the above-described compounds or compositions containing them, and the immunoregulatory or immunomodulatory agent are administered at an interval of 90 days (e.g., at an interval of 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 day(s)).

Another aspect of the invention relates to a method for producing a radioconjugate (eg, any of the radioconjugates described herein). The method includes the steps of (a) conjugating a bifunctional chelate with a biological molecule, (b) purifying the conjugate obtained in step (a), and (c) chelating one or more radionuclides (for example, one or more Ac-225 radionuclides) with the purified conjugate with step (b) at a temperature less than 35°C (eg, 20-25°C) to obtain a radioconjugate (eg, an actinium radioconjugate).

In some embodiments, the radioconjugate is a radioimmunoconjugate (eg, any of the radioimmunoconjugates described herein).

In some embodiments, the pH of the reaction mixture in conjugation step (a) is less than 6.4 (eg, 6.3, 6.2, 6.1, 6.0, 5.9, or 5.8, or less).

In some embodiments, the pH of the reaction mixture in conjugation step (c) is less than 5.5 (e.g., 5.4, 5.3, 5.2, 5.1, or 5.0, or less) or greater than 7.0 ( e.g. 7.1, 7.2, 7.3, 7.4, 7.5 or more).

In some embodiments, the temperature of the reaction mixture in conjugation step (c) is 20-34° C. (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34° WITH).

Chemical terms.

The term acyl as used herein refers to a hydrogen or alkyl group (e.g., a haloalkyl group) as defined herein, which is attached to the parent molecular group via a carbonyl group as defined herein, e.g., formyl (i.e., carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl and the like. Exemplary unsubstituted acyl groups include 1 to 7, 1 to 11, or 1 to 21 carbon atoms. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents, as described herein.

The term alkyl as used herein includes saturated groups, both straight and branched, containing from 1 to 20 carbon atoms (eg, 1 to 10 or 1 to 6), unless otherwise noted. Examples of alkyl groups are methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, neopentyl and the like, and they may be optionally substituted by one, two, three or, in the case of alkyl groups, two or more carbon atoms, four substituents independently selected from the group consisting of: (1) C1.6 alkoxy; (2) C1.6 alkylsulfinyl; (3) an amino as defined herein (e.g., a substituted amino group (i.e., -NH2) or an unsubstituted amino group (i.e., -N(R ⁿ¹ )2, wherein R ^N1 is as defined for an amino group ); (4) C6_ ₁₀ aryl-C1_ ₆ alkoxy; (5) azido; (6) halo; (7) (C ₂ - ₉ heterocyclyl)oxy; (8) hydroxy, optionally substituted with an O-protecting group; (9) nitro; (10) oxo (for example, carboxyaldehyde or acyl); (11) C _1-7 spirocyclyl; (12) thioalkoxy; (13) thiol; (14) -CO2RA, optionally substituted with an O-protecting group, where R ^a is selected from the group consisting of (a) C1_20 alkyl (for example, C1-6 alkyl), (b) C2-20 alkenyl (for example, C2_6 alkenyl), (c) C6_10 aryl, (d) hydrogen, (e) C1.6 alkyl-C6_10 aryl, (f) amino-C1_20 alkyl, (g) polyethylene glycol of the formula -(CH2)s2(OCH2CH2)s1(CH2)s3OR', where s1 is an integer from 1 to 10 (for example, from 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C1_20 alkyl, and (h) an aminopolyethylene glycol of the formula -NR ^N1 (CH2) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 , where s1 is an integer from 1 to 10 ( e.g., 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or from 1 to 10), and each R ⁿ¹ independently represents hydrogen or optionally substituted C1-6 alkyl; (15) -C(O)NR ^B R ^C wherein each of R ^B and R ^C is independently selected from the _group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) _C6-10 aryl, and (d) C1_ ₆ alkyl-C ₆ - ₁₀ aryl; (16) -SO2RD, where RD is selected from the group consisting of

- 4 045310 (a) C _1-6 alkyl, (b) C ₆ - ₁₀ aryl, (c) C _1-6 alkyl-Cb-1o aryl, and (d) hydroxy; (17) -SO2NR ^E R ^F wherein R ^e and R ^f are each independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl, and (d) C1- 6 alkyl-C6-10 aryl; (18) -C(O)R ^G , where ^RG is selected from the group consisting of (a) C1-20 alkyl (for example, C _1-6 alkyl), (b) C _2-20 alkenyl (for example, C _{2 -6} alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 alkyl-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g) polyethylene glycol formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' represents H or C _1-20 alkyl , and (h) an aminopolyethylene glycol of the formula - NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represent an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl; (19) -NR ^H C(O)R', where R ^H is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and R ¹ is selected from the group consisting of (a2) C1-20 alkyl (for example, C _1-6 alkyl), (b2) C _2-20 alkenyl (for example, C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 alkyl-C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2) polyethylene glycol of the formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer a number from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4 , from 1 to 6 or from 1 to 10), and R' represents H or C _1-20 alkyl, and (h2) aminopolyethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 represents an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl; (20) -NR ^J C(O)OR ^K , where RJ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and R ^K is selected from the group consisting of (a2) C1-20 alkyl (for example, C _1-6 alkyl), (b2) C _2-20 alkenyl (for example, C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 alkyl -C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2) polyethylene glycol of the formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, from 1 to 6 or from 1 to 10), and R' represents H or C _1-20 alkyl, and (h2) aminopolyethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 represents is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl; and (21) amidine. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C ₁ -alkylaryl may be further substituted with an oxo group to produce the corresponding aryloyl substituent.

The term alkylene and the prefix alk- as used herein refer to a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, for example, methylene, ethylene, isopropylene and the like. The term C _x-y alkylene and the prefix C _x-y alkyl- refer to alkylene groups having from x to y carbon atoms. Exemplary x values are 1, 2, 3, 4, 5, and 6, and exemplary y values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (for example, C _1-6 , C _1-10 , C _2-20 , C _2-6 , C _2-10 or C _2-20 alkylene). In some embodiments, the alkylene may be further substituted with 1, 2, 3, or 4 substituent groups, as defined herein for an alkyl group.

The term alkenyl as used herein refers to straight or branched chain monovalent groups containing, unless otherwise indicated, 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms) containing one or more carbon -carbon double bonds, examples of which are ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like. Alkenyls include both cis and trans isomers. Alkenyl groups may be optionally substituted with 1, 2, 3, or 4 substituents that are independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl) as defined herein, or any of the exemplary alkyl substituents as described in this document.

The term alkynyl as used herein refers to straight or branched chain monovalent groups of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbon atoms) containing a carbon-carbon triple bond , examples of which are ethynyl, 1-propynyl and the like. Alkynyl groups may be optionally substituted with 1, 2, 3 or 4 substituent groups independently selected from aryl, cycloalkyl or heterocyclyl (eg, heteroaryl) as defined herein, or any of the exemplary alkyl substituents described herein.

The term amino as used herein refers to -N(R ⁿ¹ )2, where each R ^N1 is independently H, OH, NO2, N(R ⁿ² )2, SO ₂ OR ⁿ² , SO ₂ R ⁿ² , SOR ⁿ² , N -protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylaryl, cycloalkyl, alkylcycloalkyl, carboxyalkyl (for example, optional

- 5 045310 optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl (for example, acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (for example, optionally substituted with O -protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), heterocyclyl (eg, heteroaryl) or alkylheterocyclyl (eg, alkylheteroaryl), wherein each of these R ^N1 groups may be optionally substituted as defined herein for each group; or two R ⁿ¹ combine to form a heterocyclyl or N-protecting group, and wherein each R ^N2 is independently H, alkyl or aryl. The amino groups according to the invention may be an unsubstituted amino group (ie -NH2) or a substituted amino group (ie -N(R ⁿ¹ )2). In a preferred embodiment, amino is -NH2 or -NHR ^N1 , wherein R ^N1 is independently OH, NO2, NH2, NR ⁿ² 2, SO ₂ OR ⁿ² , SO2R ⁿ² , SOR ⁿ² , alkyl, carboxyalkyl, sulfoalkyl, acyl (eg , acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (eg, tert-butoxycarbonylalkyl) or aryl, and each R ^N2 may be H, C1-20 alkyl (eg, _C1-6 alkyl) or _C6-10 aryl.

The term amino acid as used herein refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group -CO ₂ H or a sulfo group SO ₃ H), where the amino acid is attached to the parent molecular group via a side chain, an amino group, or an acid group (for example, side chain). In some embodiments, the amino acid is attached to the parent molecular group via a carbonyl group, wherein the side chain or amino group is attached to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkylaryl, alkylheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan , tyrosine and valine. Amino acid groups may be optionally substituted with one, two, three or, in the case of amino acid groups of two or more carbon atoms, four substituents independently selected from the group consisting of: (1) C _1-6 alkoxy; (2) C _1-6 alkylsulfinyl; (3) an amino as defined herein (e.g., an unsubstituted amino group (i.e., -NH ₂ ) or a substituted amino group (i.e., -N(R ^N1 )2, wherein R ^N1 is as defined for amino groups); (4) C6-10 aryl-C _1-6 alkoxy; (5) azido; (6) halogen; (7) (C ₂ -etherocyclyl)oxy; (8) hydroxy; (9) nitro; (10 ) oxo (for example), carboxyaldehyde or acyl); (11) C _1-7 spirocyclyl; (12) thioalkoxy; (13) thiol; (14) -CO ₂ R ^A , where R ^A is selected from the group consisting of (a) C _1-20 alkyl (for example, C _1-6 alkyl), (b) C _2-20 alkenyl (for example, C _{2- 6} alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 alkyl-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g) polyethylene glycol of the formula ( CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 represents an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents is an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C _1-20 alkyl, and (h) an aminopolyethylene glycol of the formula -NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 is independently hydrogen or optionally substituted C1-6 alkyl; (15) -C(O)NR ^B R ^C wherein each of R ^B and R ^C is independently selected from the group consisting of (a) hydrogen, (b) C _1-6 alkyl, (c) C _6-10 aryl , and (d) C1 ₆ alkyl-C _6-10 aryl; (16) -SO ₂ R ^D where R ^D is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) C1-6 alkyl-C6-10 aryl and (d) hydroxy; (17) -SO2NR ^E R ^F , wherein each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) _C6-10 aryl, and (d) C _1-6 alkyl-C _6-10 aryl; (18) C(O)R ^g , where R ^g is selected from the group consisting of (a) C _1-20 alkyl (for example, C _1-6 alkyl), (b) C _2-20 alkenyl (for example, C _{2 -6} alkenyl), (c) C _6-10 aryl, (d) hydrogen, (e) C _1-6 alkyl-C _6-10 aryl, (f) amino-C _1-20 alkyl, (g) polyethylene glycol formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' represents H or C _1-20 alkyl , and (h) an aminopolyethylene glycol of the formula NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 is independently hydrogen or optionally substituted C1-6 alkyl; (19) -NR ^H C(O)R', where R ^H is selected from the group consisting of (a1) hydrogen and (b1) C _1-6 alkyl, and R ¹ is selected from the group consisting of (a2) C _{1 -20} alkyl (for example, C _1-6 alkyl), (b2) C _2-20 alkenyl (for example, C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _{1 -6} alkyl-C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2) polyethylene glycol of the formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 represents is an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6 or from 1 to 10), and R' represents H or C _1-20 alkyl, and (h2) aminopolyethylene glycol of the formula - 6 045310

NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 where si is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 are each independently an integer 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl; (20) -NR ^J C(O)OR ^K , where RJ is selected from the group consisting of (a1) hydrogen and (b1) C1-6 alkyl, and R ^K is selected from the group consisting of (a2) C1-20 alkyl (for example, C _1-6 alkyl), (b2) C _2-20 alkenyl (for example, C _2-6 alkenyl), (c2) C _6-10 aryl, (d2) hydrogen, (e2) C _1-6 alkyl -C _6-10 aryl, (f2) amino-C _1-20 alkyl, (g2) polyethylene glycol of the formula -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 OR', where s1 is an integer 1 to 10 (for example, 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, from 1 to 6 or from 1 to 10), and R' represents H or C _1-20 alkyl, and (h2) aminopolyethylene glycol of the formula NR ^N1 (CH2)s2(CH2CH2O)s1(CH2)s3NR ^N1 , where s1 represents an integer from 1 to 10 (for example, from 1 to 6 or from 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, from 0 to 4, from 0 to 6, from 1 to 4, 1 to 6 or 1 to 10), and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl; and (21) amidine. In some embodiments, each of these groups may be further substituted as described herein.

The term aryl as used herein refers to a mono-, bicyclic or polycyclic carbocyclic ring system having one or two aromatic rings, examples of which are phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl and the like, and which may be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of: (1) C _1-7 acyl (eg, carboxyaldehyde); (2) C _1-20 alkyl (for example, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, C _1-6 alkylsulfinyl-C _1-6 alkyl, amino-C _1-6 alkyl, azido -C _1-6 alkyl, (carboxyaldehyde)-C _1-6 alkyl, halogen C _1-6 alkyl (e.g. perfluoroalkyl), hydroxy-C _1-6 alkyl, nitro-C _1-6 alkyl or C _1-6 thioalkoxy- C _1-6 alkyl); (3) C _1-20 alkoxy (for example, C _1-6 alkoxy such as perfluoroalkoxy); (4) C _1-6 alkylsulfinyl; (5) C _6-10 aryl; (6) amino; (7) C _1-6 alkyl-C _6-10 aryl; (8) azido; (9) C _3-8 cycloalkyl; (10) C _1-6 alkyl-C _3-8 cycloalkyl; (11) halogen; (12) C _1-12 heterocyclyl (eg C _1-12 heteroaryl); (13) (C _1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C _1-20 thioalkoxy (for example, C _1-6 thioalkoxy); (17) (CH ₂ ) _q CO ₂ R ^A where q is an integer from zero to four and R ^A is selected from the group consisting of (a) C1-6 alkyl, (b) C6-10 aryl, (c) hydrogen, and (d) C1-6 alkyl-C6-10 aryl; (18) -(CH2)qCONR R where q is an integer from zero to four, and where RB and RC are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 -10 aryl, and (d) C1-6alkyl-C6-10 aryl; (19) -(CH2)qSO2R ^D ', where q is an integer from zero to four, and where R ^D is selected from the group consisting of (a) alkyl, (b) C6-10 aryl, and (c) alkyl- From _6-10 aryla; (20) -(CH ₂ ) _q SO ₂ NR ^E R ^F where q is an integer from zero to four, and where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b ) C _1-6 alkyl, (c) C _6-10 aryl and (d) C _1-6 alkyl-C _6-10 aryl; (21) thiol; (22) C _6-10 aryloxy; (23) C _3-8 cycloalkoxy; (24) C _6-10 aryl-C _1-6 alkoxy; (25) C _1-6 alkyl-C _1-12 heterocyclyl (for example, C _1-6 alkyl-C _1-12 heteroaryl); (26) C _2-20 alkenyl; and (27) C _2-20 alkynyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C1-alkylaryl or C1-alkylheterocyclyl group may be further substituted with an oxo group to provide the corresponding aryloyl and (heterocyclyl)oyl substituent group.

The term arylalkyl as used herein refers to an aryl group, as defined herein, attached to the parent molecular group via an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups contain 7 to 30 carbon atoms (e.g., 7 to 16 or 7 to 20 carbon atoms, such as C _1-6 alkyl-C _6-10 aryl, C _1-10 alkyl-C _6-10 aryl or C _1-20 alkyl-C _6-10 aryl). In some embodiments, alkylene and aryl may each be further substituted with 1, 2, 3, or 4 substituent groups, as defined herein for the respective groups. Other groups preceded by the prefix alkyl- are defined in the same way, where alkyl refers to C _1-6 alkylene unless otherwise noted and the attached chemical structure is as defined herein.

The term carbonyl as used herein refers to the group C(O), which may also be represented as C=O.

The term carboxy as used herein means CO ₂ H.

The term cyano as used herein refers to the -CN group.

