IGF-1R MONOCLONAL ANTIBODIES AND USES THEREOF
20230052140 · 2023-02-16
Inventors
- Eric Steven Burak (Cambridge, CA)
- John Richard Forbes (Burlington, CA)
- Matthew David Burr Moran (Mississauga, CA)
- Ryan Wayne Simms (Toronto, CA)
- John Fitzmaurice Valliant (Ancaster, CA)
- Alla Darwish (Kitchener, CA)
Cpc classification
A61K51/0485
HUMAN NECESSITIES
C07K16/2863
CHEMISTRY; METALLURGY
A61K51/1027
HUMAN NECESSITIES
C07K16/26
CHEMISTRY; METALLURGY
A61K49/106
HUMAN NECESSITIES
A61K51/1096
HUMAN NECESSITIES
International classification
A61K51/10
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07K16/26
CHEMISTRY; METALLURGY
Abstract
The present invention relates to conjugates including a chelating moiety of a metal complex thereof and a therapeutic or targeting moiety, methods for their production, and uses thereof.
Claims
1. A compound having a structure of Formula I, or a pharmaceutically acceptable salt thereof:
A-L.sup.1-(L.sup.2).sub.n-B Formula I wherein A is a chelating moiety or a metal complex thereof; L.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted aryl or heteroaryl; B is a cross-linking group selected from the group consisting of an amino-reactive cross-linking group, a methionine-reactive cross-linking group, a thiol-reactive cross-linking group, and a sortase-mediated coupling sequence; n is 1-5; and each L.sup.2, independently, has a structure of Formula II:
(—X.sup.1-L.sup.3-Z.sup.1—) Formula II wherein X.sup.1 is C═O(NR.sup.1), C═S(NR.sup.1), OC═O(NR.sup.1), NR.sup.1C═O(O), NR.sup.1C═O(NR.sup.1), —CH.sub.2PhC═O(NR.sup.1), —CH.sub.2Ph(NH)C═S(NR.sup.1), 0, or NR.sup.1, in which R.sup.1 is H or optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted aryl or heteroaryl; L.sup.3 is optionally substituted C.sub.1-C.sub.50 alkyl or optionally substituted C.sub.1-C.sub.50 heteroalkyl or C.sub.5-C.sub.20 polyethylene glycol; and Z.sup.1 is CH.sub.2, C═0, C═S, OC═O, NR.sup.1C═O, or NR.sup.1, in which R.sup.1 is hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, or pyrrolidine-2,5-dione.
2. The compound in claim 1, wherein the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic 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-(2,6-dioxotetrahydro-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(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaacetic acid), H.sub.4octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H.sub.2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H.sub.6phospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), or porphyrin.
3. The compound of claim 2, wherein the structure of Formula I is: ##STR00006## wherein Y.sup.1 is —CH.sub.2OCH.sub.2(L.sup.2).sub.n-B, C═O(L.sup.2).sub.n-B, or C═S(L.sup.2).sub.n-B and Y.sup.2 is —CH.sub.2CO.sub.2H; or wherein Y.sup.1 is H, and Y.sup.2 is L.sup.1-(L.sup.2).sub.n-B.
4. The compound of claim 1, wherein L.sup.1 has the structure: ##STR00007## wherein R.sup.2 is hydrogen or —CO.sub.2H.
5. The compound of claim 1, wherein the metal of said metal complex is selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, a lanthanide, and an actinide; or the metal of said metal complex is a radionuclide selected from the group consisting of .sup.47Sc, .sup.55Co, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.82Rb, .sup.86Y, .sup.87Y, .sup.90Y, .sup.97Ru, .sup.99mTc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.117mSn, .sup.149Pm, .sup.149Tb, .sup.153Sm, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.199Au, .sup.201Tl, .sup.203Pb, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.225Ac, and .sup.227Th.
6. The compound of claim 1, wherein the cross-linking group is an amino-reactive, a methionine-reactive, or a thiol-reactive cross-linking group.
7. The compound of claim 6, wherein the amino-reactive, methionine-reactive, or thiol-reactive cross-linking group comprises an activated ester, imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine.
8. The compound of claim 7, wherein the activated ester is a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, or 4-nitrophenol ester.
9. The compound of claim 1, wherein the cross-linking group is selected from the group consisting of: ##STR00008##
10. The compound of claim 1, wherein the compound is selected from the group consisting of: ##STR00009##
11. A conjugate having the following structure, or a pharmaceutically acceptable salt thereof:
A-L.sup.1-(L.sup.2).sub.n-B wherein the conjugate is formed by reacting a compound of claim 1 with a human or humanized IgG antibody or an antigen binding fragment thereof, wherein, with respect to the conjugate, A is a chelating moiety or a metal complex thereof; B is a human or humanized IgG antibody or an antigen binding fragment thereof; L.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted aryl or heteroaryl; n is 1-5; and each L.sup.2, independently, has a structure of Formula II:
(—X.sup.1-L.sup.3-Z.sup.1—) Formula II wherein X.sup.1 is C═O(NR.sup.1), C═S(NR.sup.1), OC═O(NR.sup.1), NR.sup.1C═O(O), NR.sup.1C═O(NR.sup.1), —CH.sub.2PhC═O(NR.sup.1), —CH.sub.2Ph(NH)C═S(NR.sup.1), 0, or NR.sup.1, in which R.sup.1 is H or optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted aryl or heteroaryl; L.sup.3 is optionally substituted C.sub.1-C.sub.50 alkyl or optionally substituted C.sub.1-C.sub.50 heteroalkyl or C.sub.5-C.sub.20 polyethylene glycol; and Z.sup.1 is CH.sub.2, C═O, C═S, OC═O, NR.sup.1C═O, or NR.sup.1, in which R.sup.1 is hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, or pyrrolidine-2,5-dione.
12. The conjugate of claim 11, wherein the human or humanized IgG antibody is an IGF-1R antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0139]
[0140]
[0141]
[0142]
[0143]
[0144]
[0145]
[0146]
DETAILED DESCRIPTION
[0147] Radiolabelled targeting moieties (also known as radioimmunoconjugates) are designed to target a protein or receptor that is upregulated in a disease state to deliver a radioactive payload to damage and kill cells of interest (radioimmunotherapy). The process of delivering such a payload, via radioactive decay, produces an alpha, beta, or gamma particle or Auger electron that can cause direct effects to DNA (such as single or double stranded DNA breaks) or indirect effects such as by-stander or crossfire effects.
[0148] Radioimmunoconjugates typically contain a biological targeting moiety (e.g, an antibody or antigen binding fragment thereof that specifically binds to IGF-1R), a radioisotope, and a molecule that links the two. Conjugates are formed when a bifunctional chelate is appended to the biological targeting molecule so that structural alterations are minimal while maintaining target affinity. Once radiolabelled, the final radioimmunoconjugate is formed.
[0149] Bifunctional chelates structurally contain a chelate, the linker, and cross-linking group (
[0150] One the key factors of developing safe and effective radioimmunoconjugates is maximizing efficacy while minimizing off-target toxicity in normal tissue. While this statement is one of the core tenants of developing new drugs, the application to radioimmunotherapeutics presents new challenges. Radioimmunoconjugates do not need to block a receptor, as needed with a therapeutic antibody, or release the cytotoxic payload intracellularly, as required with an antibody drug conjugate, in order to have therapeutic efficacy. However, the emission of the toxic particle is an event that occurs as a result of first-order (radioactive) decay and can occur at random anywhere inside the body after administration. Once the emission occurs, damage could occur to surrounding cells within the range of the emission leading to the potential of off-target toxicity. Therefore, limiting exposure of these emissions to normal tissue is the key to developing new drugs.