The term cycloalkyl as used herein refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of three to eight carbon atoms unless otherwise indicated, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclic heptyl and the like. When the cycloalkyl group includes one carbon-carbon double bond or one carbon-carbon triple bond, the cycloalkyl group may be referred to as a cycloalkenyl group or a cycloalkynyl group, respectively. Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl, cyclohexenyl, cyclohexynyl and the like. The cycloalkyl groups according to the present invention may be

- 7 045310 optionally substituted: (1) With _1-7 acyl (for example, carboxyaldehyde); (2) With _1-20 alkyl (for example, Ci.6 alkyl, Ci.6 alkoxy-C1-b alkyl, C _1-6 alkylsulfinyl-C1-b alkyl, amino-C1-b alkyl, azido- _C16 alkyl, (carboxyaldehyde)-C _1-6 alkyl, halogen-C _1-6 alkyl (e.g. perfluoroalkyl), hydroxy-C _1-6 alkyl, nitro-C _1-6 alkyl or C _1-6 thioalkoxy-C _1-6 alkyl ); (3) C _1-20 alkoxy (eg C _1-6 alkoxy, eg perfluoroalkoxy); (4) C _1-6 alkylsulfinyl; (5) With _6-10 aryl; (6) amino; (7) C _1-6 alkyl-C _6-10 aryl; (8) azido; (9) With _3-8 cycloalkyl; (10) C _1-6 alkyl-C _3-8 cycloalkyl; (11) halogen; (12) C _1-12 heterocyclyl (eg C _1-12 heteroaryl); (13) (C _1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C _1-20 thioalkoxy (for example, C _1-6 thioalkoxy); (17) -(CH ₂ ) _q CO ₂ R ^A where q is A' an integer from zero to four, and R is selected from the group consisting of (a) C _1-6 alkyl, (b) C _{6- 10} aryl, (c) hydrogen and (d) C _1-6 alkyl-C _6-10 aryl; (18) -(CH ₂ ) _q CONR ^B RC where q is an integer from B'C' zero to four and where R and R are independently selected from the group consisting of (a) hydrogen, (b) C ₆ . ₁₀ alkyl, (c) C _6-10 aryl and (d) C _1-6 alkyl-C _6-10 aryl; (19)-(CH ₂ ) _q SO ₂ R ^D where q is an integer from D' zero to four and where R is selected from the group consisting of (a) C _6-10 alkyl, (b) C _{6- 10} aryl and (c) C _1-6 alkylC _6-10 aryl; (20) -(CH ₂ ) _q SO ₂ NR ^E R ^F where q is an integer from zero to four, and where each of R ^e and R ^f is independently selected from the group consisting of (a) hydrogen, (b ) C _6-10 alkyl, (c) C _6-10 aryl and (d) C _1-6 alkyl-C _6-10 aryl; (21) thiol; (22) C _6-10 aryloxy; (23) C _3-8 cycloalkoxy; (24) C _6-10 aryl-C _1-6 alkoxy; (25) C _1-6 alkyl-C _1-12 heterocyclyl (for example, C _1-6 alkyl-C _1-12 heteroaryl); (26) oxo; (27) C _2-20 alkenyl; and (28) C _2-20 alkynyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C ₁ alkylaryl or C ₁ -alkylheterocyclyl group may be further substituted with an oxo group to provide the corresponding aryloyl and (heterocyclyl)oyl substituent group.

The term diastereomer as used herein means stereoisomers that are not mirror images of each other and do not coincide when superimposed on each other.

The term enantiomer as used herein means each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and no more than 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

The term halogen as used herein refers to a halogen selected from bromine, chlorine, iodine or fluorine.

The term heteroalkyl as used herein refers to an alkyl group, as defined herein, in which each of one or two constituent carbon atoms is replaced by nitrogen, oxygen or sulfur. In some embodiments, the heteroalkyl group may be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. The term heteroalkyl as used herein refers to an alkyl group, as defined herein, in which each of one or two constituent carbon atoms is replaced by nitrogen, oxygen or sulfur. In some embodiments, the heteroalkenyl and heteroalkynyl groups may be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups.

The term heteroaryl as used herein refers to that subgroup of heterocyclyls, as defined herein, that are aromatic: that is, they contain 4n+2 pi electrons in a mono- or polycyclic ring system. Exemplary unsubstituted heteroaryl groups have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbon atoms. In some embodiments, the heteroaryl is substituted with 1,2,3, or 4 substituent groups, as defined for a heterocyclyl group.

The term heteroarylalkyl refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted heteroarylalkyl groups contain 2 to 32 carbon atoms (e.g., 2 to 22, 2 to 18, 2 to 17, 2 to 16, 3 to 15, 2 to 14, 2 to 13, or 2 to 12 carbon atoms, such as C _1-6 alkyl-C _1-12 heteroaryl, C _1-10 alkyl-C _1-12 heteroaryl or C _1-20 alkyl-C _1-12 heteroaryl). In some embodiments, alkylene and heteroaryl may each be further substituted with 1, 2, 3, or 4 substituent groups, as defined herein for the corresponding group. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

The term heterocyclyl as used herein refers to a 5-, 6-, or 7-membered ring, unless otherwise indicated, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. A 5-membered ring has zero to two double bonds, while a 6- and 7-membered ring has zero to three double bonds. Exemplary unsubstituted heterocyclyl groups have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbon atoms. The term heterocyclyl also refers to a heterocyclic compound having a bridged polycyclic structure in which one or more carbon atoms and/or heteroatoms connect two non-adjacent monocyclic ring members, e.g.

- 8 045310 nuclidinyl group. The term heterocyclyl includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two or three carbocyclic rings, for example, an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other monocyclic heterocyclic ring. , such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples of fused heterocyclyls include tropanes and 1,2,3,5,8,8a-hexahydroindolysine. Heterocylic compounds include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morph olinyl, thiomorpholinyl, thiazolyl, thiazolidinyl , isothiazolyl, isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl , tetrazolyl, oxadiazolyl (e.g. 1,2 ,3-oxadiazolyl), purinyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl , dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl and the like, including their dihydro and tetrahydro forms in which one or more double bonds are reduced and replaced by hydrogen atoms. Other exemplary heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1Nimidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (for example, 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1Hpyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (for example, 2,3,4,5-tetrahydro-2,4-dioxo-5methyl-5-phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (for example, 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (for example, 4,5-dihydro-3-methyl4-amino-5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (for example, 1,2,3,4-tetrahydro2,4-dioxo-3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (for example, 2,6-dioxo-3-ethyl-3phenylpiperidinyl); 1,6-dihydro-6-oxopyrimidinyl; 1,6-dihydro-4-oxopyrimidinyl (for example, 2(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (for example, 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (for example, 1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (for example, 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl); 2,3-dihydro-2-oxo-1H-indolyl (for example, 3,3-dimethyl2,3-dihydro-2-oxo-III-indolyl and 2,3-dihydro-2-oxo-3,3'-spiropropane -1H-indol-1-yl); 1,3-dihydro-1oxo-2H-isoindolyl; 1,3-dihydro-1,3-dioxo-2H-isoindolyl; 1H-benzopyrazolyl (for example, 1(ethoxycarbonyl)-III-benzopyrazolyl); 2,3-dihydro-2-oxo-1 H-benzimidazolyl (for example, 3-ethyl-2,3-dihydro-2-oxo-III-benzimidazolyl); 2,3-dihydro-2-oxobenzoxazolyl (eg 5-chloro-2,3-dihydro-2oxo-benzoxazolyl); 2,3-dihydro-2-oxobenzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo-4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (for example, 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (for example, 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (for example, 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (for example, 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl); 2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naph[1,8-c,d]isothiazolyl; and 1,8-naphthylenedicarboxamido. Additional heterocyclic compounds include 3,3a,4,5,6,6a-hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl and 2,5diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanil ), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanil, tiepanil, azocanil, oxecanil and thiocanil. Heterocyclic groups also include groups of the formula

where E' is selected from the group consisting of -N- and -CH-; F' is selected from the group consisting of -N=CH-, -NHCH2-, -NH-C(O)-, -NH-, -CH=N-, -CH2-NH-, -C(O)-NH -, -CH=CH-, -CH2-, -CH2CH2-, -CH2O-, -OCH2-, -O- and -S-; and C is selected from the group consisting of -CH- and -N-. Any of the heterocyclyl groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) C _1-7 acyl (eg, carboxyaldehyde); (2) _{C1_20} alkyl (e.g. C1-6 alkyl, C1-6 alkoxy- _{C1_6} alkyl, C1-6 alkylsulfinyl- _{C1_6} alkyl, amino- _{C1_6} alkyl, azido- _{C1_6} alkyl, (carboxyaldehyde) _{-C1_6} alkyl, halo- _{C1_6} alkyl (eg perfluoroalkyl), hydroxy- _C1-6 alkyl, nitro- _C1-6 alkyl or C1-6 thioalkoxy- _C1-6 alkyl); (3) C1 ₂₀ alkoxy (eg C1-6 alkoxy, eg perfluoroalkoxy); (4) C1-6 alkylsulfinyl; (5) C ₆ - ₁₀ aryl; (6) amino; (7) C1-6 alkyl-C ₆ - ₁₀ aryl; (8) azido; (9) C3-8 cycloalkyl; (10) C1-6 alkyl-C ₃ - ₈ cycloalkyl; (11) halogen; (12) C1. ₁₂ heterocyclyl (for example, C ₂ - ₁₂ heteroaryl); (13) (C1_ ₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) _C1-20 thioalkoxy (eg C1-6 thioalkoxy); (17) -(CH ₂ ) _q CO ₂ R ^A where q is an integer from zero to four and R ^A is selected from the group with

- 9 045310 consisting of (a) C _1-6 alkyl, (b) C ₆ - ₁₀ aryl, (c) hydrogen and (d) C _1-6 alkyl-C ₆ -10 aryl; (18) -(CH2)qCONR ^B 'R ^C ' where q is an integer from zero to four, and where R ^B and R ^C are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6-10 aryl and (d) C1-6 alkyl-C6-10 aryl; (19) -(CH2)qSO2R ^D ' where q is an integer from zero to four, and where R ^D is selected from the group consisting of (a) C1-6 alkyl, (b) C _6-10 aryl and (c ) C _1-6 alkyl-C _6-10 aryl; (20) -(CH ₂ ) _q SO ₂ NR ^E R ^F where q is an integer from zero to four, and where each of R ^E and R ^F is independently selected from the group consisting of (a) hydrogen, (b ) C _1-6 alkyl, ( _c ) C _6-10 aryl and (d) C _1-6 alkyl-C _6-10 aryl; (21) thiol; (22) C _6-10 aryloxy; (23) C _3-8 cycloalkoxy; (24) arylalkoxy; (25) C _1-6 alkyl-C _1-12 heterocyclyl (for example, C _1-6 alkyl-C _1-12 heteroaryl); (26) oxo; (27) (C _1-12 heterocyclyl)imino; (28) C _2-20 alkenyl; and (29) C _2-20 alkynyl. In some embodiments, each of these groups may be further substituted as described herein. For example, the alkylene group of a C ₁ -alkylaryl or C ₁ -alkylheterocyclyl group may be further substituted with an oxo group to provide the corresponding aryloyl and (heterocyclyl)oyl substituent group.

The term hydrocarbon as used herein refers to a group consisting of only carbon and hydrogen atoms.

The term hydroxyl as used herein refers to the -OH group. In some embodiments, the hydroxyl group may be substituted with 1,2,3, or 4 substituent groups (eg, O-protecting groups) as defined herein for alkyl.

The term isomer as used herein means any tautomer, stereoisomer, enantiomer or diastereomer of any compound of the invention. It is known that the compounds of the invention may have one or more chiral centers and/or double bonds and therefore exist as stereoisomers, such as double bond isomers (i.e. geometric E/Z isomers) or diastereomers (e.g. enantiomers ( i.e. (+) or (-)), or cis-/trans-isomers). According to the invention, the chemical structures provided herein, and therefore the compounds of the invention, cover all relevant stereoisomers, that is, both the stereomerically pure form (for example, geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures , for example, racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the invention can generally be separated into their constituent enantiomers or stereoisomers by well-known methods such as gas chromatography using a chiral phase, high performance liquid chromatography using a chiral phase, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be prepared from stereomerically or enantiomerically pure intermediates, reagents and catalysts using well-known asymmetric synthesis methods.

The term N-protected amino group as used herein refers to an amino group as defined herein to which one or two N-protecting groups as defined herein are attached.

The term N-protecting group as used herein refers to groups designed to protect the amino group from unwanted reactions during synthesis procedures. Common N-protecting groups are disclosed in Greene, Protective Groups in Organic Synthesis, ^3rd Edition (John Wiley & Sons, New York, 1999), incorporated herein by reference. N-protecting groups include acyl, acryloyl or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, ortho-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D-, L-, or D-L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate-forming groups such as benzyloxycarbonyl, para-chlorobenzyloxycarbonyl, para-methoxybenzyloxycarbonyl, para-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, rbromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4dimethoxybenzyloxycarbonyl, 4-methoxyben zyloxycarbonyl, 2-nitro-4,5dimethoxybenzyloxycarbonyl , 3,4,5-trimethoxybenzyloxycarbonyl, 1-(para-biphenyl)-1methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, tertbutyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2 ,2 -trichloroethoxycarbonyl, phenoxycarbonyl, 4nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like, alkyl aryl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like, and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

- 10 045310

The term O-protecting group as used herein refers to groups designed to protect an oxygen-containing group (eg, phenolic, hydroxyl or carbonyl) from unwanted reactions during synthesis procedures. Common O-protecting groups are disclosed in Greene, Protective Groups in Organic Synthesis, ^3rd Edition (John Wiley & Sons, New York, 1999), incorporated herein by reference. Exemplary O-protecting groups include acyl, acryloyl or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, orthonitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4 -chlorobenzoyl, 4-bromobenzoyl, tert-butyldimethylsilyl, tri-isopropylsiloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4isopropylphenoxyacetyl, dimethylformamidino and 4-nitrobenzoyl; alkylcarbonyl groups such as acyl, acetyl, propionyl, pivaloyl and the like; optionally substituted arylcarbonyl groups such as benzoyl; silyl groups such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), triisopropylsilyl (TIPS) and the like; hydroxyl ester groups such as methyl, methoxymethyl, tetragyropyranyl, benzyl, para-methoxybenzyl, trityl and the like; alkoxycarbonyls such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, tert-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl and the like; alkoxyalkoxycarbonyl groups such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl and the like; haloalkoxycarbonyls such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl and the like; optionally substituted arylalkoxycarbonyl groups such as benzyloxycarbonyl, paramethylbenzyloxycarbonyl, para-methoxybenzyloxycarbonyl, para-nitrobenzyloxycarbonyl, 2,4dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, para-chlorobenzyloxycarbonyl, parabromobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and the like; and optionally substituted aryloxycarbonyl groups such as phenoxycarbonyl, para-nitrophenoxycarbonyl, orthonitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, para-methylphenoxycarbonyl, metamethylphenoxycarbonyl, ortho-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, parachlorophenoxycarbonyl, 2-chloro-4-nitrophenoxycarbonyl silt and the like ; substituted alkyl, aryl and alkylaryl ethers (e.g. trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimtylsilyl)ethoxy]ethyl; 2- trimethylsilylethyl; tert-butyl ether; para-chlorophenyl, para-methoxyphenyl, para-nitrophenyl, benzyl, para-methoxybenzyl and nitrobenzyl); silyl ethers (eg, trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); carbonates (eg, methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl); carbonyl group protecting groups (eg, acetal and ketal groups such as dimethyl acetal, 1,3-dioxolane and the like; acyl groups; and dithian groups such as 1,3-dithiolane, 1,3-dithiolane and the like); carboxylic acid protecting groups (for example, ester groups such as methyl ester, benzyl ester, tert-butyl ester, orthoesters and the like; and oxazoline groups.

The term oxo as used herein means =O.

The term polyethylene glycol as used herein means an alkoxy chain consisting of one or more monomer units, each of which consists of -OCH ₂ CH ₂ -. Polyethylene glycol (PEG) is also sometimes called polyethylene oxide (PEO) or polyoxyethylene (POE), and these terms may be considered interchangeable for the purposes of the present invention. For example, polyethylene glycol may have the structure -(CH ₂ ) _s2 (OCH ₂ CH ₂ ) _s1 (CH ₂ ) _s3 O-, where s1 is an integer from 1 to 10 (for example, 1 to 6 or 1 to 4) , and each of s2 and s3 independently represents an integer from 0 to 10 (eg, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10). The polyethylene glycol may also be considered to include the aminopolyethylene glycol of the formula - NR ^N1 (CH ₂ ) _s2 (CH ₂ CH ₂ O) _s1 (CH ₂ ) _s3 NR ^N1 -, where s1 is an integer from 1 to 10 (for example, from 1 to 6 or 1 to 4), each of s2 and s3 independently represents an integer from 0 to 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10) and each R ^N1 independently represents hydrogen or optionally substituted C1-6 alkyl.

The term stereoisomer as used herein refers to all possible different isomeric as well as conformational forms that a compound (e.g., a compound of any formula described herein) may have, in particular, all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Certain compounds of the present invention may exist in different tautomeric forms, all of which are included within the scope of this invention.

- 11 045310 shaving.

The term sulfonyl as used herein refers to the group -S(O)2-.

The term thiol as used herein refers to the -SH group.

Definitions

As used herein, the term administered in combination or combined administration means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of the effects of each agent on the patient. In some embodiments, they are administered at intervals of 90 days (e.g., at intervals of 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)), at intervals at 28 days intervals (for example, 14, 7, 6, 5, 4, 3, 2 or 1 day(s) apart), at 24 hour intervals (for example, 12, 6, 5, 4, 3, 2 or 1 hour (hours)), or at intervals of approximately 60, 30, 15, 10, 5, or 1 minute. In some embodiments, administration of the agents occurs at a short interval sufficient to achieve combinatorial (eg, synergistic) effects.

As used herein, the term antibody refers to a polypeptide whose amino acid sequence includes immunoglobulins and fragments thereof that specifically bind to the specified antigen, or fragments thereof. The antibodies of the present invention can be of any type (eg, IgA, IgD, IgE, IgG, or IgM) or subtype (eg, IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those skilled in the art will appreciate that the characteristic sequence or portion of an antibody may include amino acids contained in one or more regions of the antibody (eg, variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). In addition, those skilled in the art will appreciate that the characteristic sequence or portion of an antibody may include one or more polypeptide chains and may include sequence elements contained in the same polypeptide chain or in different polypeptide chains.

As used herein, the term antigen binding fragment refers to a portion of an antibody that retains the binding characteristics of the parent antibody.

The terms bifunctional chelate or bifunctional conjugate, used interchangeably herein, refer to a compound containing a chelating moiety or a metal complex thereof, a linker moiety, and a therapeutic moiety, targeting moiety, or crosslinking moiety.

The term cancer refers to any cancer caused by the proliferation of malignant tumor cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias and lymphomas. Solid cancer is a cancer characterized by a mass of abnormal tissue, such as sarcoma, carcinoma and lymphoma. The terms hematologic cancer or hematologic malignancy, used interchangeably herein, refer to cancers present in body fluids, such as lymphomas and leukemias.

The term chelate as used herein refers to an organic compound or part thereof that may be bonded to a central metal or radiometal atom at two or more points.

The term conjugate as used herein refers to a molecule containing a chelating group or a metal complex thereof, a linker group, and optionally containing a therapeutic moiety, targeting moiety, or cross-linking group.

As used herein, the term compound is intended to include all stereoisomers, geometric isomers and tautomers of the structures depicted.

The compounds described herein may be asymmetric (eg, have one or more stereogenic centers). All stereoisomers, such as enantiomers and diastereomers, are intended to be included unless otherwise noted. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art and include, for example, resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds and the like may also occur in the compounds described herein, and all such stable isomers are contemplated by the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as individual isomeric forms.

The compounds of the present invention also include tautomeric forms. Tautomeric forms arise from the exchange of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonated states having the same empirical formula and overall charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and ring forms where the proton can occupy two or more positions of the heterocyclic system, such as 1H- and 3H-imidazole, 1H-,

- 12 045310

2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

At various places throughout the present specification, substituents of the compounds of the present invention are disclosed in groups or ranges. In particular, the present invention is intended to include any and all individual subcombinations of members of such groups and ranges. For example, the term _{C1_6} alkyl is intended to individually cover methyl, ethyl, _C3 alkyl, _C4 alkyl, _C5 alkyl, and _C6 alkyl. As used herein, a phrase of the form optionally substituted X (eg, optionally substituted alkyl) is intended to be equivalent to X where X is optionally substituted (eg, alkyl, wherein said alkyl is optionally substituted). This phrase does not imply that feature X (eg, alkyl) is itself optional.

As used herein, the term detecting agent refers to a molecule or atom useful for diagnosing a disease by locating cells containing an antigen. Various methods for labeling polypeptides with detection agents are known in the art. Examples of detection agents include, but are not limited to, radioisotopes and radionuclides, dyes (eg, biotin-streptavidin complex), contrast agents, luminescent agents (eg, FITC (fluorescein-5-isothiocyanate), rhodamine, lanthanide phosphors, cyanine and near-field dyes). infrared) and magnetic agents such as gadolinium chelates.

As used herein, the term radionuclide refers to an atom capable of undergoing radioactive decay (e.g., ³ H, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³⁵ S, ⁴⁷ Sc, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸⁹ Zr, ⁸⁶ Y, ⁸⁷ Y, ⁹⁰ Y, ⁹⁷ Ru,Tc, ^99m Tc ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹ⁿ In, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰³ Pb, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁷ Th, ^229Th , ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸² Rb, ^117m Sn, ²⁰¹ TI). The terms radioactive nuclide, radioisotope, or radioactive isotope may also be used to describe a radionuclide. Radionuclides can be used as detection agents as described above. In some embodiments, the radionuclide may be an alpha-emitting radionuclide.

The term effective amount of an agent (e.g., any of the above-mentioned conjugates) as used herein is that amount that is sufficient to achieve beneficial or desired results, such as clinical results, and as such, the effective amount depends on the context in which it is used .

The term immunoconjugate as used herein refers to a conjugate comprising a targeting moiety, such as an antibody, nanobody, affibody, or consensus sequence from a fibronectin type III domain. In some embodiments, the immunoconjugate contains an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 5 or 8 conjugates per targeting moiety).

The term radioconjugate as used herein refers to any conjugate comprising a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

The term radioimmunoconjugate as used herein refers to any immunoconjugate comprising a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

The term radioimmunotherapy as used herein refers to the method of using a radioimmunoconjugate to achieve a therapeutic effect. In some embodiments, radioimmunotherapy may include administering a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate produces a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include introducing a radioimmunoconjugate into a cell, wherein administration of the radioimmunoconjugate kills said cell. In cases where radioimmunotherapy involves selective killing of a cell, in some embodiments, said cell is a cancer cell in a subject having cancer.

The term pharmaceutical composition as used herein refers to a composition containing a compound described herein combined with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a government regulatory agency as part of a treatment regimen for treating a disease in a mammal. The pharmaceutical compositions may be prepared, for example, for oral administration in unit dosage form (eg, tablet, capsule, caplet, gelatin capsule, or syrup); for topical use (eg, as a cream, gel, lotion, or ointment); for intravenous administration (eg, as a sterile solution free of embolic particles in a solvent system suitable for intravenous use); or as part of any other drug described herein.

The term pharmaceutically acceptable excipient as used herein refers to any ingredient other than the compounds described herein

- 13 045310 (for example, a carrier capable of suspending or dissolving the active compound), which is non-toxic and does not cause inflammation in the patient. Excipients may include, for example: release agents, antioxidants, binders, coatings, pressing aids, disintegrants, colorants, emollients, emulsifiers, fillers, film-forming agents or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotective agents, sorbents, suspending or dispersing agents, sweeteners or water of hydration. Exemplary excipients include, but are not limited to: ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin , hydroxypropylcellulose, hydroxymethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, collapse small glycolate sodium, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C and xylitol.

The term pharmaceutically acceptable salt as used herein refers to those salts of the compounds described herein which, within the bounds of sound medical judgment, are suitable for use in contact with tissues of humans and animals without undue toxicity, irritation or allergic reaction. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base group with a suitable organic acid.

The compounds of the invention may have ionic groups to enable their preparation as pharmaceutically acceptable salts. These salts may be acid addition salts prepared with inorganic or organic acids, or salts, in the case of the acid forms of the compounds of the invention, may be prepared with inorganic or organic bases. Often the compounds are prepared or used in the form of pharmaceutically acceptable salts obtained as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art and include, for example, hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric or tartaric acids for the formation of acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine , various amines for the formation of basic salts. Methods for preparing the corresponding salts are well known in the art.

Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, g eptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate and others. Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium and magnesium salts, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine and ethylamine .

The term therapeutic moiety as used herein refers to any molecule or any part of a molecule that provides a beneficial therapeutic outcome. In some embodiments, the therapeutic moiety is a protein or polypeptide, such as an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule.

The term targeting moiety as used herein refers to any molecule or any part of a molecule that is bound by a given target. In some embodiments, the targeting moiety is a protein or polypeptide, such as an antibody or an antigen binding fragment thereof, a nanobody, an affibody, or a fibronectin type III domain consensus sequence.

The term cross-linking group as used herein refers to any reactive group that is capable of joining two or more molecules by a covalent bond. In some embodiments, the cross-linking group is an amino-reactive or thiol-reactive cross-linking group. In some embodiments, an amino- or thiol-reactive sulfide

- 14 045310 This group contains an activated ester, such as hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester, or imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate. In some embodiments, the crosslinking group may be glycine-glycine-glycine and/or leucine-proline(any amino acid)-threonine-glycine, which are recognition sequences for binding targeting agents to the linker using a sortase-mediated binding reaction. One skilled in the art will appreciate that the use of crosslinking groups in the practice of the invention is not limited to the specific designs disclosed herein, but may instead include other known crosslinking groups.

The term polypeptide as used herein refers to a chain of at least two amino acids linked to each other by a peptide bond. In some embodiments, the polypeptide may include at least 3-5 amino acids, each of which is linked to the others by at least one peptide bond. Those skilled in the art will appreciate that polypeptides may include one or more unnatural amino acids or other compounds that may nonetheless be incorporated into the polypeptide chain. In some embodiments, the polypeptide may be glycosylated, for example, the polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single polypeptide (eg, an antibody polypeptide) may contain two or more separate polypeptide chains, which in some cases may be linked to each other, for example, by one or more disulfide bonds or other means.

Subject means a human or non-human animal (eg, mammal).

By substantially identical or substantially identical is meant a polypeptide sequence that has the same polypeptide sequence, respectively, as the reference sequence, or has a certain percentage of amino acid residues, respectively, that are identical to the residues at the corresponding position in the reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence substantially identical to a reference sequence has at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to the reference amino acid sequence. For polypeptides, the sequence length being compared is typically at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90. 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., full-length sequence). Sequence identity can be measured using sequence analysis software at default settings (eg, sequence analysis software package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software can match similar sequences, assigning degrees of homology to various substitutions, deletions and other modifications.

As used herein, and as is well known in the art, treating a condition or treating a condition (eg, the conditions described herein, such as cancer) is an approach to obtain positive or desired results, such as clinical results. Positive or desirable results may include, but are not limited to, reduction or improvement of one or more symptoms or conditions; reduction in the severity of a disease, disorder or condition; a stable (i.e., not worsening) state of a disease, disorder, or condition; preventing the spread of a disease, disorder or condition; delaying or slowing the progression of a disease, disorder or condition; improvement or alleviation of a disease, disorder or condition; and remission (partial or complete), detectable or undetectable. Alleviation of a disease, disorder or condition means that the extent and/or adverse clinical manifestations of the disease, disorder or condition are reduced and/or the progression is slowed or prolonged compared to the extent or progression in the absence of treatment.

Brief description of graphic materials

Fig. 1 is a diagram depicting the general structure of a conjugate containing a chelate, a linker and a cross-linking group (top) and a conjugate containing a chelate, a linker and a targeting moiety (bottom).

Fig. 2 is a scheme depicting the synthesis of the bifunctional chelate 4-{[11-oxo11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4 ,7,10tetraazacyclododecan-1-yl]butanoic acid (compound B). The synthesis of compound B is described in example 3.

Fig. 3 is a diagram depicting the synthesis of the bifunctional chelate, 4-{[2-(2-{2-[3oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2 -[4,7,10

- 15 045310 tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (compound C). The synthesis of compound C is described in example 4.

Fig. 4 is a graph showing the percentage residue of three bifunctional chelated antibodies (Compound A, Compound B, and Compound C), defined as cpm (lysate)/cpm (efflux+recycle+lysate). The residue analysis used is described in detail in Example 6.

Fig. 5 is a series of graphs depicting the metabolic profile of excretion of untargeted human IgG antibody conjugates [ ¹⁷⁷ Lu]-compound B-HuMIGF-1R and [ ¹⁷⁷ Lu] compound C-HuMIGF-1R compared to [ ¹⁷⁷ Lu]-compound A-HuMIGF- 1R, the methods and results for which are described in detail in Example 9.

Fig. 6. Pharmacokinetics of total radioactivity in the blood of CD-1 nude mice. The results and methods are described in Example 9.

Fig. 7. Therapeutic efficacy of [ ²²⁵ Ac]-HuMIGF-1R compounds (dose 200 nCi). The results and methods are described in Example 10.

Fig. 8 is a series of graphs depicting the metabolic excretion profile of untargeted human IgG antibody conjugates [ ¹⁷⁷ Lu]-compound B-HuMIgG and [ ¹⁷⁷ Lu]-compound C-HuMIgG compared to [ ¹⁷⁷ Lu]-compound A-HuMIgG, methods and results for which are described in detail in example 14.

Detailed Description of the Invention

Radiolabeled targeting moieties (also known as radioimmunoconjugates) are designed to target a protein or receptor that is activated by a disease state to deliver a radioactive payload to target and kill cells of interest (radioimmunotherapy). The process of delivering such a payload through radioactive decay produces alpha, beta or gamma particles, or Auger electrons, which can have direct effects on DNA (such as single- or double-strand breaks in DNA), or indirect effects such as effects bystander or crossfire.

Radioimmunoconjugates typically contain a biological targeting moiety (eg, an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R), a radioisotope, and a binding molecule thereof. Conjugates are formed by the addition of a bifunctional chelate to a biological targeting molecule so that structural changes are minimal while affinity for the target is maintained. After attaching the radiolabel, the final radioimmunoconjugate is formed.

The structure of bifunctional chelates contains a chelate, a linker and a cross-linking group (Fig. 1). In the development of new bifunctional chelates, most efforts have focused on the chelating portion of the molecule. Several examples of bifunctional chelates with various cyclic and acyclic structures conjugated to a targeting moiety have been described. [Bioconjugate Chem. 2000, 11, 510-519, Bioconjugate Chem.2012, 23, 1029-1039, Mol Imaging Biol (2011) 13:215-221, Bioconjugate Chem.2002,13,110-115].

One of the key factors in developing safe and effective radioimmunoconjugates is maximizing efficacy while minimizing off-target toxicity in normal tissue. Although this statement is one of the basic principles of new drug development, its application in the field of radioimmunotherapy presents new challenges. To be therapeutically effective, radioimmunoconjugates do not need to block a receptor, unlike therapeutic antibodies, or release a cytotoxic payload intracellularly, unlike an antibody-drug conjugate. However, the emission (emission) of a toxic particle is an event that occurs as a result of first order (radioactive) decay and can occur randomly anywhere in the body after administration. Once emission occurs, damage to surrounding cells within the amplitude of the emission may occur, leading to potential off-target toxicity. Therefore, limiting the exposure of normal tissue to these emissions is a key factor in the development of new drugs.

One potential way to reduce off-target exposure is to more efficiently remove the radioactive substance from the body (eg, from normal body tissue). The most obvious mechanism is to increase the rate of clearance of the biological targeting agent. This approach also likely requires identifying ways to reduce the half-life of a biological targeting agent, which is a topic not well described for biological targeting agents. Regardless of the mechanism, increasing drug clearance will also negatively impact pharmacodynamics/efficacy because faster clearance of the drug from the body will result in lower effective concentrations at the site of action, which in turn will require a higher total dose and will not result in to achieve the desired results of reducing the total dose of radioactivity to normal tissue.