[0151] One potential method for reducing off-target exposure is to remove the radioactivity more effectively from the body (e.g., from normal tissue in the body). The most obvious mechanism is to increase the rate of clearance of the biological targeting agent. This approach also likely requires identifying ways to shorten the half-life of the 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 the pharmacodynamics/efficacy in that the more rapid removal of drug from the body will lower the effective concentration at the site of action, which, in turn, would require a higher total dose and would not achieve the desired results of reducing total radioactive dose to normal tissue.
[0152] Other efforts have focused on accelerating the metabolism of the portion of the molecule that contains the radioactive moiety. To this end, some efforts have been made to increase the rate of cleavage of the radioactivity from the biological targeting agents using what have been termed “cleavable linkers”. Cleavable linkers, however, have been taken on different meaning as it relates to radioimmunoconjugates. Cornelissen, et al. has described cleavable linkers as those by which the bifunctional conjugate attaches to the biologic targeting agent through a reduced cysteine, whereas others have described the use of enzyme-cleavable systems that require the co-administration of the radioimmunoconjugate with a cleaving agent/enzyme to 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 the cysteine linkage, or are not practical from a drug development perspective (enzyme cleavable systems) since, in the case of the citations provided, require the administration of 2 agents.
[0153] The focus of the embodiments described herein centers on more effectively eliminating radioactivity from the body after catabolism and/or metabolism of the radioimmunoconjugate by making modifications to the linker region of the bifunctional chelate.
[0154] This is a novel approach since little information appears to exist describing the in vivo impact of the linker, especially as it applies to radioimmunoconjugates. One potential reason is that following catabolism/metabolism of the radioimmunoconjugate, one would expect the radiolabelled conjugate to undergo rapid systemic elimination. The supposition was furthered experimentally when the bifunctional chelate was administered alone; it cleared the bloodstream faster than the radioimmunoconjugate with that same bifunctional chelate. Based on these data, one would expect that following catabolism/metabolism of the radioimmunoconjugate, the metabolite containing the bifunctional chelate would also be rapidly eliminated.
[0155] However, rapid clearance of the metabolites containing the radiolabelled conjugate does not necessarily occur in vivo. Based on the results described below, the linker region of bifunctional chelates can directly impact the elimination of the radioactivity from the body following catabolism of the radioconjugate while not having a detrimental impact to the overall in vitro properties or the in vivo pharmacokinetics and pharmacodynamics of the radioimmunoconjugate. Data are presented below that demonstrates that the certain bifunctional chelates available commercially produce a slower rate and a lower extent of elimination of the total radioactivity from the body when compared to the embodiments described herein.
[0156] The excretion profiles of the embodiments described in the Examples represent unexpected findings. As previously reported, Quadri and Vriesendorp [Q. J. Nucl. Med. 1998, 42, 250-261], simple modifications to the linker region of the bifunctional chelate, regardless of their hydrophobicity, did not impact urinary excretion of the radioactivity. The results provided below clearly indicate that both hydrophobic and hydrophilic linkers can impact excretion patterns. In addition, the Examples below demonstrate that hepatobiliary clearance also plays a role in excretion.
[0157] Therefore, through the embodiments described herein, bifunctional chelates, when attached to biological targeting moieties or therapeutic agents, have been identified that achieve a reduction of total body radioactivity by increasing the extent of excretion of the catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule when compared to similar bifunctional chelates in the public domain. This reduction in total body radioactivity has been determined to be due to the clearance of catabolic/metabolic by-products and does not impact the other in vitro and in vivo properties such as degree of specificity (in vitro binding), cellular retention, and tumor uptake in vivo. When taken in whole, these embodiments achieve the desired properties of radioimmunoconjugates by reducing the body burden of radioactivity while maintaining on-target activity.
Therapeutic Moieties and Targeting Moieties
[0158] Therapeutic or targeting moieties include any molecule or any part of a molecule that confers a therapeutic benefit. In some embodiments, the therapeutic moiety is a protein or polypeptide, e.g., an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule. Targeting moieties include any molecule or any part of a molecule that binds to 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 (e.g., Centyrins or Adnectins).
[0159] Polypeptides
[0160] Polypeptides include, for example, any of a variety of hematologic agents (including, for instance, erythropoietin, blood-clotting factors, etc.), interferons, colony stimulating factors, antibodies, enzymes, and hormones. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest can be a polypeptide in the present methods.
[0161] A reference polypeptide described herein can include a target-binding domain that binds to a target of interest (e.g., binds to an antigen). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor). Exemplary molecular targets (e.g., antigens) for 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 the EGF receptor (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophage receptors such as CRIg; tumor necrosis factors such as TNFα or TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Maci, p150,95, VLA-4, ICAM-1, VCAM and αvβ3 integrin including either α or β subunits thereof (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE; cytokines such as IL1; cytokine receptors such as IL2 receptor; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C; neutropilins; ephrins and receptors; netrins and receptors; slit and receptors; chemokines and chemokine receptors such as CCL5, CCR4, CCR5; amyloid beta; complement factors, such as complement factor D; lipoproteins, such as oxidized 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.
[0162] Antibodies
[0163] An IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody binding specificities found in each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions. For 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).
[0164] Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (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 (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[0165] The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include a Fab fragment, a F(ab′).sub.2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR). These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.
[0166] Antibodies or fragments described herein can be produced by any method known in the art for the synthesis of antibodies (see, e.g., 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 the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.
[0167] Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).
[0168] Insulin-Like Growth Factor 1 (IGF-1R) Antibodies
[0169] Insulin-like growth factor 1 receptor is a transmembrane protein found on the surface of human cells activated by insulin-like growth factor 1 (IGF-1) and 2 (IGF-2). Radioimmunoconjugates of the invention may include the insulin-like growth factor-1 receptor (IGF-1R). Although not a typical oncogene, IGF-1R promotes initiation and progression of cancer, playing a critical role in mitogenic transformation and maintenance of the transformed phenotype. IGF-1R has been associated with development of multiple common cancers including breast, lung (e.g., non-small lung), liver, prostate, pancreas, ovarian, colon, melanoma, adrenocortical carcinoma, and various types of sarcomas. IGF-1R signaling stimulates tumour cell proliferation and metabolism, supports angiogenesis, and confers protection from apoptosis. It affects metastatic factors (e.g. HIF-1 dependent hypoxia signaling), anchorage independent growth, as well as growth and survival of tumour metastases after extravasation. IGF-1R has also been implicated in the development, maintenance and enrichment of therapeutic resistant cancer stem cell populations.