Other efforts have been aimed at speeding up the metabolism of the part of the molecule that contains the radioactive fragment. To this end, certain efforts have been made to increase

- 16 045310 methods of cleaving radioactive substances from biological targeting agents using so-called cleavable linkers. However, cleavable linkers have taken on a different meaning in relation to radioimmunoconjugates. Cornelissen, et al. described cleavable linkers as those by which a bifunctional conjugate is attached to a biological targeting agent via a reduced cysteine, while other authors have described the use of enzymatically cleavable systems that require coadministration of the radioimmunoconjugate with the cleavage agent/enzyme for release. [Mol Cancer Ther; 12(11) November 2013, Methods in Molecular Biology, 2009, 539, 191-211, Bioconjugate chemistry, Volume 14, Issue 5, p.927-33 (2003)]. These methods either change the nature of the biological targeting moiety, in the case of a cysteine linkage, or are impractical from a drug development perspective (enzymatically degradable systems) because in the case of the references cited, administration of 2 agents is required.

The primary focus of the embodiments described herein is to more efficiently eliminate radioactive material from the body following catabolism and/or metabolism of the radioimmunoconjugate by making modifications to the linker region of the bifunctional chelate.

This approach is novel because there appears to be little information describing the effects of the linker in vivo, particularly with respect to radioimmunoconjugates. One proposed reason is that, following catabolism/metabolism of the radioimmunoconjugate, the radiolabeled conjugate can be expected to undergo rapid systemic elimination. The assumption was complemented experimentally by introducing the bifunctional chelate separately; it was cleared from the bloodstream more quickly than a radioimmunoconjugate with the same bifunctional chelate. Based on these data, it can be expected that after catabolism/metabolism of the radioimmunoconjugate, the metabolite containing the bifunctional chelate will also be rapidly eliminated.

However, rapid clearance of metabolites containing the radiolabeled conjugate does not necessarily occur in vivo. Based on the results described below, the linker region of bifunctional chelates can directly influence the elimination of radioactive material from the body after catabolism of the radioconjugate without adversely affecting the overall properties of the radioimmunoconjugate in vitro or the pharmacokinetics and pharmacodynamics in vivo. Data is presented below demonstrating that certain commercially available bifunctional chelates provide a slower rate and lower degree of elimination of total radioactivity from the body compared to the embodiments described herein.

The excretion profiles in the embodiments described in the examples present unexpected results. As previously reported, Quadri and Vriesendorp [Q. J. Nucl. Med. 1998, 42, 250261], simple modifications of the linker region of the bifunctional chelate, regardless of their hydrophobicity, did not affect the excretion of the radioactive substance in the urine. The results presented below clearly show that both hydrophobic and hydrophilic linkers can influence excretion patterns. Additionally, the examples below demonstrate that hepatobiliary clearance also plays a role in excretion.

Therefore, through the embodiments described herein, bifunctional chelates have been identified that, when attached to biological targeting moieties or therapeutic agents, provide a reduction in total radioactivity in the body by increasing the rate of excretion of catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule compared to similar bifunctional chelates known from open sources of information. This reduction in total body radioactivity was determined to be due to the clearance of catabolic/metabolic by-products and did not affect other in vitro and in vivo properties such as degree of specificity (in vitro binding), cellular retention, and in vivo tumor accumulation. In general, these embodiments achieve the desired properties of the radioimmunoconjugates by reducing the radioactive load on the body while maintaining the target activity.

Therapeutic moieties and targeting moieties.

Therapeutic or targeting moieties include any molecule or any portion of a molecule that provides a beneficial therapeutic outcome. In some embodiments, the therapeutic moiety is a protein or polypeptide, such as an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule. Targeting moieties include any molecule or any portion of a molecule that is bound by a given target. In some embodiments, the targeting moiety is a protein or polypeptide, such as antibodies or antigen-binding fragments thereof, nanobodies, affibodies, and consensus sequences from fibronectin type III domains (eg, centirins or adnectins).

Polypeptides.

Polypeptides include, for example, any of a variety of hematological agents (including, for example, erythropoietin, coagulation factors, etc.), interferons, colony-stimulating factors

- 17 045310 tors, antibodies, enzymes and hormones. The present invention is not intended to limit a particular polypeptide in any particular way, and the polypeptide of interest in the present methods can be any polypeptide.

A reference polypeptide described herein may include a target binding domain that binds to a target of interest (eg, binds an antigen). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (eg, receptor) or ligand (eg, growth factor). Exemplary molecular targets (e.g., antigens) for the polypeptides described herein (e.g., antibodies) include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40 , CD52; members of the ErbB receptor family, such as EGF receptor (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 receptor (ErbB4); macrophage receptors such as CRIg; tumor necrosis factors such as TNFa or TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM and integrin αΎβ3, including its α- or β-subunits (for example, anti-CDUa, anti-CD18 or anti- CDHb antibodies); growth factors and receptors such as EGF, FGFR (eg FGFR3) and VEGF; IgE; cytokines such as IL-1; cytokine receptors such as IL-2 receptor; blood group antigens; flk2/flt3 receptor; obesity receptor (OB); mpl receptor; CTLA-4; C-reactive protein; neutropilins; ephrins and receptors; netrins and receptors; slit protein and receptors; chemokines and chemokine receptors such as CCL5, CCR4, CCR5; amyloid beta; complement factors such as complement factor D; lipoproteins such as oxidized low-density lipoprotein (LDL) (oxLDL); lymphotoxins such as lymphotoxin alpha (LTa). Other molecular targets include Tweak, B7RP-1, proprotein convertase subtilisin/kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Antibodies.

An IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The sequence of the first domain, located at the amino terminus of each chain, is variable, providing antibody binding specificity that characterizes each individual antibody. These are known as the heavy (VH) and light (VL) chain variable regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as the heavy chain (CH) and light chain (CL) constant regions. In the case of an IgG antibody, the light chain includes one variable region (VL) and one constant region (CL). An IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2) and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).

Antibodies described herein may include, for example, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single chain Fv (scFv), disulfide-linked Fvs (sdFv) and anti-idiotypic (anti -Id) antibodies, and antigen-binding fragments of any of the above. Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The term antigen-binding antibody fragment as used herein refers to one or more antibody fragments that retain the ability to specifically bind an antigen. Examples of binding fragments encompassed by the term antigen binding fragment of an antibody include Fab fragment, F(ab') ₂ fragment, Fd fragment, Fv fragment, scFv fragment, dAb fragment (Ward et al., (1989) Nature 341 :544-546), and a dedicated complementarity determining region (CDR). These antibody fragments can be prepared using conventional techniques known to those skilled in the art, and the fragments can be screened for suitability in the same manner as intact antibodies.

The antibodies or fragments described herein can be prepared by any antibody synthesis method known in the art (see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988 ); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using methods described, for example, in Morrison, 1985, Science 229:1202, and humanized antibodies using methods described, for example, in US patent No. 6,180,370.

Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described, for example, in Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147:60 (1991).

Antibodies to insulin-like growth factor 1 (IGF-1R).

Insulin-like growth factor 1 receptor is a transmembrane protein present on the surface of human cells, activated by insulin-like growth factor 1 (IGF-1) and 2 (IGF-2). The radioimmunoconjugates of the invention may include insulin-like growth factor-1 receptor (IGF-1R). Although it is not a typical oncogene, IGF-1R promotes the initiation of

- 18 045310 development and progression of cancer, playing a key role in mitogenic transformation and maintenance of the transformed phenotype. IGF-1R has been associated with the development of a variety of common cancers, including breast, lung (eg, non-small cell lung cancer), liver, prostate, pancreatic, ovarian, colon, melanoma, adrenocortical carcinoma, and various types of sarcomas. IGF-1R signaling stimulates tumor cell proliferation and metabolism, supports angiogenesis, and provides protection against apoptosis. It influences metastatic factors (eg, HIF-1-dependent hypoxia signaling), free cell growth, and the growth and survival of tumor metastases after extravasation. IGF-1R has also been shown to be involved in the development, maintenance, and enrichment of therapy-resistant cancer stem cell populations.

Despite the abundance of evidence suggesting a role for IGF-1R in cancer, therapeutics targeting IGF-1R have so far failed to demonstrate significant effects on the disease. Many speculations have been made about the reason for this lack of effectiveness, including the failure to identify suitable biomarkers to identify patients, the complexity and interdependence of the IGF-1/IR signaling pathway, and the development of other compensatory mechanisms of growth hormone [Beckwith and Yee, Mol Endocrinol, November 2015, 29( 11):1549-1557]. Radioimmunotherapy, however, may provide a promising mechanism for treating cancers characterized by overexpression of the IGF-1 receptor by exploiting the ability of the IGF-1R to undergo antibody-induced internalization and lysosomal degradation to deliver targeted radioisotopes inside cancer cells. Internalization and lysosomal degradation of the radioimmunoconjugate targeting IGF-1R prolongs the residence time of the delivered radioisotope inside cancer cells, thereby maximizing the likelihood of cell-killing emission. In the case of actinium-225, which produces 4 alpha particles per decay chain, cell death can be achieved by as little as one radionuclide atom delivered per cell [Sgouros, et al. J Nucl Med. 2010, 51:311-2]. Cell destruction due to direct effects on DNA and destruction by alpha particles can occur in the target cell or within a radius of 2 or 3 non-target cells for a given alpha decay. In addition to having very high potential antitumor activity, radioimmunoconjugates targeting the IGF-1R cannot induce resistance to the mechanism of action because they, unlike a therapeutic antibody, do not rely on blocking ligand binding to the receptor to inhibit the oncological process.

Several anti-IGF-1R antibodies have been developed and studied for the treatment of various cancers, including figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab and teprotumumab. Once bound to IGF-1R, these antibodies are internalized into the cell and are degraded by lysosomal enzymes. The combination of overexpression on tumor cells and internalization allows detection agents to be delivered directly to the tumor site while limiting the exposure of normal tissue to toxic agents.

The AVE1642 light chain variable region CDRs have the following sequences:

SEQ ID NO: 1 (CDR-L1) RSSQSIVHSNVNTYLE,

SEQ ID NO: 2 (CDR-L2) KVSNRFS,

SEQ ID NO: 3 (CDR-L3) FQGSHVPPT.

The AVE1642 light chain variable region has the following sequence: SEQ ID NO: 4 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRF SGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAK .

The AVE1642 heavy chain variable region CDRs have the following sequences:

SEQ ID NO: 5 (CDR-H1) SYWMH,

SEQ ID NO: 6 (CDR-H2) GEINPSNGRTNY NQKFQG,

SEQ ID NO: 7 (CDR-H3) GRPDYYGSSKWY FDV.

The heavy chain variable region of AVE1642 has the following sequence: SEQ ID NO: 8

QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNY NQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALG.

Nanobodies.

Nanobodies are antibody fragments consisting of a single monomeric antibody variable domain. Nanobodies may also be called single-domain antibodies. Like antibodies, nanobodies selectively bind to a specific antigen. Nanobodies may be heavy chain variable domains or light chain domains. Nanobodies can occur naturally or be the product of biological engineering. Nanobodies can be biologically engineered using site-directed mutagenesis or screening for mutagenic factors (eg, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

- 19 045310

Affitel.

Affibodies are polypeptides or proteins designed to bind to a specific antigen. Thus, affibodies can be considered to mimic certain functions of antibodies. Affibodies may be engineered variants of the B domain in the immunoglobulin binding region of staphylococcal protein A. Affibodies may be engineered variants of the Z domain, a B domain having lower affinity for the Fab region. Affibodies can be biologically engineered using site-directed mutagenesis or screening for mutagenic factors (eg, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

Affibody molecules have been produced that exhibit specific binding to various proteins (e.g., insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2, and EGFR), demonstrating affinities (Kd) ranging from micromolar to picomolar.

Type III fibronectin domains.

The fibronectin type III domain is an evolutionarily conserved protein domain present in a variety of extracellular proteins. The fibronectin type III domain has been used as a molecular scaffold to produce molecules that can selectively bind a specific antigen. Variants of fibronectin type III (FN3) domains that have been engineered to bind selectively may also be referred to as monobodies. FN3 domains can be biologically engineered using site-directed mutagenesis or screening for mutagenic factors (eg, CIS display, phage display, yeast display, bacterial display, mRNA display, ribosomal display).

Modified polypeptides.

The polypeptides of the invention may have a modified amino acid sequence. Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid identity sequence of the reference polypeptide). In certain embodiments, the modification does not significantly interfere with the desired biological activity (eg, binding to IGF-1R). The modification may reduce (e.g., by at least 5, 10, 20, 25, 35, 50, 60, 70, 75, 80, 90, or 95%), may have no effect, or may increase (e.g., at least by 5, 10, 25, 50, 100, 200, 500 or 1000%) biological activity of the original polypeptide. The modified polypeptide may have the characteristics of a polypeptide or optimized characteristics of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity and conjugation properties.

Modifications include those carried out by natural means, such as post-translational processing, or by chemical modification methods known in the art. Modifications can occur anywhere in the polypeptide, including the polypeptide backbone, amino acid side chains, and the amino or carboxy terminus. The same type of modification may occur to the same or varying degrees at multiple sites on a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can be the result of post-translational natural processes or can be produced synthetically. Other modifications include PEGylation, acetylation, acylation, addition of an acetamidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to a flavin, covalent attachment to a dark moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent addition of drug, covalent addition of marker (eg, fluorescent or radioactive), covalent addition of lipid or lipid derivative, covalent addition of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking, cystine formation, pyroglutamate formation, formylation, gamma -carboxylation, glycosylation, formation of GPI anchors, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

A modified polypeptide may also include insertion, deletion, or substitution of an amino acid, conservative or non-conservative (eg, D-amino acids, deamino acids) in the polypeptide sequence (eg, when such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy terminus of any of the polypeptides of the invention may facilitate conjugation of the polypeptides, for example, by disulfide bonding. For example, a polypeptide may be modi

- 20 045310 is modified by including one cysteine residue at the amino terminus or one cysteine residue at the carboxy terminus. Amino acid substitutions can be conservative (that is, in which one residue is replaced by another of the same basic type or group) or non-conservative (that is, in which a residue is replaced by a different type of amino acid). In addition, a naturally occurring amino acid may be replaced with a non-naturally occurring amino acid (ie, a conservative non-natural amino acid substitution or a non-conservative non-natural amino acid substitution).

Synthetically produced polypeptides may include substitutions for amino acids not naturally encoded by DNA (eg, non-naturally occurring or unnatural amino acids). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid having an acetylaminomethyl group attached to a cysteine sulfur atom, a PEGylated amino acid, omega amino acids of the formula NH2(CH2)nCOOH, where n is 2-6, neutral non-polar amino acids, such as sarcosine, tert-butylalanine, tert-butylglycine, N-methylisoleucine and norleucine. Phenylglycine can replace Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral non-polar, cysteic acid is acidic, and ornithine is basic. Proline can be replaced by hydroxyproline and retain its conformational properties.

Analogs can be obtained using replacement mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions defined as conservative substitutions are given in Table. 1. If such substitutions lead to an undesirable change, then a different type of substitution is introduced, designated as illustrative substitutions in the table. 1, or as further described herein with reference to amino acid classes, and the resulting products are screened.

Table 1

Amino acid substitutions

Original balance Illustrative Substitution Conservative substitution Ala (A) Vai, Leu, Ile Vai Arg(R) Lys, Gin, Asn Lys Asn(N) Gin, His, Lys, Arg Gin Asp (D) Glu Glu Cys(C) Ser Ser Gin (Q) Asn Asn Glu(E) Asp Asp Gly (G) Pro Pro His(H) Asn, Gin, Lys, Arg Arg He (1) Leu, Vai, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Vai, Met, Ala, Phe Ile Lys (K) Arg, Gin, Asn Arg Met(M) Leu, Phe, Ile Leu Phe(F) Leu, Vai, Ile, Ala Leu Pro (P) Gly Gly Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr Tyr Tyr(Y) Trp, Phe, Thr, Ser Phe Vai (V) Ile, Leu, Met, Phe, Ala, norleucine Leu

Significant modifications of function or immunological identity are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or ( c) side chain size.

Stitching groups.

A cross-linking group is a reactive group capable of joining two or more molecules by a covalent bond. Crosslinking groups can be used to attach a linker and chelating moiety to a therapeutic or targeting moiety. Crosslinking groups can also be used to attach a linker and chelating moiety to a target in vivo. In some embodiments, the cross-linking group is an amino-reactive, methionine-reactive, or thiol-reactive cross-linking group, or provides sortase-mediated binding. In some embodiments, the amino- or thiol-reactive cross-linking group comprises an activated ester, such as a hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenol ester, a 4-nitrophenol ester, or an imidate, anhydride, thiol,

- 21 045310 disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate or oxaziridine. In some embodiments, the sortase recognition sequence may consist of a terminal amino acid sequence of glycine-glycine-glycine (GGG) and/or LPTXG, where X is any amino acid. One skilled in the art will appreciate that the use of crosslinking groups in the practice of the invention is not limited to the specific designs disclosed herein, but may instead include other known crosslinking groups.

Detection agents.

A detection agent is a molecule or atom administered as a conjugate to a polypeptide, such as an antibody or an antigen-binding fragment thereof, and is useful for diagnosing a disease by locating cells containing the antigen, planning radiation therapy, or treating the disease. Suitable detection agents include, but are not limited to, radioisotopes, dyes (eg, biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents, and contrast enhancing agents (eg, paramagnetic ions) for magnetic resonance imaging (MRI) . To load a polypeptide moiety with a detection agent, it may need to be reacted with a reagent having a linker to which a detection agent or multiple detection agents are attached.

Radioisotopes and radionuclides.

Radioisotopes and radionuclides known in the art to be suitable for use as detection agents include, but are not limited to, ^{3 H} , ¹⁴ C, ¹⁵ N, 18 F, ³⁵ S, ⁴⁷ Sc, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu , ⁶² Cu, ⁶⁴ Cu, ^{67 Cu} , ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸⁹ Zr, 86 Y, ⁸⁷ Y, ⁹⁰ Y, ⁹⁷ Ru, Tc, ^99m Tc ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰³ Pb, ²¹¹ At, ²¹² Pb, ²¹² Bi, ²¹³ Bi , ²²³ Ra, ²²⁵ Ac, ²²⁷ Th, ²²⁹ Th, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸² Rb, ^117m Sn, ²⁰¹ Ti.

Chelating moieties.

Chelating moieties known in the art to be suitable for use as detection agents include, but are not limited to, DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R ,7R,10R)-α,α',α,α'-tetramethyl1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl)- 1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), OO3AM-acetic acid (2-(4,7,10-tris(2 -amino-2-oxoethyl)1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2'-(10-(2,6dioxotetrahydro-2H-pyran- 3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid), DOTMP (1,4,6,10tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), SV -TE2A (1,4,8,11tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7triacetic acid), NOTP (1,4,7 -triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETRA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11tetraazacyclotetradecane-1 ,4,8,11-tetraacetic acid), HENA (1,4,7,10,13,16hexaazacyclohexadecane-1,4,7,10,13,16-hexacetic acid), PEPA (1,4,7,10 ,13pentaazacyclopentadecane-N,N',N,N', N''''-pentaacetic acid), H ₄ Octapa (N,N'-bis(6-carboxy-2pyridylmethyl)-ethylenediamine-N,N'-diacetic acid ), H ₂ Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]methylamino]ethane), H ₆ phospa (N,N'-(methylenephosphonate)-N,N'-[6- (methoxycarbonyl)pyridin-2-yl]-methyl1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N,N',N'-hexacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A ( hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid bismethylamide), HOPO (hydroxypyridinone octadentate) or porphyrins. Chelating groups can be used in combinations with metal chelates with metals such as manganese, iron and gadolinium, and isotopes (eg isotopes in the general energy range from 60 to 4000 keV) such as ⁴⁷ Sc, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu , ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, 68 Ga ^{, 82} Rb, ⁸⁶ Y, ⁸⁷ Y, ⁹⁰ Y, ⁹⁷ Ru, ^99m Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ^117m Sn, 149 ^,14^, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁹ Au, ²⁰¹ TI, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th.

Linkers.

Linkers according to the invention may have the structure of formula I:

AL ¹ -(L ² )nB

Formula I, where A is a chelating moiety or a metal complex thereof;

L ¹ is optionally substituted _{1 -6} alkyl, substituted _{1 -6} heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety targeting moiety or cross-linker

- 22 045310 pu, or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L ² independently has the structure:

(-X ¹ -L ³ -Z ¹ -)

Formula II, where X ¹ is C=O(NR ¹ ), C=S(NR ¹ ), OC=O(NR ¹ ), NR ¹ C=O(O), NR ¹ C=O(NR ¹ ) , -CH ₂ PhC=O(NR1), -CH ₂ Ph(NH)C=S(NR ¹ ), O, NR ¹ , and R ¹ is H or optionally substituted GG alkyl, or optionally substituted GG heteroalkyl, substituted aryl or heteroaryl;

L ³ is optionally substituted GG ₀ alkyl or optionally substituted

G-Go heteroalkyl, or C ₅ -C ₂₀ polyethylene glycol;

Z ¹ is CH2, C=O, C=S, OC=O, NR ¹ C=O, NR ¹ , and R ¹ is hydrogen or optionally substituted C1-C6 alkyl, pyrrolidine-2,5-dione.

The conjugates according to the invention contain three separate modules, which together lead to increased efficiency of these conjugates compared to those known in the art.

1. Chelating fragment or its complex compound with a metal.

Module A is included to include a detecting agent (eg, a chelating moiety or a metal complex thereof). The metal complex may include a radionuclide for imaging.

2. Linkers.

The linkers according to the invention have the structure of formula I:

AL ¹ -(L ² ) _n -B

Formula I, where A is a chelating moiety or a metal complex thereof;

L ¹ represents optionally substituted C ₁ -C6 alkyl, substituted C ₁ -C6 heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety targeting moiety or cross-linking group, or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L ² independently has the structure:

(-X'-L ³ -Z ¹ -)

Formula II, where X ¹ is C=O(NR ¹ ), C=S(NR ¹ ), OC=O(NR ¹ ), NR ¹ C=O(O), NR ¹ C=O(NR ¹ ) , CH2PhC=O(NR ¹ ), -CH2Ph(NH)C=S(NR ¹ ), O, NR ¹ , and R ¹ is H or optionally substituted GG alkyl, or optionally substituted GG heteroalkyl, substituted aryl or heteroaryl;

L ³ represents optionally substituted C ₁ -C ₅₀ alkyl or optionally substituted C ₁ C ₅₀ heteroalkyl, or C ₅ -C ₂₀ polyethylene glycol;

Z ¹ represents CH ₂ , C=O, C=S, OC=O, NR ¹ C=O, NR ¹ , and R ¹ represents hydrogen or optionally substituted GG alkyl, pyrrolidine-2,5-dione.

3. Therapeutic moiety, targeting moiety, or crosslinker.

Module B is a therapeutic moiety (e.g., antibodies, antigen-binding moieties), a targeting moiety (e.g., nanobodies, affibodies, consensus sequences from fibronectin type III domains), or a cross-linker (e.g., an amino-reactive, thiol-reactive cross-linker, or enabling sortase-mediated binding) .

Administration and dosage.

The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of a compound of the invention. The composition can be prepared for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition to obtain the appropriate preparation. Suitable formulations for use in the present invention are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. A summary of drug delivery methods is given in, for example, Langer (Science 249:15271533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral or topical administration, for example by the transdermal route, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions may be administered parenterally (eg, by intravenous, intramuscular, or subcutaneous injection) or by oral administration, or by topical application or intra-articular injection into areas affected by vascular disease or cancer. Additional routes of administration include intravascular, intraarterial, intratumoral, intraperitoneal, intraventricular, epidural, as well as nasal, ocular, intrascleral, intraorbital, rectal, topical, or inhalation.

- 23 045310 introduction of aerosol. Sustained release administration is also included in the invention and is accomplished by means such as depot injections or erodible implants or components. Thus, the invention relates to compositions for parenteral administration, which include, but are not limited to, the above agents dissolved or suspended in a suitable vehicle, preferably an aqueous vehicle, for example water, buffered water, saline or PBS (sodium phosphate buffer). The compositions may contain pharmaceutically acceptable excipients necessary to mimic physiological conditions, such as, among others, pH adjusting agents and buffering agents, tonicity adjusting agents, wetting agents or detergents. The invention also relates to compositions for oral delivery, which may contain inert ingredients, such as binders or excipients, for the preparation of a unit dosage form, such as a tablet or capsule. In addition, this invention relates to compositions for topical use, which may contain inert ingredients, such as solvents or emulsifiers, for the preparation of a cream, ointment, gel, paste or eye drops.

These compositions may be sterilized by conventional sterilization methods or may be subjected to sterilizing filtration. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the preparations is usually from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 6 to 7, for example from 6 to 6.5. The resulting compositions in solid form may be packaged in multiple single-dose containers, each containing a fixed amount of the above agent or agents, such as those contained in a sealed package of tablets or capsules. The composition in solid form may also be packaged in a customizable quantity container, such as a compressible tube for a topically applied cream or ointment.

Compositions containing an effective amount may be administered for radiation therapy planning, diagnosis, or therapeutic treatment. When administered for radiation therapy planning or diagnostic purposes, the conjugate is administered to the subject at a diagnostically effective dose and/or in an amount effective to determine a therapeutically effective dose. In therapeutic applications, the compositions are administered to a subject (eg, a human) already suffering from a condition (eg, cancer) in an amount sufficient to cure or at least partially relieve the symptoms of the disorder and its complications. An amount sufficient to achieve this purpose is defined as a therapeutically effective amount, being an amount of a compound sufficient to significantly improve at least one symptom associated with a disease or medical condition. For example, in the treatment of cancer, an agent or compound that reduces, prevents, delays, suppresses or relieves any symptom of a disease or condition will be therapeutically effective. A therapeutically effective amount of the agent or compound is not required to cure the disease or condition, but will provide treatment of the disease or condition such that the onset of the disease or condition is delayed, delayed or prevented, or the symptoms of the disease or condition are improved, or the duration of the disease or condition is changed, or, for example, it was less severe, or the individual had a faster recovery. The conjugates of the invention can be used to treat cancer by administering to a subject a first dose of any of the foregoing conjugates or compositions in an amount effective for radiation treatment planning, followed by a second dose of any of the above conjugates or compositions in a therapeutically effective amount.

Amounts effective for these applications may depend on the severity of the disease or condition, as well as the body weight and general condition of the subject. The therapeutically effective amount of the compositions of the invention used in the methods of the invention in mammals (eg, humans) can be determined by one skilled in the art, taking into account individual differences in the age, body weight and condition of the mammal. Because individual conjugates of the invention exhibit increased ability to target and retain residual amounts in cancer cells, the dosage of the compounds of the invention may be lower (e.g., less than or equal to about 90, 75, 50, 40, 30, 20, 15, 12, 10, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or 0.1% of the equivalent dose of the unconjugated agent required to achieve a therapeutic effect. The agents of the invention are administered to a subject (eg, a mammal, such as a human) in an effective amount, which is an amount that produces the desired result in the subject being treated. Therapeutically effective amounts can also be determined empirically by those skilled in the art.

Compositions of the invention comprising an effective amount may be administered single or multiple times in accordance with the dosage size and schedule selected by the attending physician. The dose and schedule of administration may be determined and adjusted depending on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment in accordance with methods commonly used by physicians or described herein

- 24 045310 those.

The conjugates of the present invention may be used in combination with conventional treatments or therapies, or may be used separately from conventional treatments or therapies.

When the compounds of this invention are administered in combination therapy with other agents, they may be administered to an individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions of the present invention may consist of a combination of a compound of the present invention in combination with a pharmaceutically acceptable excipient as described herein and another therapeutic or prophylactic agent known in the art.

The terms antiproliferative or antiproliferative agent, used interchangeably herein, mean any anticancer agent, including the antiproliferative agents listed in Table 2, any of which may be used in combination with a conjugate of the invention to treat the medical conditions listed herein. Antiproliferative agents also include organic platinum derivatives, naphthoquinone and benzoquinone derivatives, chrysophanic acid and its anthraquinone derivatives.

The terms immunoregulatory agent or immunomodulatory agent, used interchangeably herein, mean any immunomodulator, including those listed in table. 2, any of which may be used in combination with a conjugate of the invention to treat the medical conditions listed herein.

As used herein, the term radiosensitizer includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiosensitizers may include, but are not limited to, 5-fluorouracil, platinum analogs (eg, cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (eg, cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.

table 2 Alkylating agents busulfan dacarbazine ifosfamide hexamethylmelamine thiotepa dacarbazine lomustine cyclophosphamide chlorambucil procarbazine alteramine estramustine phosphate mechlorethamine streptozocin temozolomide semustine Platinum based agents spiroplatin tetraplatin ormaplatin iproplatin picoplatin oxaliplatin carboplatin lobaplatin (Aeterna) satraplatin (Johnson Matthey) BBR-3464 (Hoffmann-La Roche) miriplatin AP-5280 (Access) cisplatin Antimetabolites azacitidine floxuridine 2-chlorodeoxyadenosine 6-mercaptopurine 6-thioguanine cytarabine 2-fluorodeoxycytidine methotrexate tomudex fludarabine raltitrexed trimetrexate deoxycoformycin pentostatin hydroxyurea decitabine (SuperGen) clofarabine (Bioenvision) irofulven (MGI Pharma) DMDC (2'-deoxy-2'methylidenecytidine) (HoffmannLa Roche) ethynylcytidine (Taiho) gemcitabine capecitabine

- 25 045310

Inhibitors amsacrine exatecan mesylate (Daiichi) topoisomerases epirubicin etoposide teniposide or mitoxantrone 7-ETHYL-10-hydroxy camptothecin dexrazoxanet (TopoTarget) pixantrone (Novuspharma) rebeccamycin analogue (Exelixis) BBR-3576 (Novuspharma) rubitecan (SuperGen) irinotecan (CPT-11) topotecan Quinamed (ChemGenex) Hymatecan (Sigma-Tau) Diphlomothecan (Beaufour-lpsen) TAS-103 (Taiho) elsamitrucin (Spectrum) edotecarin cositecan belotecan hydroxycamptothecin (SN-38) Antitumor antibiotics valrubicin terarubicin idarubicin rubidazone plicamycin porphyromycin mitoxantrone (novantrone) amonafide azonafide anthrapyrazole oxantrazole losoxantrone sabarubicin epirubicin mitoxantrone doxorubicin Antimitotic agents colchicine vinblastine vindesine dolastatin 10 (NCI) rhizoxin (Fujisawa) mivobulin (Warner-Lambert) cemadotin (BASF) RPR 109881A (Aventis) TXD 258 (Aventis) epothilone B (epothilone B) T 900607 (Tula ri k) T 138067 (Tula ri k) cryptophycin 52 (Eli Lilly) vinflunine (Fabre) auristatin PE (Teikoku Hormone) BMS 247550 (BMS) BMS 184476 (BMS) BMS 188797 (BMS) taxoprexin (Protarga) SB 408075 (GlaxoSmithKline) vinorelbine trichostatin A E7010 (Abbott) PG-TXL (Cell Therapeutics) IDN5109 (Bayer) A 105972 (Abbott) A 204197 (Abbott) LU 223651 (BASF) D 24851 (ASTAMedica) ER-86526 (Eisai) combretastatin-A4 (BMSC) isohomohalichondriin-B (PharmaMar) ZD 6126 (AstraZeneca) AZ10992 (Asahi) IDN-5109 (Indena) AVLB (Prescient NeuroPharma) azaepothilone B (BMS) BNP-7787 (BioNumerik) CA-4 prodrug (OXiGENE) dolastatin-10 (NIH) CA-4 (OXiGENE) docetaxel vincristine paclitaxel Aromatase inhibitors aminoglutethimide atamestane (BioMedicines) letrozole anastrozole YM-511 (Yamanouchi) formestane exemestane Thymidylate synthase inhibitors pemetrexed (Eli Lilly) ZD-9331 (BTG) nolatrexed (Eximias) CoFactor™ (BioKeys)