[0170] Despite the abundance of data implicating IGF-1R's role in cancer, therapeutics targeting IGF-1R have yet to demonstrate a significant impact on disease. There has been much speculation for this lack of efficacy including the inability to identify appropriate biomarkers for patient identification, complexity and interdependency of the IGF-1/IR signaling pathway and the development of other growth hormone compensatory mechanisms [Beckwith and Yee, Mol Endocrinol, November 2015, 29(11):1549-1557]. Radioimmunotherapy, however, may provide a viable mechanism for treating cancers over expressing the IGF-1 receptor by utilizing the ability of IGF-1R to undergo antibody triggered internalization and lysosomal degradation to deliver targeted radioisotopes inside cancer cells. Internalization and lysosomal degradation of an IGF-1R targeted radioimmunoconjugate prolongs the residence time of the delivered radioisotope inside cancer cells thereby maximizing the potential for a cell killing emission to occur. In the case of actinium-225 which yields 4 alpha particles per decay chain, cell death can be accomplished by as little as 1 atom of radionuclide delivered per cell [Sgouros, et al. J Nucl Med. 2010, 51:311-2]. Cell killing due to direct DNA impact and breakage by an alpha particle may occur in the targeted cell or in a radius of 2 or 3 non-targeted cells for a given alpha particle decay. In addition to having very high potential anti-tumour potency, IGF-1R targeted radioimmunoconjugates may not generate mechanistic resistance as they do not rely on blocking ligand binding to the receptor to inhibit the oncologic process, as needed with a therapeutic antibody.
[0171] Several IGF-1R antibodies have been developed and investigated for the treatment of various types of cancers including figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. After binding to IGF-1R, these antibodies are internalized into the cell and degraded by lysosomal enzymes. The combination of overexpression on tumor cells and internalization offers the possibility of delivering detection agents directly to the tumor site while limiting the exposure of normal tissues to toxic agents.
[0172] The CDRs of the light chain variable region of AVE1642 have the sequences:
TABLE-US-00001 SEQ ID NO: 1 (CDR-L1) RSSQSIVHSNVNTYLE SEQ ID NO: 2 (CDR-L2) KVSNRFS SEQ ID NO: 3 (CDR-L3) FQGSHVPPT
[0173] The light chain variable region of AVE1642 has the sequence:
TABLE-US-00002 SEQ ID NO: 4 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYL QKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVE AEDLGIYYCFQGSHVPPTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAK
[0174] The CDRs of the heavy chain variable region of AVE1642 have the sequences:
TABLE-US-00003 SEQ ID NO: 5 (CDR-H1) SYWMH SEQ ID NO: 6 (CDR-H2) GEINPSNGRTNY NQKFQG SEQ ID NO: 7 (CDR-H3) GRPDYYGSSKWY FDV
[0175] The heavy chain variable region of AVE1642 has the sequence:
TABLE-US-00004 SEQ ID NO: 8 QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGL EWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSED SAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPL APSSKSTSGGTAALG
[0176] Nanobodies
[0177] Nanobodies are antibody fragments consisting of a single monomeric variable antibody domain. Nanobodies may also be referred to as single-domain antibodies. Like antibodies, nanobodies bind selectively to a specific antigen. Nanobodies may be heavy-chain variable domains or light chain domains. Nanobodies may occur naturally or be the product of biological engineering. Nanobodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).
[0178] Affibodies
[0179] Affibodies are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may 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 that has lower affinity for the Fab region. Affibodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).
[0180] Affibody molecules showing specific binding to a variety of different proteins (e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2 and EGFR) have been generated, demonstrating affinities (K.sub.d) in the μM to pM range.
[0181] Fibronectin Type III Domains
[0182] The Fibronectin type III domain is an evolutionarily conserved protein domain found in a wide-variety of extracellular proteins. The Fibronectin type III domain has been used as a molecular scaffold to produce molecules capable of selectively binding a specific antigen. Variants of the Fibronectin type III domains (FN3) that have been engineered for selective-binding may also be referred to as monobodies. FN3 domains may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., CIS-display, phage display, yeast display, bacterial display, mRNA display, ribosome display).
[0183] Modified Polypeptides
[0184] 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% identity to the amino acid sequence of the reference polypeptide). In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g., 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., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize a characteristic of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.
[0185] Modifications include those by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-terminus. The same type of modification may be present in the same or varying degrees at several sites in 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 may result from post-translational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, 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.
[0186] A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where 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 can facilitate conjugation of these polypeptides by, e.g., disulfide bonding. For example, a polypeptide can be modified to include a single cysteine residue at the amino-terminus or a single cysteine residue at the carboxy-terminus. Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).
[0187] Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). 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 sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
[0188] Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated “exemplary substitutions” in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened.
TABLE-US-00005 TABLE 1 Amino acid substitutions Original Conservative residue Exemplary substitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, 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 Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu
[0189] Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
[0190] Cross-Linking Groups
[0191] A cross-linking group is a reactive group that is able to join two or more molecules by a covalent bond. Cross-linking groups may be used to attach the linker and chelating moiety to a therapeutic or targeting moiety. Cross-linking groups may also be used to attach the 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 a sortase-mediated coupling. In some embodiments, the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 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 comprise of a terminal glycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence, where X is any amino acid. The person having ordinary skill in the art will understand that the use of cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.
Detection Agents
[0192] A detection agent is a molecule or atom which is administered conjugated to a polypeptide, e.g., an antibody or antigen-binding fragment thereof, and is useful in diagnosing a disease by locating the cells containing the antigen, radiation treatment planning, or treatment of a disease. Useful detection agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). In order to load a polypeptide component with a detection agent it may be necessary to react it with a reagent having a linker to which are attached the detection agent or multiple detection agents.
[0193] Radioisotopes and Radionuclides
[0194] Radioisotopes and radionuclides known in the art for their utility as detection agents include, but are not limited to, .sup.3H, .sup.14C, .sup.15N, .sup.18F, .sup.35S, .sup.47Sc, .sup.55Co, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.75Br, .sup.76Br, .sup.77Br, .sup.89Zr, .sup.86Y, .sup.87Y, .sup.90Y, .sup.97Ru, .sup.99Tc, .sup.99mTc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.149Pm, .sup.149Tb, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.198Au, .sup.199Au, .sup.203Pb, .sup.211At, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.225Ac, .sup.227Th, .sup.229Th, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.82Rb, .sup.117mSn, .sup.201Tl.
[0195] Chelating Moieties
[0196] Chelating moieties are known in the art for their utility as detection agents include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic 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-(2,6-dioxotetrahydro-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(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaacetic acid), H.sub.4octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H.sub.2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H.sub.6phospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones), or porphyrins. Chelating groups may be used in metal chelate combinations with metals, such as manganese, iron, and gadolinium and isotopes (e.g., isotopes in the general energy range of 60 to 4,000 keV), such as .sup.47Sc, .sup.55Co, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.82Rb, .sup.86Y, .sup.87Y, .sup.90Y, .sup.97Ru, .sup.99mTc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.117mSn, .sup.149Tb, .sup.149Pm, .sup.153Sm, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.199Au, .sup.201Tl, .sup.203Pb, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.225Ac, and .sup.227Th.
[0197] Linkers
[0198] Linkers of the invention may have the structure of Formula I:
A-L.sup.1-(L.sup.2).sub.n-B Formula I
wherein A is chelating moiety or a metal complex thereof;
L.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl;
B is a is a therapeutic moiety, a targeting moiety, or cross-linking group,
or a pharmaceutically acceptable salt thereof;
n is 1-5;
each L.sup.2, independently, has the structure:
(—X.sup.1-L.sup.3-Z.sup.1—) Formula II
wherein is X.sup.1 is C═O(NR.sup.1), C═S(NR.sup.1), OC═O(NR.sup.1), NR.sup.1C═O(O), NR.sup.1C═O(NR.sup.1), —CH.sub.2PhC═O(NR.sup.1), —CH.sub.2Ph(NH)C═S(NR.sup.1), O, NR.sup.1 and R.sup.1 is H or optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl;
L.sup.3 is optionally substituted C.sub.1-C.sub.0 alkyl or optionally substituted C.sub.1-C.sub.0 heteroalkyl or C.sub.5-C.sub.20 polyethylene glycol;
Z.sup.1 is CH.sub.2, C═O, C═S, OC═O, NR.sup.1C═O, NR.sup.1 and R.sup.1 is a hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, pyrrolidine-2,5-dione.
[0199] The conjugates of the invention comprise three distinct modules that together result in their increased effectiveness compared to those known in the art.
[0200] 1. Chelating Moiety or Metal Complex Thereof:
Module A is included for incorporation of a detection agent (e.g., a chelating moiety or metal complex thereof). A metal complex may include an imaging radionuclide.
[0201] 2. Linkers:
Linkers of the invention have the structure of Formula I:
A-L.sup.1-(L.sup.2).sub.n-B Formula I
[0202] wherein A is chelating moiety or a metal complex thereof;
[0203] L.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl;
[0204] B is a is a therapeutic moiety, a targeting moiety, or cross-linking group,
[0205] or a pharmaceutically acceptable salt thereof;
[0206] n is 1-5;
[0207] each L.sup.2, independently, has the structure:
(—X.sup.1-L.sup.3-Z.sup.1—) Formula II
[0208] wherein is X.sup.1 is C═O(NR.sup.1), C═S(NR.sup.1), OC═O(NR.sup.1), NR.sup.1C═0(0), NR.sup.1C═O(NR.sup.1), —CH.sub.2PhC═O(NR.sup.1), —CH.sub.2Ph(NH)C═S(NR.sup.1), O, NR.sup.1 and R.sup.1 is H or optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl;
[0209] L.sup.3 is optionally substituted C.sub.1-C.sub.50 alkyl or optionally substituted C.sub.1-C.sub.50 heteroalkyl or C.sub.5-C.sub.20 polyethylene glycol;
[0210] Z.sup.1 is CH.sub.2, C═O, C═S, OC═O, NR.sup.1C═O, NR.sup.1 and R.sup.1 is a hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, pyrrolidine-2,5-dione.
[0211] 3. Therapeutic Moiety, Targeting Moiety, or Cross-Linking Group:
Module B is a therapeutic moiety (e.g., antibodies, antigen-binding fragments), a targeting moiety (e.g. nanobodies, affibodies, consensus sequences from Fibronectin type III domains), or a cross-linking group (e.g. amino-reactive, thiol-reactive cross-linking group, or a sortase-mediated coupling).
Administration and Dosage
[0212] The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a compound of the invention. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).
[0213] The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.
[0214] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
[0215] The compositions containing an effective amount can be administered for radiation treatment planning, diagnostic, or therapeutic treatments. When administered for radiation treatment planning or diagnostic purposes, the conjugate is administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose. In therapeutic applications, compositions are administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount adequate to accomplish this purpose is defined as a “therapeutically effective amount,” an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of cancer, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual. The conjugates of the invention can be used for the treatment of 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 administering a second dose of any of the foregoing conjugates or compositions in a therapeutically effective amount.
[0216] Amounts effective for these uses may depend on the severity of the disease or condition and the weight and general state of the subject. The therapeutically effective amount of the compositions of the invention and used in the methods of this invention applied to mammals (e.g., humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain conjugates of the invention exhibit an enhanced ability to target cancer cells and residualize, the dosage of the compounds of the invention can be lower than (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 required for a therapeutic effect of the unconjugated agent. The agents of the invention are administered to a subject (e.g., a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject. Therapeutically effective amounts can also be determined empirically by those of skill in the art.
[0217] Single or multiple administrations of the compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
[0218] The conjugates of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy.
[0219] When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a compound of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.
[0220] By “antiproliferative” or “antiproliferative agent,” as used interchangeably herein, is meant any anticancer agent, including those antiproliferative agents listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein. Antiproliferative agents also include organo-platinum derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.
[0221] By “immunoregulatory agent” or “immunomodulatory agent,” as used interchangeably herein, is meant any immuno-modulator, including those listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein.
[0222] As used herein, “radiation sensitizer” includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiation sensitizers may include, but are not limited to, 5-fluorouracil, analogs of platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF anatgonists.
TABLE-US-00006 TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazine ifosfamide altretamine hexamethyl- estramustine melamine phosphate thiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomide cyclophosphamide Semustine Platinum agents spiroplatin lobaplatin (Aeterna) tetraplatin satraplatin (Johnson ormaplatin Matthey) iproplatin BBR-3464 picoplatin (Hoffmann-La Roche) oxaliplatin Miriplatin carboplatin AP-5280 (Access) cisplatin Antimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorodeoxy- pentostatin adenosine hydroxyurea 6-mercaptopurine decitabine (SuperGen) 6-thioguanine clofarabine cytarabine (Bioenvision) 2-fluorodeoxy irofulven cytidine (MGI Pharma) methotrexate DMDC tomudex (Hoffmann-La Roche) fludarabine ethynylcytidine raltitrexed (Taiho) gemcitabine capecitabine Substitute Specification (Clean) Topoisomerase amsacrine exatecan mesylate inhibitors epirubicin (Daiichi) etoposide quinamed teniposide or (ChemGenex) mitoxantrone gimatecan 7-ethyl- (Sigma-Tau) 10-hydroxy- diflomotecan camptothecin (Beaufour-Ipsen) dexrazoxanet TAS-103 (Taiho) (TopoTarget) elsamitrucin pixantrone (Spectrum) (Novuspharma) Edotecarin rebeccamycin Cositecan analogue (Exelixis) Belotecan BBR-3576 hydroxycamptothecin (Novuspharma) (SN-38) rubitecan (SuperGen) irinotecan (CPT-11) topotecan Antitumor antibiotics valrubicin azonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycin Sabarubicin porfiromycin Epirubicin mitoxantrone mitoxantrone (novantrone) doxorubicin amonafide Antimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (Cell vindesine Therapeutics) dolastatin 10 (NCI) IDN 5109 (Bayer) rhizoxin (Fujisawa) A 105972 (Abbott) mivobulin A 204197 (Abbott) (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 RPR 109881A (ASTAMedica) (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatin epothilone B A4 (BMS) (Novartis) isohomohalichondrin- T 900607 (Tularik) B T 138067 (Tularik) (PharmaMar) cryptophycin ZD 6126 52 (Eli Lilly) (AstraZeneca) vinflunine (Fabre) AZ10992 (Asahi) auristatin PE IDN-5109 (Indena) (Teikoku Hormone) AVLB (Prescient BMS 247550 (BMS) NeuroPharma) BMS 184476 (BMS) azaepothilone BMS 188797 (BMS) B (BMS) taxoprexin (Protarga) BNP-7787 SB 408075 (BioNumerik) (GlaxoSmithKline) CA-4 prodrug Vinorelbine (OXiGENE) Trichostatin A dolastatin-10 (NIH) CA-4 (OXiGENE) docetaxel vincristine paclitaxel Aromatase inhibitors aminoglutethimide YM-511 atamestane (Yamanouchi) (BioMedicines) formestane letrozole exemestane anastrazole Thymidylate pemetrexed nolatrexed (Eximias) synthase inhibitors (Eli Lilly) CoFactor ™ ZD-9331 (BTG) (BioKeys) DNA antagonists trabectedin edotreotide (Novartis) (PharmaMar) mafosfamide glufosfamide (Baxter International) (Baxter apaziquone (Spectrum International) Pharmaceuticals) albumin + 32P O6 benzyl guanine (Isotope Solutions) (Paligent) thymectacin (NewBiotics) Farnesyltransferase arglabin tipifarnib (Johnson inhibitors (NuOncology Labs) & Johnson) Ionafarnib perillyl alcohol (Schering-Plough) (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA zosuquidar Pharma) trihydrochloride tariquidar (Xenova) (Eli Lilly) MS-209 biricodar dicitrate (Schering AG) (Vertex) Histone tacedinaline (Pfizer) pivaloyloxymethyl acetyltransferase SAHA (Aton butyrate (Titan) inhibitors Pharma) depsipeptide MS-275 (Fujisawa) (Schering AG) Metalloproteinase Neovastat (Aeterna CMT-3 (CollaGenex) inhibitors Laboratories) BMS-275291 marimastat (Celltech) (British Biotech) Ribonucleoside gallium maltolate tezacitabine (Aventis) reductase inhibitors (Titan) didox (Molecules triapine (Vion) for Health) TNF alpha virulizin (Lorus revimid (Celgene) agonists/antagonists Therapeutics) CDC-394 (Celgene) Endothelin A atrasentan (Abbott) YM-598 receptor antagonist ZD-4054 (Yamanouchi) (AstraZeneca) Retinoic acid fenretinide (Johnson alitretinoin (Ligand) receptor agonists & Johnson) LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy oncophage (Anosys) (Antigenics) pentrix (Australian GMK (Progenies) Cancer adenocarcinoma Technology) vaccine (Biomira) ISF-154 (Tragen) CTP-37 (AVI cancer vaccine BioPharma) (Intercell) IRX-2 (Immuno-Rx) norelin (Biostar) PEP-005 BLP-25 (Peplin Biotech) (Biomira) synchrovax vaccines MGV (CTL Immuno) (Progenies) melanoma vaccine β-alethine (CTL Immuno) (Dovetail) p21 RAS vaccine CLL therapy (GemVax) (Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCam abatacept (BMS) Biotherapeutics) pembrolizumab atezolizumab (Merck) (Genentech) Hormonal and estrogens dexamethasone antihormonal agents conjugated estrogens prednisone ethinyl estradiol methylprednisolone chlortrianisen prednisolone idenestrol aminoglutethimide hydroxy- leuprolide progesterone octreotide caproate mitotane medroxy- P-04 (Novogen) progesterone 2-methoxyestradiol testosterone (EntreMed) testosterone arzoxifene (Eli Lilly) propionate; tamoxifen fluoxymesterone toremofine methyltestosterone goserelin diethylstilbestrol Leuporelin megestrol bicalutamide bicalutamide flutamide nilutamide Photodynamic talaporfin Pd-bacterio- agents (Light Sciences) pheophorbide (Yeda) Theralux Motexafin (Theratechnologies) lutetium motexafin hypericin gadolinium (Pharmacyclics) Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth) leflunomide kahalide F (Sugen/Pharmacia) (PharmaMar) ZD1839 CEP-701 (Cephalon) (AstraZeneca) CEP-751 (Cephalon) erlotinib (Oncogene MLN518 (Millenium) Science) PKC412 (Novartis) canertinib (Pfizer) Phenoxodiol squalamine (Novogen) (Genaera) C225 (ImClone) SU5416 (Pharmacia) rhu-Mab (Genentech) SU6668 (Pharmacia) MDX-H210 ZD4190 (Medarex) (AstraZeneca) 2C4 (Genentech) ZD6474 MDX-447 (Medarex) (AstraZeneca) ABX-EGF (Abgenix) vatalanib (Novartis) IMC-1C11 (ImClone) PKI166 (Novartis) Tyrphostins GW2016 Gefitinib (Iressa) (GlaxoSmithKline) PTK787 (Novartis) EKB-509 (Wyeth) EMD 72000 (Merck) trastuzumab Emodin (Genentech) Radicinol OSI-774 Vemurafenib (Tarceva ™) (B-Raf enzyme CI-1033 (Pfizer) inhibitor, Daiichi SU11248 Sankyo) (Pharmacia) RH3 (York Medical) Genistein Radicinol Met-MAb (Roche) SR-27897 (CCK ceflatonin (apoptosis A inhibitor, promotor, ChemGenex) Sanofi-Synthelabo) BCX-1777 (PNP inhibitor, BioCryst) tocladesine (cyclic AMP ranpirnase (ribonuclease stimulant, Alfacell) agonist, Ribapharm) galarubicin (RNA synthesis alvocidib (CDK inhibitor, Dong-A) inhibitor, Aventis) tirapazamine (reducing agent, SRI CV-247 (COX-2 International) inhibitor, Ivy Medical) N-acetylcysteine (reducing agent, Zambon) P54 (COX-2 R-flurbiprofen (NF-kappaB inhibitor, Phytopharm) inhibitor, Encore) CapCell ™ (CYP450 3CPA (NF-kappaB stimulant, inhibitor, Active Biotech) Bavarian Nordic) seocalcitol (vitamin D GCS-100 receptor agonist, Leo) (gal3 antagonist, 131-I-TM-601 (DNA antagonist, GlycoGenesys) TransMolecular) G17DT immunogen eflornithine (ODC inhibitor, (gastrin inhibitor, Aphton) ILEX Oncology) efaproxiral (oxygenator, minodronic acid (osteoclast inhibitor, Allos Therapeutics) Yamanouchi) PI-88 (heparanase indisulam (p53 stimulant, Eisai) inhibitor, Progen) aplidine (PPT inhibitor, PharmaMar) tesmilifene (histamine gemtuzumab (CD33 antagonist, YM antibody, Wyeth Ayerst) BioSciences) PG2 (hematopoiesis enhancer, histamine (histamine H2 Pharmagenesis) receptor agonist, Maxim) Immunol ™ (triclosan oral rinse, Endo) tiazofurin (IMPDH triacetyluridine (uridine prodrug , Wellstat) inhibitor, Ribapharm) SN-4071 (sarcoma agent, Signature cilengitide (integrin BioScience) antagonist, TransMID-107 ™ (immunotoxin, Merck KGaA) KS Biomedix) SR-31747 (IL-1 antagonist, PCK-3145 (apoptosis promotor, Sanofi-Synthelabo) Procyon) CCI-779 (mTOR kinase doranidazole (apoptosis promotor, Pola) inhibitor, Wyeth) CHS-828 (cytotoxic agent, Leo) exisulind (PDE V trans-retinoic acid (differentiator, NIH) inhibitor, Cell Pathways) MX6 (apoptosis promotor, MAXIA) CP-461 (PDE V inhibitor, apomine (apoptosis promotor, Cell Pathways) ILEX Oncology) AG-2037 (GART urocidin (apoptosis promotor, Bioniche) inhibitor, Pfizer) Ro-31-7453 (apoptosis WX-UK1 (plasminogen promotor, La Roche) activator inhibitor, Wilex) brostallicin (apoptosis PBI-1402 (PMN stimulant, promotor, Pharmacia) ProMetic LifeSciences) β-lapachone bortezomib (proteasome gelonin inhibitor, Millennium) cafestol SRL-172 (T cell kahweol stimulant, SR Pharma) caffeic acid TLK-286 (glutathione S Tyrphostin AG transferase inhibitor, Telik) PD-1 inhibitors PT-100 (growth factor CTLA-4 inhibitors agonist, Point sorafenib Therapeutics) midostaurin (PKC inhibitor, Novartis) bryostatin-1 (PKC stimulant, GPC Biotech) CDA-II (apoptosis promotor, Everlife) SDX-101 (apoptosis promotor, Salmedix) rituximab (CD20 antibody, Genentech carmustine Mitoxantrone Bleomycin Absinthin Chrysophanic acid Cesium oxides BRAF inhibitors, PD-L1 inhibitors MEK inhibitors bevacizumab angiogenesis inhibitors dabrafenib
[0223] The following Examples are intended to illustrate the synthesis of a representative number of conjugates and the use of these conjugates for the treatment of cancer. Accordingly, the Examples are intended to illustrate but not to limit the invention. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.