- 26 045310

DNA antagonists trabectedin (PharmaMar) glufosfamide (Baxter International) albumin + 32P (Isotope Solutions) timektacin (NewBiotics) edotreotide (Novartis) mafosfamide (Baxter International) apaziquone (Spectrum Pharmaceuticals) 6-O-benzylguanine (Paligent) Farnesyltransferase inhibitors arglabin (NuOncology Labs) lonafarnib (Schering-Plough) BAY-43-9006 (Bayer) tipifarnib (Johnson & Johnson) perillyl alcohol (DOR BioPharma) Proton pump inhibitors CBT-1 (CBA Pharma) tariquidar (Xenova) MS-209 (Schering AG) zosuquidar trihydrochloride (Eli Lilly) biricodar dicitrate (Vertex) Hitone acetyltransferase inhibitors tacedinaline (Pfizer) SAHA (Aton Pharma) MS-275 (Schering AG) pivaloyloxymethyl butyrate (Titan) depsipeptide (Fujisawa) Metalloproteinase inhibitors neovastat (Aeterna Laboratories) marimastat (British Biotech) SMT-3 (CollaGenex) BMS-275291 (Celltech) Ribonucleoside reductase inhibitors gallium maltolate (Titan) triapine (Vion) tezacitabine (Aventis) didox (Molecules for Health) Agonists/antagonists TNF-a virulysin (Lorus Therapeutics) CDC-394 (Celgene) revimid (Celgene) Endothelin A receptor antagonist atrasentan (Abbott) ZD-4054 (AstraZeneca) YM-598 (Yamanouchi) Retinoic acid receptor agonists fenretinide (Johnson & Johnson) LGD-1550 (Ligand) alitretinoin (Ligand) Immunomodulators Interferon oncophage (Antigenics) GMK (Progenies) adenocarcinoma vaccine (Biomira) CTP-37 (AVI BioPharma) IRX-2 (Immuno-Rx) PEP-005 (Peplin Biotech) synchrovax vaccines (CTL Immuno) melanoma vaccine (CTL Immuno) p21 RAS vaccine (GemVax) MAGE-A3 (GSK) nivolumab (BMS) abatacept (BMS) pembrolizumab (Merck) dexosomal therapy (Anosys) pentrix (Australian Cancer technology) ISF-154 (Tragen) cancer vaccine (Intercell) norelin (Biostar) BLP-25 (Biomira) MGV (Progenies) beta-alethine (Dovetail) CLL therapy (Vasogen) ipilimumab (BMS), CM-10 (cCam Biotherapeutics) atezolizumab (Genentech) Hormonal and antihormonal agents estrogens conjugated estrogens ethinyl estradiol chlorotrianisene idenestrol hydroxyprogesterone caproate medroxyprogesterone testosterone testosterone propionate; fluoxymesterone methyltestosterone diethylstilbestrol megestrol bicalutamide flutamide nilutamide dexamethasone prednisone methylprednisone prednisolone aminoglutethimide leuprolide octreotide mitotane R-04 (Novogen) 2-methoxyestrad iol (EntreMed) arzoxifene (Eli Lilly) tamoxifen toremofin goserelin leuprorelin bicalutamide Agents for photodynamic therapy talaporphine (Light Sciences) teralux (Theratechnologies) gadolinium motexafine (Pharmacyclics) Pd-bacteriopheophorbide (Yeda) lutetium motexafin hypericin

- 27 045310

Kinase inhibitors Imatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) cahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4 (Genentech) PK1166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774 (Tarceva™) gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) emodin genistein radicinol radicinol vemurafenib (inhibitor Met-MAb (Roche) B-Raf enzyme, Daiichi Sankyo)

SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) tocladesine (cyclic AMP agonist, Ribapharm) alvocidib (CDK inhibitor, Aventis) CV-247 (COX-2 inhibitor, Ivy Medical) P54 (COX-2 inhibitor, Phytopharm)

CapCell™ (CYP450 stimulator, Bavarian Nordic) GCS-100 (gal3 antagonist, GlycoGenesys) immunogen G17DT (gastrin inhibitor, Aphton) efaproxiral (oxygenator, Alios Therapeutics) PI-88 (heparanase inhibitor, Progen) tesmilifen (histamine antagonist, YM BioSciences) histamine (H2-histamine receptor agonist, Maxim) thiazofurin (IMPDG inhibitor, Ribapharm) cilengitide (integrin antagonist, Merck KGaA) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo)

CCI-779 (mTOR kinase inhibitor, Wyeth) exisulind (PDE5 inhibitor, Cell Pathways) CP-461 (PDE5 inhibitor, Cell Pathways) AG-2037 (GART inhibitor, Pfizer)

WX-UK1 (plasminogen activator inhibitor, Wilex) PBI-1402 (PMN stimulator, ProMetic LifeSciences) bortezomib (proteasome inhibitor, Millennium)

SRL-172 (T cell stimulator, SR Pharma)

TLK-286 (glutathione 5-transferase inhibitor, Telik) PT-100 (growth factor agonist, Point Therapeutics) midostaurin (PKC inhibitor, Novartis) bryostatin-1 (PKC stimulator, GPC Biotech) CDA-II (apoptosis promoter, Everlife) SDX-101 (Apoptosis promotor, Salmedix) Rituximab (antibodies to CD20, Genentiech karmustin mitoxantron Bleomycin Herisophanic acid cesium oxides BREF inhibitors, PD-L1 inhibitors MEC BVACOMAB Inhibitors Dubrafeniba Cyphe Latonin (apoptosis promotor, Chemgenex)

BCX-1777 (PNP inhibitor, BioCryst) ranpirnase (ribonuclease stimulator, Alfacell) galarubicin (RNA synthesis inhibitor, Dong-A) tirapazamine (reducing agent, SRI International)

N-acetylcysteine (reducing agent, Zambon) R-flurbiprofen (NF-kB inhibitor, Encore)

ZCPA (NF-κB inhibitor, Active Biotech) seocalcitol (vitamin D receptor agonist, Leo) 131-I-TM-601 (DNA antagonist, TransMolecular) eflornithine (ODC inhibitor, ILEX Oncology) minodronic acid (osteoclast inhibitor, Yamanouchi) indisulam ( p53 stimulator, Eisai) aplidine (PPT inhibitor, PharmaMar) gemtuzumab (anti-CD33 antibody, Wyeth Ayerst)

PG2 (hematopoiesis enhancer, Pharmagenesis)

Immunol™ (triclosan mouthwash, Endo) triacetyluridine (uridine prodrug, Wellstat)

SN-4071 (sarcoma agent, Signature BioScience) TransMID-107™ (immunotoxin, KS Biomed ix)

RCK-3145 (apoptosis promoter, Procyon) doranidazole (apoptosis promoter, Pola) CHS-828 (cytotoxic agent, Leo) trans-retinoic acid (differentiating agent, NIH) MX6 (apoptosis promoter, MAXIA) apomin (apoptosis promoter, ILEX Oncology) urocidin (apoptosis promoter, Bioniche) Ro-31-7453 (apoptosis promoter, La Roche) brostallicin (apoptosis promoter, Pharmacia) β-lapachone gelonin cafestol kahweol caffeic acid tyrphostin AG PD-1 inhibitors CTLA-4 inhibitors sorafenib

The following examples are intended to illustrate the synthesis of a number of illustrative conjugates and the use of these conjugates for the treatment of cancer. Accordingly, the following examples are intended to illustrate and not limit the invention. Additional compounds not shown in the examples can be synthesized using conventional methods in combination with the methods described herein.

Examples.

- 28 045310

Example 1: General materials and methods.

The antibodies used were HuMIgG (Aldrich, I4506) and HuMIGF-1R (AVE1642). Lutetium-177 was obtained from Perkin Elmer as 0.05N lutetium chloride. hydrochloric acid solution.

Analytical HPLC-MS (high performance liquid chromatography with mass spectrometric detection) was performed using a Waters Acquity HPLC-MS system consisting of a Waters Acquity binary pump, a Waters Acquity sample manager (samples cooled to 10°C), a Waters Acquity column manager (column temperature 30 °C), Waters Acquity photodiode array detector (monitoring at 254 nm and 214 nm), Waters Acquity tandem quadrupole mass detector (TQD) with electrospray ionization and Waters Acquity VEN C18 column, 2.1x50 (1.7 µm) . Preparative HPLC was performed using a Waters HPLC system consisting of a Waters 1525 binary HPLC pump, a Waters 2489 UV/Vis detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C18 19x100 mm (5 μm) column.

HPLC elution method 1: Waters Acquity BEH C18 column 2.1x50 mm (1.7 µm); mobile phase A: H ₂ O (0.1 vol.% TFA (trifluoroacetic acid)); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate = 0.3 ml/min; initially = 90% A, 3-3.5 min = 0% A, 4 min = 90% A, 5 min = 90% A.

HPLC elution method 2: Waters XBridge Prep Phenyl 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 80% A, 13 min = 0% A.

HPLC elution method 3: Waters Acquity BEH C18 column 2.1x50 mm (1.7 µm); mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate = 0.3 ml/min; initially = 90% A, 8 min = 0% A, 10 min = 0% A, 11 min = 90% A, 12 min = 90% A.

HPLC elution method 4: Waters XBridge Prep C18 OBD 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 80% A, 3 min = 80% A, 13 min = 20% A, 18 min = 0% A.

HPLC elution method 5: Waters XBridge Prep C18 OBD 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 90% A, 3 min = 90% A, 13 min = 0% A, 20 min = 0% A.

HPLC elution method 6: Waters XBridge Prep C18 OBD 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 75% A, 13 min = 0% A, 15 min = 0% A.

HPLC elution method 7: Waters XBridge Prep C18 OBD 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 80% A, 12 min = 0% A, 15 min = 0% A.

HPLC elution method 8: Waters XBridge Prep C18 OBD 19x100 mm (5 µm) column; mobile phase A: H ₂ O (0.1 vol.% TFA); mobile phase B: acetonitrile (0.1 vol.% TFA); flow rate: 10 ml/min; initially = 90% A, 12 min = 0% A, 15 min = 0% A.

Analytical size exclusion chromatography (SEC) was performed using a Waters system consisting of a Waters 1525 binary HPLC pump, a Waters 2489 UV/Vis detector (monitored at 280 nm), a Bioscan Flow Count radiation detector (FC-3300), and a TOSOH column. TSKgel G3000SWxI, 7.8x300 mm. For the isocratic SEC method, flow rate = 1 mL/min was used with the mobile phase being 0.1 M phosphate, 0.6 M NaCl, 0.025% sodium azide, pH = 7.

MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) (positive ion mode) was performed using a Bruker Ultraflextreme MALDI spectrometer.

Radiothin layer chromatography (radioTLC) performed using a Bioscan AR-2000 imaging scanner was performed on iTLC-SG glass microfiber chromatography paper plates (Agilent Technologies, SGI0001) using citrate buffer (0.1 M, pH 5.5 ).

Example 2 Synthesis of [ ¹⁷⁷ Lu] compound A-HuMIGF-1R (commercial standard).

Bifunctional chelating agent 2,2',2-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA anhydride -GA, Compound A) was obtained from CheMatech.

Compound A (3.0 μmol) was dissolved in sodium acetate buffer (0.228 ml, pH 6.5). An aliquot of Compound A solution (8 μL, 106 nmol) was added to a solution containing HuMIGF-1R antibody (6.7 nmol, AVE1642) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate (compound A)-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion mode): (compound A)-HuMIGF-1R: found m/z 151759; HuMIGF-1R: m/z 149835 found.

As a typical reaction, Lu-177 (1.1 mCi, 14 μl) was added to a solution of (compound A)HuMIGF-1R (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate bottle

- 29 045310 ferre (pH 6.5)). The reaction of adding a radioactive label was carried out by incubation at 37°C for 30 minutes. The crude product, [ ¹⁷⁷ Lu] compound A-HuMIGF-1R, was purified through a column packed with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid). SEC retention time: 8.1 min; radiochemical purity in radioTLC: 99%; radiochemical yield: 74%; specific radioactivity: 8.2 mCi/mg.

Example 3. Synthesis of 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]butanoic acid (compound B).

Bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2[4,7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl]butanoic acid (compound B) was synthesized according to the scheme presented in Fig. 2. To a solution of 5-(tert-butoxy)-5-oxo4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1- yl)pentanoic acid (DOTA-GA-(tBu) ₄ , 50 mg, 0.07 mmol) in ACN (acetonitrile) (2.0 ml) was added DSC (50 mg, 0.21 mmol), followed by pyridine (0 .20 ml, 2.48 mmol). The reaction mixture was stirred at room temperature for 1 hour. 11-Aminoundecanoic acid (70 mg, 0.36 mmol) was added to the reaction mixture, followed by PBS solution (1.0 mL) at room temperature. The reaction mixture was stirred for 72 hours at room temperature. The reaction mixture was filtered through a syringe filter and purified directly by preparative HPLC using method 6 to give intermediate 2-A (71 mg, 74.8%).

Pyridine (0 .05 ml, 50 mg, 0.62 mmol) at room temperature. The solution was stirred at room temperature for 24 hours. The reaction mixture was purified directly by preparative HPLC using Method 7 to give intermediate 2-B (33 mg, 82.5%) as a wax after concentration using a Biotage V10 flash evaporation apparatus.

Intermediate 2-B (33 mg, 0.022 mmol) was dissolved in DCM/TFA (1.0 mL/2.0 mL) and left stirring at room temperature for 24 hours. The reaction mixture was concentrated with a stream of air and purified directly by preparative HPLC using Method 8 to give Compound B (14 mg, 50.0%) as a colorless wax after concentration. An aliquot was analyzed by HPLC-MS using elution method 3; retention time: 4.15 minutes; MS (positive ESI): found m/z 808.1 [M+H]+; C3 ₆ H54F4N5On (calc. 808.8).

^1H NMR (600 MHz, DMSO-d6) δ 7.99 - 7.88 (m, 1H), 7.82 (t, J = 5.5 Hz, 1H), 3.78 (broad s, 4H) , 3.43 (wide s, 12H), 3.08 (wide s, 4H), 3.00 (m, 3H), 2.93 (wide s, 3H), 2.77 (t, J = 7, 2 Hz, 2H), 2.30 (wide s, 2H), 1.88 (wide s, 2H), 1.66 (p, J = 7.3 Hz, 2H), 1.36 (m, 4H) , 1.32 - 1.20 (m, 9H).

Example 4. Synthesis of [ ¹⁷⁷ Lu]-compound B-HuMIGF-1R.

Compound B (0.7 µmol) was dissolved in sodium acetate buffer (69 µl, pH 6.5). An aliquot of compound B solution (4 μl, 40 nmol) was added to a solution containing HuMIGF-1R antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF (time of flight mass spectrometry)-MS (in positive ion detection mode): compound BHuMIGF-1R: found m/z 152988 [M+H]+; HuMIGF-1R: found m/z 149835 [M+H] ⁺ .

As a typical reaction, Lu-177 (1.15 mCi, 14 μl) was added to a solution of compound BHuMIGF-1R (75 μg in acetate buffer (pH 6.5)) and ascorbic acid (1 μl, 0.1 M in acetate buffer (pH 6.5)). The reaction of adding a radioactive label was carried out by incubation at 37°C for 30 minutes. The crude product, [ ¹⁷⁷ Lu] compound C-HuMIGF-1R, was purified using a column packed with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid). Radiochemical purity in radioTLC: 99%; radiochemical yield: 75%; specific radioactivity: 11.9 mCi/mg.

Example 5. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7, 10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododecan-1-yl]butanoic acid (compound C).

Bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10 -Tris(carboxymethyl)-1,4,7,10tetraazacyclododecan-1-yl]butanoic acid (compound C) was synthesized according to the scheme shown in FIG. 3. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2oxoethyl)-1,4,7,10-tetraazacyclododecane-1- yl)pentanoic acid (DOTA-GA(tBu) ₄ , 100 mg, 0.143 mmol) in ACN (8.0 ml) was added DSC (73 mg, 0.285 mmol) and pyridine (0.80 ml, 9.89 mmol). The reaction mixture was stirred for 90 minutes at room temperature. This solution was added to a half-solution of amino-PEG3 acid (63 mg, 0.285 mmol in 1.2 ml DMF (N,N-dimethylformamide)) in a 100 ml round bottom flask. After 4 hours at ambient temperature the product was isolated

- 30 045310 reactions, carrying out concentration to dryness in a stream of air. The crude material was purified by HPLC using elution method 2 (crude product dissolved in 6 ml of 20% ACN/H ₂ O). Fractions containing product were combined and concentrated in vacuo and then co-evaporated with ACN (3 x 2 ml). Intermediate 1-A was obtained in 82% yield.

ACN (2 mL), NEt ₃ (50 μL, 360 μmol, 6 eq.), HBTU (23 mg, 60 μmol, 1 eq.) were added to the tube containing intermediate 1-A (82 mg, 60 µmol). and TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μl ACN). The resulting clear solution was stirred at ambient temperature for 3 hours. The reaction product was isolated by concentrating the solution to dryness in a stream of air and then diluted with ACN/H ₂ O (1:1, 3 ml total) and purified by preparative HPLC using elution method 4. Fractions containing the product were combined and concentrated in vacuo. and then co-evaporated with ACN (3x2 ml). Intermediate 1-B was obtained as a clear residue (67 mg, 74% yield).

DCM (2 mL) and TFA (2 mL) were added to the tube containing intermediate 1-B (67 mg, 64 μmol) and the resulting solution was stirred at ambient temperature for 16 hours. Additional TFA (2 ml) was added and the reaction mixture was stirred at ambient temperature for 6 hours. The reaction mixture was concentrated to dryness under a stream of air, after which the crude product was finally dissolved in ACN/H ₂ O (1 ml 10% ACN/H ₂ O). The crude reaction solution was then purified by preparative HPLC using elution method 5. Product containing fractions were pooled, frozen and lyophilized. Compound C was obtained as a white solid (36 mg, 63% yield). An aliquot was analyzed by HPLC-MS using elution method 3; retention time: 3.11 min; MS (positive ESI): found m/z 828.4 [M+H]+; C ₃₄ H ₅₀ F ₄ N ₅ O ₁₄ (calc. 828.3).

^1H NMR (DMSO-d6, 600 MHz) δ 7.97-7.91 (m, 2H), 3.77 (t, 2H, J = 6.0 Hz), 3.58-3.55 (m , 2H), 3.53-3.48 (m, 8H), 3.44-3.38 (m, 10H), 3.23-3.08 (m, 11H), 3.02 (t, 2H , J = 6.0 Hz), 2.93 (wide s, 4H), 2.30 (wide s, 2H), 1.87 (wide s, 2H).

Example 6. Synthesis of [ ¹⁷⁷ Lu] compound C-HuMIGF-1R.

Compound C (17.5 μmol) was dissolved in sodium acetate buffer (1.32 ml, pH 6.5). An aliquot of compound C solution (8 μl, 91 nmol) was added to a solution containing HuMIGF-1R antibody (13.4 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (in positive ion detection mode): compound C-HuMIGF-1R: found m/z 152166 [M+H]+; HuMIGF-1R: found m/z 149724 [M+H] ⁺ .

As a typical Lu-177 reaction (1.6 mCi, 16 μl), CHuMIGF-1R (150 μg in acetate buffer (pH 6.5)) and ascorbic acid (1 μl, 0.1 M in acetate buffer) were added to the solution. pH 6.5).The radiolabel addition reaction was carried out by incubating at ambient temperature for 20 minutes. [ ¹⁷⁷ Lu]-compound C-HuMIGF-IR was purified using a column packed with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid) Radiochemical purity by radioTLC: 99%, radiochemical yield: 91%, specific radioactivity: 15.6 mCi/mg.

Example 7: Saturation Binding Experiments.

In saturation binding experiments, the specific binding at steady state of a radioconjugate is measured at various concentrations to determine K _d (the concentration of ligand that binds half of the receptor sites at steady state) and B _max (the maximum number of binding sites). In this type of binding assay, both total and nonspecific binding are measured, where receptor specific binding is calculated by subtracting the difference. Nonspecific binding is typically assessed by measuring radioconjugate binding in the presence of a fixed concentration of HumIGF-1R, which binds to virtually all receptors. Since all receptors are occupied by HumIGF-1R, the radioconjugate binds only nonspecifically. Kd and _Bmax values are calculated using nonlinear regression analysis and computer curve fitting.

The goal of this analysis was to verify that these new radioconjugates retain binding characteristics consistent with the native antibody in the IGF-1R-expressing A431 cell line. Twenty-four hours before the start of the experiment, 1.5 x 105 A431 cells were seeded into 48-well microplates in 500 μl of medium with supplemented nutrients. The radioconjugate was diluted with binding buffer (PBS + 0.5% BSA) to a concentration range of 0.08 to 40 nM; the final concentration during the analysis ranged from 0.04 to 20 nM. At the beginning of the assay, the medium was aspirated, discarded, and 500 μl of serum-free DMEM (Dulbecco's modified Eagle's medium) was added to each well. The plates were incubated at 37°C for 1 hour. After incubation, the medium was aspirated from each well and discarded. The cells were washed and 100 μl of binding buffer (complete binding) or 4 μM cold antibody (nonspecific binding) was added to the given wells. The plates were incubated

- 31 045310 at 4°C for 1 hour with gentle shaking. After the blocking step, 100 μl of radioconjugate was added to each well. The plates were then incubated at 4°C for 2 hours. After incubation, the contents of each well were aspirated and discarded. Cells were washed twice with PBS and then lysed with 1% Triton-X-100. Lysates were transferred to radioactivity count tubes and measured with radioconjugate standards on a Wizard 1470 gamma counter to determine the amount of radioactivity (in counts per minute (cpm)) for each lysate. The remaining lysate from each well (25 μl) was transferred to a 96-well plate, and the protein content of each lysate was determined using a standard quantitative protein assay. Total, nonspecific, and specific ligand binding results and the mass of bound conjugate in each lysate were calculated by converting the cpm of the lysate to fmol using the specific activity of the conjugate standards and then normalizing the bound quantity in fmol to the protein content of each lysate (in milligrams). Specific binding was determined by subtracting nonspecific binding from total binding. Total, specific and nonspecific binding values (fmol/mg) were plotted (Y-axis) as a function of conjugate concentration (nM, X-axis) as shown in the table below. K _d and B _max were obtained by curve fitting of the specific binding data according to the hyperbolic single-site binding model (GraphPad Prism Software, version 7).

The results showed that changes in the linker did not result in a change in binding affinity. In addition, these changes did not alter binding or target specificity.

Design Binding affinity (Kd) [ ¹⁷⁷ bh]-compound A-HuMIGF-1R 2.9 nM [ ¹⁷⁷ bh]-compound B-HuMIGF-1R 2.0 nM [ ¹⁷⁷ bh]-compound C-HuMIGF-1R 2.2 nM

Example 8: Residue experiments.

The residue assay was designed to determine the extent to which radiolabeled antibody derivatives with linkers are retained in cells. This assay relies on the inherent ability of the IGF-1 receptor to be internalized upon ligand binding and the ability to track radiolabeled compounds. In this type of binding experiment, a constant amount of radioconjugate is incubated with an IGF-1R expressing cell line for a fixed period of time. After incubation, cells are cleaned with a mild acid buffer to remove any external or membrane-bound radioconjugate. Fresh medium is reintroduced and the cells are again incubated for the specified amount of time. It is during this period that cellular processes degrade the radioconjugate and thereby efflux of radioactive fragments back into the culture medium or retention of radioactive fragments in the cell occurs. The residual content is determined by calculating the amount of internalized radioactive material as a percentage of the total activity associated with the cells after the acid wash.

A431 cells were seeded in 24-well plates at a concentration of 2.5x105 cells/well in complete medium (DMEM). After overnight incubation, the medium containing the cells was replaced with serum-free DMEM and incubated for 1 h at 37°C. The medium was discarded and the plates were washed once with sterile PBS. The radioconjugate was diluted in serum-free DMEM to a concentration of 2 nM. Each well was loaded with 500 μl of radioconjugate and incubated for 4 hours at 37°C. After incubation, the plates were immediately placed on ice and the medium was discarded into pre-labeled (unbound) tubes for gamma counting. Cells were washed once with sterile PBS, gently vortexed, and poured into (unbound) gamma counting tubes. Mild acidic wash buffer (pH 4.6, 500 μl) was added to all wells. The plates were incubated at 4°C for 15 min and the buffer was collected into pre-labeled gamma counting tubes (membrane bound). 1 ml of warmed serum-free medium was added to all wells and the plates were incubated at 37°C for 0 and 24 hours. After the prescribed incubation, the plates were placed on ice and processed as follows. The medium was decanted and collected into labeled (efflux) tubes for gamma measurement. The plates were then washed once with 1 ml of cold PBS and collected into efflux tubes. Acid wash buffer (pH 2.5, 500 μl) was added to all wells, and the plates were incubated for 5 min on ice. The acid wash fraction was then collected into labeled (recirculated) tubes for gamma measurement. Cells were lysed with 300 μl of 1% Triton X-100 for 30 min at room temperature. 250 μl of cell lysate was transferred to gamma counting tubes and counted for 10 min. 25 μl of the cell lysate fraction was transferred to a 96-well plate for protein quantitation (Pierce BCA protein assay). The percentage of residue (Figure 4) was determined as cpm (lysate)/cpm (efflux+recirculated+lysate).

In vitro residue experiments demonstrated that conjugation with

- 32 045310 different linkers provided radioconjugates that were virtually identical in terms of cellular internalization and retention, indicating that these properties of the monoclonal antibody were not altered by conjugation. In addition, these data suggest that radioimmunoconjugates are likely to undergo similar catabolic degradation after internalization into tumor cells in vivo, regardless of the attached linker structure.

Example 9. Results of studies of the pharmacokinetics and metabolism of HuMIGF-1R compounds.

Groups of 4 or 5 mice (CD-1 normal or CD-1 athymic nude) were intravenously injected with approximately 15 μCi of radiolabeled test compound. Immunoconjugates with various linkers were synthesized and radiolabeled with lutetium-177. For pharmacokinetic studies, animals were sacrificed at specified time points and blood and tumor (if applicable) were analyzed for total radioactivity. For metabolic studies, animals were housed in metabolic cages (4–5 per cage) to collect urine and feces every 24 h for up to 7 days. The radioactive content of urine and stool samples was quantified and converted to total urine or stool based on body weight. Excretion profiles for urine, feces, or total excretion (urine + feces) were obtained by plotting the total % administered dose (% ID) versus time.

The metabolic excretion profile of [ ¹⁷⁷ Lu]-compound B-HuMIGF-1R and [ ¹⁷⁷ Lu]-compound CHuMIGF-1R were compared with [ ¹⁷⁷ Lu]-compound A-HuMIGF-1R. It was found that although the type of linker influenced the route, rate and extent of excretion of the compound (Figure 5), it did not affect the overall pharmacokinetics of the total radioactivity associated with the radioimmunoconjugate (Figure 6). The [ ¹⁷⁷ Lu] compound AHuMIGF-1R was eliminated slowly, and only 13% of the administered dose (ID) was eliminated within 7 days through low-level urinary excretion. In contrast, excretion of the [ ^177Lu ]-compound B-HuMIGF-1R resulted in a 210% increase, and excretion of the [ ^177Lu ]-compound C-HuMIGF-1R was 310% higher. This rank order of excretion was similar for several antibodies tested with Compound C providing the highest excretion. In addition, compounds B and C exhibit completely different excretion pathways: [ ¹⁷⁷ Lu]-compound B-HuMIGF1R was eliminated predominantly through feces, while elimination of [ ¹⁷⁷ Lu]-compound C-HuMIGF-1R was distributed approximately equally between urine and feces. This excretion pattern was also similar among several biological targeting vectors tested.

Example 10 Radiotherapeutic efficacy.

The therapeutic efficacy of [ ²²⁵ Ac] compound A-HuMIGF-1R, [ ²²⁵ Ac] compound B-HuMIGF1R and [ ²²⁵ Ac] compound C-HuMIGF-1R were compared with HuMIGF-1R alone and vehicle control. The synthesis routes for radiolabeled actinium-225 (Ac-225) compounds were similar to those for the corresponding Lu-177 analogues. Therapeutic efficacy studies were performed using the IGF-1R overexpressing colon cancer cell line Colo-205 (ATCC #CCL-222). Tumor xenografts were engrafted into female Balb/c athymic nude mice aged 5–7 weeks (Charles River Laboratories). Two (2) million cells mixed with PBS and Matrigel (Becton Dickinson) in a volume ratio of 50:50 were injected subcutaneously into the lower right quadrant over the thigh of each animal. The tumors were allowed to grow for 7-10 days to an initial volume of approximately 200 mm ³ . Groups of animals with tumors (n=4-8) were injected with 200 μl of the test drug intravenously through the lateral tail vein. The studied drugs with an Ac-225 labeled compound were administered at an activity dose of 20-400 nanocuries (nCi) in the form of a mixture with 20 mM sodium citrate, pH 5.5, 0.82% NaCl and 0.01% Tween 80. As a control, unconjugated the antibody (HuMIGF-1R) without a radioactive label was administered at an equivalent protein mass corresponding to the highest dose of radioactivity of the studied radioimmunoconjugates with actinium-225. Tumor measurements were performed 2-3 times a week using vernier calipers in two dimensions. Tumor length was defined as the longest dimension, and width was measured perpendicular to the length of the tumor. Along the way, the animals were weighed. General physical condition and general behavior were assessed daily. A typical study lasted 28 days. Tumor volume (mm ³ ) was calculated as an ellipsoid using caliper measurements. Tumor growth was expressed as relative tumor volume (RTV), which is the tumor volume measured on day X divided by the tumor volume measured on dosing day.

The therapeutic efficacy of [ ²²⁵ Ac] compound A-HuMIGF-1R, [ ²²⁵ Ac] compound B-HuMIGF1R and [ ²²⁵ Ac] compound C-HuMIGF-1R was almost the same for all compounds; however, all radioimmunoconjugates containing actinium-225 demonstrate higher efficiency than the non-radioactive control HuMIGF-1R.

Example 11. Synthesis of [ ¹⁷⁷ Lu]-compound A-human-IgG.

Compound A (1.34 μmol) was dissolved in sodium acetate buffer (20 μl, pH 6.5) and added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature, the resulting immunoconjugate was purified using a SEC HPLC column (1 ml/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). Antibody conjugate compound A-human-IgG. MALDI-TOF-MS (in registration mode, put

- 33 045310 body ions): compound A-human-IgG: found m/z 150360 [M+H]+; human-IgG: found m/z 148339 [M+H] ⁺ .

As a typical reaction, Lu-177 (1.1 mCi, 5 μl) was added to a solution of human A-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( ^pH 6.5). 50, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid) Radiochemical purity in radioTLC: 98%, radiochemical yield: 45%, specific radioactivity: 15.1 mCi/mg.

Example 12. Synthesis of [ ¹⁷⁷ Lu]-compound B-human-IgG.

Compound B (1.17 μmol) was dissolved in sodium acetate buffer (0.117 ml, pH 6.5). An aliquot of compound B solution (2 μl, 10 nmol) was added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). The human IgG preparation used consisted of a purified mixture of all IgG isotypes (IgG1-4). After 1 hour at ambient temperature, the antibody conjugate product was purified using a column packed with Sephadex G-50 resin. The Human-IgG compound was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (in positive ion detection mode): compound B-human-IgG: found m/z 149949 [M+H]+; human-IgG: found m/z 148540 [M+H] ⁺ .

As a typical reaction, Lu-177 (1.1 mCi, 5 μl) was added to a solution of Bhuman-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH ^6.5 ). buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa) Radiochemical purity by radioTLC: 98%, radiochemical yield: 51%, specific radioactivity: 9.68 mCi/mg.

Example 13. Synthesis of [ ¹⁷⁷ Lu]-compound C-human-IgG.