EXAMPLES
Example 1. General Materials and Methods
[0224] The antibodies used were HuMIgG (Aldrich, 14506) and HuMIGF-1R (AVE1642). Lutetium-177 was received from Perkin Elmer as lutetium chloride in a 0.05 N hydrochloric acid solution.
[0225] Analytical HPLC-MS was performed using a Waters Acquity HPLC-MS system comprised of a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (samples cooled to 10° C.), a Water Acquity Column Manager (column temperature 30° C.), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD with electrospray ionization and a Waters Acquity BEH C18, 2.1×50 (1.7 μm) column. Preparative HPLC was performed using a Waters HPLC system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C18 19×100 mm (5 μm) column.
[0226] HPLC elution method 1: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 3-3.5 min=0% A, 4 min=90% A, 5 min=90% A.
[0227] HPLC elution method 2: Waters XBridge Prep Phenyl 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 13 min=0% A.
[0228] HPLC elution method 3: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 8 min=0% A, 10 min=0% A, 11 min=90% A, 12 min=90% A.
[0229] HPLC elution method 4: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 3 min=80% A, 13 min=20% A, 18 min=0% A.
[0230] HPLC elution method 5: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 3 min=90% A, 13 min=0% A, 20 min=0% A.
[0231] HPLC elution method 6: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=75% A, 13 min=0% A, 15 min=0% A.
[0232] HPLC elution method 7: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 12 min=0% A, 15 min=0% A.
[0233] HPLC elution method 8: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 12 min=0% A, 15 min=0% A.
[0234] Analytical Size Exclusion Chromatography (SEC) was performed using a Waters system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiodetector (FC-3300) and TOSOH TSKgel G3000SWxl, 7.8×300 mm column. The isocratic SEC method had a flow rate=1 mL/min, with a mobile phase of 0.1 M phosphate, 0.6M NaCl, 0.025% sodium azide, pH=7.
[0235] MALDI-MS (positive ion) performed using a MALDI Bruker Ultraflextreme Spectrometer.
[0236] Radio thin-layer chromatography (radioTLC) performed with Bioscan AR-2000 Imaging Scanner, carried out on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SG10001) plates using citrate buffer (0.1M, pH 5.5).
Example 2. Synthesis of [.SUP.177.Lu]-Compound A-HuMIGF-1R (Commercial Standard)
[0237] The bifunctional chelating agent 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-GA anhydride, Compound A) was obtained from CheMatech.
[0238] The Compound A (3.0 moles) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 μL, 106 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. 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): (Compound A)-HuMIGF-1R found m/z 151759; HuMIGF-1R found m/z 149835.
[0239] As a typical reaction, the 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 buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [.sup.177Lu]-Compound A-HuMIGF-1R, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). SEC retention time: 8.1 min; radioTLC radiochemical purity: 99%; radiochemical yield: 74%; specific activity: 8.2 mCi/mg.
Example 3. Synthesis of 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)
[0240] A 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 provided in
[0241] To a solution of Intermediate 2-A (40 mg, 0.03 mmol), TFP (90 mg, 0.54 mmol) and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL) was added pyridine (0.05 mL, 50 mg, 0.62 mmol) at room temperature. The solution was stirred at room temperature for 24 hours. The reaction was purified directly by Prep-HPLC using method 7 to provide Intermediate 2-B (33 mg, 82.5%) as a wax after concentration using a Biotage V10 Rapid Evaporator.
[0242] Intermediate 2-B (33 mg, 0.022 mmol) was dissolved DCM/TFA (1.0 mL/2.0 mL) and allowed to stir at room temperature for 24 hours. The reaction was concentrated by air stream and purified directly by Prep-HPLC using method 8 to yield Compound B (14 mg, 50.0%) as a clear wax after concentration. An aliquot was analyzed by HPLC-MS elution method 3; retention time: 4.15 minutes; MS (positive ESI): found m/z 808.1 [M+H].sup.+; C.sub.36H.sub.54F.sub.4N.sub.5O.sub.11 (calc. 808.8).
[0243] .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ 7.99-7.88 (m, 1H), 7.82 (t, J=5.5 Hz, 1H), 3.78 (broad s, 4H), 3.43 (broad s, 12H), 3.08 (broad s, 4H), 3.00 (m, 3H), 2.93 (broad s, 3H), 2.77 (t, J=7.2 Hz, 2H), 2.30 (broad s, 2H), 1.88 (broad s, 2H), 1.66 (p, J=7.3 Hz, 2H), 1.36 (m, 4H), 1.32-1.20 (m, 9H).
Example 4. Synthesis of [.SUP.177.Lu]-Compound B-HuMIGF-1R
[0244] Compound B (0.7 moles) was dissolved in sodium acetate buffer (69 μL, pH 6.5). An aliquot of Compound B solution (4 μL, 40 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-HuMIGF-1R: found m/z 152988 [M+H].sup.+; HuMIGF-1R: found m/z 149835 [M+H].sup.+.
[0245] As a typical reaction, the Lu-177 (1.15 mCi, 14 μL) was added to a solution of Compound B-HuMIGF-1R (75 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [.sup.177Lu]-Compound C-HuMIGF-1R, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 75%; specific activity: 11.9 mCi/mg.
Example 5. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C)
[0246] A bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C), was synthesized according to the scheme provided in
[0247] To vial containing Intermediate 1-A (82 mg, 60 μmol) was added ACN (2 mL), NEt.sub.3 (50 μL, 360 μmol, 6 equiv.), HBTU (23 mg, 60 μmol, 1 equiv) and a TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μL of ACN). The resulting clear solution was stirred at ambient temperature for 3 hours. The reaction was worked up by concentrating the solution to dryness under an air stream and was then diluted with ACN/H.sub.2O (1:1, 3 mL total) and purified on preparative HPLC using elution method 4. The fractions containing product were pooled and concentrated under vacuum and then co-evaporated with ACN (3×2 mL). Intermediate 1-B was obtained as a clear residue (67 mg, 74% yield).
[0248] To a vial containing Intermediate 1-B (67 mg, 64 μmol) was added DCM (2 mL) and TFA (2 mL) and the resulting solution was stirred at ambient temperature for 16 hour. Additional TFA (2 mL) was added and the reaction was stirred at ambient temperature for 6 hour. The reaction was concentrated to dryness under an air stream with the crude product being finally dissolved in ACN/H.sub.2O (1 mL of 10% ACN/H.sub.2O). The crude reaction solution was then purified by preparative HPLC using elution method 5. The fractions containing product were pooled, frozen and lyophilized. Compound C was obtained as a white solid (36 mg, 63% yield). An aliquot was analyzed by HPLC-MS elution method 3; retention time: 3.11 minutes; MS (positive ESI): found m/z 828.4 [M+H].sup.+; C.sub.34H.sub.50F.sub.4N.sub.5O.sub.14 (calc. 828.3).
[0249] .sup.1H NMR (DMSO-d.sub.6, 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 (broad s, 4H), 2.30 (broad s, 2H), 1.87 (broad s, 2H).
Example 6. Synthesis of [.SUP.177.Lu]-Compound C-HuMIGF-1R
[0250] The Compound C (17.5 moles) was dissolved in sodium acetate buffer (1.32 mL, pH 6.5). An aliquot of Compound C solution (8 μL, 91 nmoles) was added to a solution containing the antibody HuMIGF-1R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-HuMIGF-1R found m/z 152166 [M+H].sup.+; HuMIGF-1R found m/z 149724 [M+H].sup.+.
[0251] As a typical reaction, the Lu-177 (1.6 mCi, 16 μL) was added to a solution of Compound C-HuMIGF-1R (150 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at ambient temperature for 20 minutes. [.sup.177Lu]-Compound C-HuMIGF-1R was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 91%; specific activity: 15.6 mCi/mg.
Example 7. Saturation Binding Experiments
[0252] Saturation binding experiments measure the specific binding at equilibrium of a radioconjugate at various concentrations in order to determine the K.sub.d (ligand concentration that binds to half the receptor sites at equilibrium) and Bmax (maximum number of binding sites). In this type of binding assay, both total and nonspecific binding are measured, where specific binding to the receptor 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 that binds to essentially all the receptors. Since all the receptors are occupied by the HumIGF-1R, the radioconjugate only binds nonspecifically. K.sub.d and Bmax values are calculated by nonlinear regression analysis and computerized curve fitting.
[0253] The purpose of this assay was to ensure that these new radioconjugates maintained binding characteristics consistent with the native antibody in an IGF-1R expressing A431 cell line. Twenty-four hours prior to the start of the experiment, 1.5×10.sup.5 A431 cells were seeded in 48-well microplates in 500 μl supplemented medium. The radioconjugate was diluted with binding buffer (PBS+0.5% BSA) to a range of concentrations from 0.08 nM to 40 nM; final assay concentration 0.04 to 20 nM. At the start of the assay, the media is aspirated, discarded and 500 μl of serum-free DMEM was added to each well. The plates were incubated at 37° C. for 1 hour. Following incubation, media was aspirated from each well and discarded. The cells were washed and 100 μl of binding buffer (total binding) or 4 μM cold-antibody (non-specific binding) added to designated wells. Plates were incubated at 4° C. for 1 hour with mild shaking. Following the blocking step, 100 μl of radioconjugate was added to each well. The plates were then incubated at 4° C. for 2 hour. Following incubation, the contents of each well was aspirated and discarded. The cells were washed twice with PBS and were then lysed with 1% Triton-X-100. The lysates were transferred to counting tubes and run with radioconjugate standards on the Wizard 1470 gamma counter to determine the radioactivity content (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 determined using a standard protein quantification assay. Total, non-specific and specific ligand binding determinations, mass of bound conjugate in each lysate were calculated by converting lysate CPM to fmol bound using the specific activity of the conjugate standards and then normalizing the fmol bound to the protein content of each lysate (in milligrams). Specific binding was determined by subtracting the non-specific binding from total binding. Total, specific and non-specific binding values (fmol/mg) were plotted (y-axis) against conjugate concentration (nM, x-axis) as shown in Table 1. The K.sub.d and Bmax were derived by curve fitting of the specific binding data to a single-site hyperbola model (GraphPad Prism Software, version 7).
[0254] Results indicated that binding affinity was not changed through the changes in the linker. In addition, these changes did not alter the binding and specificity to the target.
TABLE-US-00007 TABLE 1 Binding Affinity Construct (K.sub.d) [.sup.177Lu]-Compound A-HuMIGF-1R 2.9 nM [.sup.177Lu]-Compound B-HuMIGF-1R 2.0 nM [.sup.177Lu]-Compound C-HuMIGF-1R 2.2 nM
Example 8. Residualization Experiments
[0255] The residualization assay was designed to determine the degree of cell retention of radiolabeled-linker-antibody derivatives. The assay relies on the inherent ability of the IGF-1 receptor to internalize when bound to ligand and the ability to track radiolabelled 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. Following incubation, the cells are stripped with a mild acid buffer to remove any external or membrane-bound radioconjugate. Fresh medium is re-applied and the cells are again incubated for a pre-determined amount of time. It is during this period that cell processes degrade the radioconjugate and thereby efflux radioactive fragments back into the culture medium or retain the radioactive fragments in the cell. Residualization is determined by calculating the amount of internalized radioactivity as a percentage of the total cell-associated activity following acid wash.
[0256] A431 cells were plated in 24-well plates at a concentration of 2.5×10.sup.5 cells/well in full medium (DMEM). Following overnight incubation, the cells were changed to serum-free DMEM and incubated for 1 hour at 37° C. Media was decanted and plates were washed once with sterile PBS. The radioconjugate was diluted in serum-free DMEM to a concentration of 2 nM. 500 uL of radioconjugate was loaded into each well and incubated for 4 hours at 37° C. After incubation, plates were immediately placed on ice and medium was discarded into pre-labeled (non-bound) gamma counting tubes. Cells were washed once with sterile PBS, gently shaken and decanted into the (non-bound) gamma tubes. Mild acid wash buffer (pH 4.6, 500 μL) was added into all wells. Plates were incubated at 4° C. for 15 minutes and buffer was collected into pre-labeled gamma-counting tubes (membrane-bound). 1 ml of warmed serum-free media was added to all wells and plates were incubated at 37° C. for 0 and 24 hours. Following the prescribed incubation, plates were placed on ice and processed in the following manner. Media was decanted and collected into labeled (efflux) gamma tubes. Plates were then washed once with 1 ml cold PBS and collected into efflux tubes. Acid wash buffer (pH 2.5, 500 μL) was added to all wells and plates were incubated for 5 minutes on ice. The acid wash fraction was then collected into labeled (recycled) gamma tubes. Cells were lysed with 300 μL 1% Triton X-100 for 30 minutes at room temperature. 250 μL of the cell lysate was transferred into gamma counting tubes and counted for 10 minutes. 25 μL of the cell lysate fraction was transferred to a 96-well plate for protein quantification (Pierce BCA Protein Assay). Percent residualization (
[0257] In vitro residualization experiments demonstrated that conjugation with the different linkers resulted in radioconjugates that were effectively identical in terms of cellular internalization and retention indicating that these properties of the monoclonal antibody were not altered through conjugation. Furthermore, these data indicate that the radioimmunoconjugates are likely to undergo similar catabolic degradation after internalization into tumor cells in vivo irrespective of the appended linker structure.
Example 9. Pharmacokinetic and Metabolism Study Results for HuMIGF-1R Compounds
[0258] Groups of 4 or 5 mice (normal CD-1 or athymic CD-1 nude) were injected intravenously with approximately 15 microcuries of radiolabelled test compound. Immunoconjugates with various linkers were synthesized and radiolabelled with lutetium-177. For pharmacokinetic studies, animals were sacrificed at specific timepoints, and blood and tumor (when applicable) were analyzed for total radioactivity. For metabolism studies, animals were placed in metabolic cages (4-5 per cage) for urine and feces collection every 24 hours for up to 7 days. The radioactive content of urine and feces samples was quantified and converted to total urine or feces output based on weight. Excretion profiles for urine, feces, or total excretion (urine+feces) were generated by plotting cumulative % injected dose (% ID) overtime.