Compound C (0.96 μmol) was dissolved in sodium acetate buffer (95 μl, pH 6.5). An aliquot of compound C solution (2 μl, 20 nmol) was added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate product was purified using a column packed with Sephadex G-50 resin. Compound C-human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (in positive ion mode): compound C-human-IgG: found m/z 150095 [M+H]+; human-IgG: found m/z 148540 [M+H] ⁺ .

As a typical reaction, Lu-177 (1.1 mCi, 5 μl) was added to a solution of Human C-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH 6.5 ⁾ . eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa) Radiochemical purity by radioTLC: 98%, radiochemical yield: 37%, specific radioactivity: 9.99 mCi/mg.

Example 14. Results of studies of the pharmacokinetics and metabolism of compounds based on HuMIgG.

Untargeted human IgG antibodies were used for metabolic excretion studies to demonstrate that changes in radioactive excretion profiles caused by linker compound B and compound C conjugation represent a general process, demonstrating that these data are not limited to the HuMIGF-1R antibody. Pharmacokinetics and metabolism studies were performed using [ ¹⁷⁷ Lu] compound A-HuMIgG, [ ¹⁷⁷ Lu] compound B-HuMIgG and [ ¹⁷⁷ Lu] compound C-HuMIgG as described for the HuMIGF-1R antibody compounds described previously.

The metabolic excretion profile of untargeted human IgG radioimmunoconjugates [ ¹⁷⁷ Lu]-compound B-HuMIgG and [ ¹⁷⁷ Lu]-compound C-HuMIgG were compared with [ ¹⁷⁷ Lu]-compound AHuMIgG. As described for HuMIGF-1R-based radioimmunoconjugates, it was found that although the type of linker influenced the path, rate and extent of excretion of the compound (Fig. 8), it did not affect the overall pharmacokinetics of the total radioactivity associated with the radioimmunoconjugate. HuMIgG-based compounds showed the same rank order of excretion rates as HuMIGF-IR-based compounds; that is, the radioimmunoconjugate containing compound C provided the greatest degree of excretion. The [ ¹⁷⁷ Lu] compound A-HuMIgG was excreted slowly, and only 13% of the administered dose (ID) was eliminated within 7 days through low-level urinary excretion. In contrast, excretion of the [ ¹⁷⁷ Lu]-compound B-HuMIGF-1R resulted in a 196% increase, and excretion of the [ ¹⁷⁷ Lu]-compound C-HuMIGF-1R was 216% higher. In addition, compounds B and C have completely different excretion pathways. [ ¹⁷⁷ Lu] compound B-HuMIgG was eliminated preferentially

- 34 045310 venously through feces, while the elimination of [^T^-compound C-HuMIgG was distributed approximately equally between urine and feces. This metabolic profile was essentially equivalent to HuMIGF-1R-based compounds, indicating that the improved excretion profile of Compound B or Compound C when conjugated to antibodies is a general and reproducible effect.

Example 15. Synthesis of [ ²²⁵ Ac]-compound A-human-IgG.

Compound A (1.34 μmol) was dissolved in sodium acetate buffer (20 μl, pH 6.5) and added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 45 min at ambient temperature, the antibody conjugate product was purified using a SEC HPLC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). MALDITOF-MS (positive ion mode): Compound A -human-IgG: found m/z 150360 [M+H]+; human-IgG: found m/z 148339 [M+H] ⁺ .

As a typical reaction, Ac-225 (1.1 mCi, 5 μl) was added to a solution of human A-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH ^6.5 ). using a column packed with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 16. Synthesis of [ ²²⁵ Ac]-compound B-human-IgG.

Compound B (1.17 μmol) was dissolved in sodium acetate buffer (0.117 ml, pH 6.5). An aliquot of compound B solution (2 μl, 10 nmol) was added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified using a column packed with Sephadex G-50 resin. The compound A-human-IgG antibody conjugate was eluted from the column with acetate buffer (pH 6.5). MALDITOF-MS (in positive ion detection mode): B-human-IgG compound: found m/z IgG [M+H]+; human-IgG: found m/z 148540 [M+H] ⁺ .

As a typical reaction, Ac-225 (1.1 mCi, 5 μl) was added to a solution of human IgG compound (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH 6.5) ^. HPLC SEC (1 ml/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa).

Example 17. Synthesis of [ ²²⁵ Ac]-compound C-human-IgG.

Compound C (0.96 μmol) was dissolved in sodium acetate buffer (95 μl, pH 6.5). An aliquot of compound C solution (2 μl, 20 nmol) was added to a solution containing human-IgG antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the antibody conjugate product was purified using a column packed with Sephadex G-50 resin. The human C-IgG conjugate antibody was eluted from the column with acetate buffer (pH 6.5). MALDITOF-MS (in positive ion detection mode): compound C-human-IgG: found m/z 150095 [M+H]+; human-IgG: found m/z 148540 [M+H] ⁺ .

As a typical reaction, Ac-225 (1.1 mCi, 5 μl) was added to a solution of human IgG compound (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH 6.5) ^. HPLC SEC (1 ml/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa).

Example 18. Synthesis of [ ²²⁵ Ac]-compound A-HuMIGF-1R.

Compound A (3.0 μmol) was dissolved in sodium acetate buffer (0.228 ml, pH 6.5). An aliquot of Compound A solution (8 μL, 106 nmol) was added to a solution containing HuMIGF-1R antibody (6.7 nmol, AVE1642) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate (compound A)-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion mode): (compound A)-HuMIGF-1R: found m/z 151759; HuMIGF-1R: m/z 149835 found.

As a typical reaction, Ac-225 (1.1 mCi, 14 μl) was added to a solution of (compound A)HuMIGF-1R (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μl, 0.1 M in acetate buffer (pH ^6.5 )). using a column packed with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid).

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Claims (40)

Example 19. Synthesis of [ ²²⁵ Ac]-compound B-HuMIGF-1R. Compound B (0.7 µmol) was dissolved in sodium acetate buffer (69 µl, pH 6.5). An aliquot of compound B solution (4 μl, 40 nmol) was added to a solution containing HuMIGF-1R antibody (6.7 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (in positive ion detection mode): compound B-HuMIGF-1R: found m/z 152988 [M+H]+; HuMIGF-1R: found m/z 149835 [M+H] ⁺ . As a typical Ac-225 reaction (1.15 mCi, 14 μl), compound BHuMIGF-1R (75 μg in acetate buffer (pH 6.5)) and ascorbic acid (1 μl, 0.1 M in acetate buffer ( pH ^6.5 ). , filled with Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid). Example 20. Synthesis of [ ²²⁵ Ac]-compound C-HuMIGF-1R. Compound C (17.5 μmol) was dissolved in sodium acetate buffer (1.32 ml, pH 6.5). An aliquot of compound C solution (8 μl, 91 nmol) was added to a solution containing HuMIGF-1R antibody (13.4 nmol) in bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified using a column packed with Sephadex G-50 resin. The immunoconjugate compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (in positive ion detection mode): compound C-HuMIGF-1R: found m/z 152166 [M+H]+; HuMIGF-1R: found m/z 149724 [M+H] ⁺ . As a typical Ac-225 reaction (1.6 mCi, 16 μl), CHuMIGF-1R (150 μg in acetate buffer (pH 6.5)) and ascorbic acid (1 μl, 0.1 M in acetate buffer) were added to the solution. pH ^6.5 ). Sephadex G-50 resin, eluting with acetate buffer (pH 6.5, 1 mM ascorbic acid). Other embodiments. While the present invention has been described in connection with specific embodiments thereof, it is to be understood that further modifications thereof are possible, and the invention is intended to cover any variations, uses or adaptations of the invention following generally the principles of the invention, and including such deviations from the present invention that arise as a result of known or common practice in the field of technology to which the invention relates and which can be applied to the essential features set forth earlier herein. CLAIM 1. A compound having the structure of formula I, or a pharmaceutically acceptable salt thereof: AL ¹ -(L ² )nB Formula I, where A is a chelating moiety or a metal complex thereof; L ¹ represents C ₁ -C ₆ alkyl or C ₁ -C ₆ heteroalkyl; B is an antibody or antigen binding fragment thereof that specifically binds to IGF-1R, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of figitumumab, cixutumumab, ganitumab, AVE1642, BIIB002, robatumumab and teprotumumab; n is 1; each L ² independently has the structure: (-X'-L'-Z ¹ -) Formula II, where X ¹ represents C=O(NR1) or NR ¹ in which R ¹ represents H, or C ₁ -C ₆ alkyl, or C ₁ -C ₆ heteroalkyl; L ³ represents C1- _C50 alkyl or C1- _C50 heteroalkyl or _C5 - _C20 polyethylene glycol; And Z ¹ represents CH ₂ , C=O, C=S, OC=O, NR1C=O or NR ¹ ; in which R ¹ is hydrogen or C1-C6 alkyl or pyrrolidine-2,5-dione, where the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid), DOTMA (1R ,4R,7R,10R)-α,α',α,α'-tetramethyl-1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis (carbamoylmethyl)-1,4,7,10tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), - 36 045310 DOSAM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1yl)acetic acid), DOTA-GA anhydride (2,2 ',2-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7, 10-tetraazacyclododecane-1,4,7,10tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclododecane-1,4,7,10tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10- tetraazacyclododecane-1,4,7,10tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7- triazacyclononane-1,4,7triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETRA (1,4,8,11-tetraazacyclotetradecane-1,4,8, 11-tetrapropionic acid), TETA (1,4,8,11tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NENA (1,4,7,10,13,16hexaazacyclohexadecane-1,4,7,10, 13,16-hexacetic acid), PEPA (1,4,7,10,13pentaazacyclopentadecane-N,N',N,N',N-pentaacetic acid), H ₄ Octapa (N,N'-bis(6-carboxy -2pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H ₂ Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]methylamino]ethane), H ₆ phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl1,2-diaminoethane), TTNA (triethylenetetramine-N,N,N',N,N',N'-hexacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid bismethylamide) or porphyrin; and wherein for each embodiment, the alkyl may be optionally substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _2-6 alkenyl, C _6-10 aryl, polyethylene glycol and -CO2RA, is optionally substituted with an O-protecting group, and where RA is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and polyethylene glycol, the heteroalkyl may be optionally substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkyl, C _2-6 alkenyl, C _6-10 aryl, polyethylene glycol and -CO2RA, optionally substituted with an O-protecting group, and where RA is selected from the group consisting of hydrogen, C _1-6 alkyl, C _2-6 alkenyl and polyethylene glycol, aryl may be optionally substituted with one, two or three substituents selected from the group consisting of C _1-6 alkoxy, amino, halogen, hydroxy, nitro, C _1-6 alkyl, C _2-6 alkenyl, C ₆₁₀ aryl, polyethylene glycol and -CO2RA, optionally substituted with an O-protecting group, and where RA is selected from the group consisting of hydrogen, C _1-6 alkyl , C _2-6 alkenyl and polyethylene glycol, and wherein for each embodiment the heteroalkyl contains a heteroatom independently selected from the group consisting of nitrogen, oxygen and sulfur. 2. The compound according to claim 1, where the specified chelating moiety is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R, 10R)a,a',a,a'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10tetrakis(carbamoylmethyl)-1, 4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetrapropionic acid), OO3AM-acetic acid (2-(4,7,10-tris(2-amino -2-oxoethyl)1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2-(10-(2,6dioxotetrahydro-2H-pyran-3-yl )-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)) and DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid). 3. A compound according to claim 2, wherein the structure of formula I is: where Y ¹ represents -CH2OCH2(L ² )nB, C=O(L ² )nB or C=S(L ² )nB, and Y ² represents -CH2CO ₂ H; or where Y ¹ represents H, and Y ² represents L1-(L ² ) _n -B, where L ² , n, B and L ¹ are as defined in claim 1. 4. The connection according to any one of claims 1-3, where L ¹ is: Formula III, where R ² represents hydrogen or -CO ₂ H. 5. Compounds according to claims 1-4, where the metal of said metal complex compound is selected from Bi, - 37 045310 Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, lanthanide or actinide; or the metal of said metal complex compound is a radionuclide selected from the group consisting of, ⁴⁷ Sc, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸² Rb , ⁸⁶ Y, ⁸⁷ Y, ⁹⁰ Y, ⁹⁷ Ru, ^99m Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ In, ^117m Sn, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁹ Au, ²⁰¹ TI, ²⁰³ Pb, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Ti. 6. A compound according to any one of claims 3-5, where Y ¹ represents H. 7. A compound according to any one of claims 1 to 6, where X ¹ represents C=O(NR1), and R ¹ represents H. 8. A compound according to any one of claims 1 to 7, where Z ¹ represents -CH2. 9. A connection according to any one of claims 1 to 8, where the connection is: But But where variable B is as defined in paragraph 1. 10. A compound according to any one of claims 1 to 9, where the metal is a radionuclide. 11. The compound according to claim 10, where the radionuclide is In ¹¹¹ . 12. The compound according to claim 10, where the radionuclide is Ga ⁶⁸ . 13. A compound according to any one of claims 1 to 9, where the metal is a radionuclide emitting alpha radiation. 14. The compound according to claim 13, where the radionuclide is Ac ²²⁵ . 15. A compound according to any one of claims 1 to 14, wherein said antibody or antigen binding fragment thereof comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO: 4. 16. A compound according to any one of claims 1 to 14, wherein said antibody or antigen binding fragment thereof comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 8. 17. A compound according to any one of claims 1 to 16, wherein said antibody or antigen binding fragment thereof is AVE1642. 18. A pharmaceutical composition containing a compound according to any one of claims 1 to 17 and a pharmaceutically acceptable excipient. 19. A method of planning and/or conducting radiation therapy, comprising administering to a subject in need thereof a diagnostically effective amount of a compound according to claim 1 and/or administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 17 or a composition according to claim 1. 18, the compound in each case containing a radionuclide. 20. A method of treating cancer, comprising administering to a subject in need thereof a first dose of a compound of any one of claims 1 to 17 or a composition of claim 18 in an amount effective for planning radiation therapy, followed by additional doses of a compound of any one of claims 117 or a composition according to claim 18 in a therapeutically effective amount. 21. The method of claim 20, wherein the compound or composition administered in the first dose and the compound or composition administered in the second dose are the same. 22. The method of claim 20, wherein the compound or composition administered in the first dose and the compound or composition administered in the second dose are different. 23. The method according to any one of claims 20-22, wherein the cancer is a solid tumor or hematological cancer. 24. The method of claim 23, wherein the solid tumor is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, Ewing's sarcoma, multiple myeloma or acute myeloid leukemia. 25. The method according to any one of claims 20-24, further comprising the administration of an antiproliferative agent, a radiosensitizer, an immunoregulatory or immunomodulatory agent. - 38 045310 26. The method according to claim 25, wherein the compound according to any one of claims 1 to 17 or the composition according to claim 18, and the antiproliferative agent or radiosensitizer are administered with an interval of 28 days from each other. 27. The method according to claim 25, where the compound according to any one of claims 1 to 17 or the composition according to claim 18, and the immunoregulatory or immunomodulatory agent are administered with an interval of 90 days from each other. 28. The compound according to claim 1, where L ³ represents C ₅ -C ₂₀ polyethylene glycol. 29. The compound of claim 1, wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising at least one complementarity determining region (CDR) selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3. 30. The compound of claim 29, wherein said antibody or antigen binding fragment thereof comprises a light chain variable domain containing at least two of said CDRs. 31. The compound of claim 30, wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain containing all three of said CDRs. 32. A compound according to claim 1, wherein said antibody or antigen-binding fragment thereof contains a heavy chain variable domain containing at least one CDR selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 5; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 7. 33. The compound of claim 32, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain containing at least two of said CDRs. 34. The compound of claim 33, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain containing all three of said CDRs. 35. A compound according to claim 1, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing at least one CDR selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2; (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3; (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 5; (e) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6; and (f) CDR-H3 containing the amino acid sequence of SEQ ID NO: 7. 36. The compound of claim 35, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing at least two of said CDRs. 37. The compound of claim 36, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing at least three of said CDRs. 38. The compound of claim 37, wherein said antibody or antigen binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing at least four of said CDRs. 39. The compound of claim 38, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing at least five of said CDRs. 40. The compound of claim 39, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain containing all six of said CDRs. -