[0259] The metabolic excretion profile of [.sup.177Lu]-Compound B-HuMIGF-1R, and [.sup.177Lu]-Compound C-HuMIGF-1R was compared was compared to [.sup.177Lu]-Compound A-HuMIGF-1R. It was found that while the linker type impacted the route, rate, and extent of compound excretion (
Example 10. Radiotherapeutic Efficacy
[0260] Therapeutic efficacy of [.sup.225Ac]-Compound A-HuMIGF-1R, [.sup.225Ac]-Compound B-HuMIGF-1R, and [.sup.225Ac]-Compound C-HuMIGF-1R was compared was compared to HuMIGF-1R alone and vehicle control. The route of synthesis of the actinium-225 (Ac-225) radiolabeled compounds were similar to that for the corresponding Lu-177 analogs. Therapeutic efficacy studies were carried out using the IGF-1R overexpressing colon cancer cell line Colo-205 (ATCC #CCL-222). Tumor xenografts are established in 5-7 week old female Balb/c athymic nude mice (Charles River Laboratories). Two (2) million cells mixed in 50:50 v/v in PBS and Matrigel (Becton Dickinson) were injected subcutaneously into the lower right quadrant above the thigh of each animal. Tumours are allowed to grow for 7-10 days to an initial volume of −200 mm.sup.3. Groups of tumor bearing animals (n=4-8) were injected intravenously via the lateral tail vein with 200 μL of test article. Ac-225 radiolabelled compound test articles were dosed at 20-400 nanocuries (nCi) of activity formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween-80. As a control, non-radiolabelled, non-conjugated antibody (HuMIGF-1R) was administered at a protein mass equivalent corresponding to the highest radioactivity dose of the actinium-225 radioimmunoconjugates tested in a study. Tumor measurements were taken 2-3 times per week with vernier calipers in two dimensions. Tumor length was defined as the longest dimension, width was measured perpendicular to the tumor length. At the same time animals were weighed. Overall body condition and general behavior were assessed daily. A typical study had a duration of 28 days. Tumor volume (mm.sup.3) was calculated from caliper measurements as an ellipsoid: Tumor growth was expressed as relative tumor volume (RTV) which is tumor volume measured on day X divided by the tumor volume measured on the day of dosing.
[0261] The therapeutic efficacy of [.sup.225Ac]-Compound A-HuMIGF-1R, [.sup.225Ac]-Compound B-HuMIGF-1R, and [.sup.225Ac]-Compound C-HuMIGF-1R was effectively equal across all compounds; with all of the actinium-225-containing radioimmunoconjugates demonstrating higher efficacy than the non-radioactive HuMIGF-1R control.
Example 11. Synthesis of [.SUP.177.Lu]-Compound A-Human-IgG
[0262] The compound Compound A (1.34 moles) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature, the resulting immunoconjugate was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). The antibody conjugate Compound A-Human-IgG. MALDI-TOF-MS (positive ion): Compound A-Human-IgG: found m/z 150360 [M+H].sup.+; Human-IgG: found m/z 148339 [M+H].sup.+.
[0263] As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 90 minutes. The crude product, [.sup.177Lu]-Compound A-Human-IgG, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid. RadioTLC radiochemical purity: 98%; radiochemical yield: 45%; specific activity: 15.1 mCi/mg.
Example 12. Synthesis of [.SUP.177.Lu]-Compound B-Human-IgG
[0264] Compound B (1.17 moles) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL, 10 nmoles) was added to a solution containing the antibody Human-IgG (6.7 nmoles) in a 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 via a Sephadex G-50 resin packed column. Compound A-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-Human-IgG found m/z 149949 [M+H].sup.+; Human-IgG found m/z 148540 [M+H].sup.+.
[0265] As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, .sup.177Lu-Compound B-Human-IgG, was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa). RadioTLC radiochemical purity: 98%; radiochemical yield: 51%; specific activity: 9.68 mCi/mg.
Example 13. Synthesis of [.SUP.177.Lu]-Compound C-Human-IgG
[0266] The compound Compound C (0.96 moles) was dissolved in sodium acetate buffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20 nmoles) was added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate product was purified via a Sephadex G-50 resin packed column. Compound C-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095 [M+H].sup.+; Human-IgG: found m/z 148540 [M+H].sup.+.
[0267] As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [.sup.177Lu]-Compound C-Human-IgG, was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa). RadioTLC radiochemical purity: 98%; radiochemical yield: 37%; specific activity: 9.99 mCi/mg.
Example 14. Pharmacokinetic and Metabolism Study Results for HuMIgG Based Compounds
[0268] Non-targeted human IgG antibodies were used for metabolic excretion studies in order to demonstrate that the alterations in radioactivity excretion profiles directed by conjugation with linker Compound B and Compound C is a general process demonstrating that these finding are not-limited to HuMIGF-1R antibody. Pharmacokinetic and metabolism studies were carried out using [.sup.177Lu]-Compound A-HuMIgG, [.sup.177Lu]-Compound B-HuMIgG, and [.sup.177Lu]-Compound C-HuMIgG as described for the HuMIGF-1R antibody based compounds described previously.
[0269] The metabolic excretion profile of a non-targeted human IgG radioimmunoconjugates [.sup.177Lu]-Compound B-HuMIgG, and [.sup.177Lu]-Compound C-HuMIgG were compared to [.sup.177Lu]-Compound A-HuMIgG. As described for the HuMIGF-1R-based radioimmunoconjugates, it was found that while the linker type impacted the route, rate, and extent of compound excretion (
[0270] This metabolic profile was fundamentally equivalent for the HuMIGF-1R based compounds demonstrating that improved excretion profile of the Compound B or Compound C, when conjugated to antibodies, is a general and reproducible effect.
Example 15: Synthesis of [.SUP.225.Ac]-Compound A-Human-IgG
[0271] The compound Compound A (1.34 moles) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature the antibody conjugate product was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): Compound A-Human-IgG: found m/z 150360 [M+H].sup.+; Human-IgG: found m/z 148339 [M+H].sup.+.
[0272] As a typical reaction, Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 90 minutes. The crude product, [.sup.225Ac]-Compound A-Human-IgG, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
Example 16. Synthesis of [.SUP.225.Ac]-Compound B-Human-IgG
[0273] The compound Compound B (1.17 moles) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL, 10 nmoles) was added to a solution containing the antibody Human-IgG (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound A-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-Human-IgG found m/z 149949 [M+H].sup.+; Human-IgG found m/z 148540 [M+H].sup.+.
[0274] As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [.sup.225Ac]-Compound B-Human-IgG, is purified via a HPLC SEC column (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 [.SUP.225.Ac]-Compound C-Human-IgG
[0275] The compound Compound C (0.96 moles) was dissolved in sodium acetate buffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20 nmoles) was added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound C-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095 [M+H].sup.+; Human-IgG: found m/z 148540 [M+H].sup.+.
[0276] As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [.sup.225Ac]-Compound C-Human-IgG, is purified via a HPLC SEC column (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 [.SUP.225.Ac]-Compound A-HuMIGF-1R
[0277] The Compound A (3.0 moles) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 μL, 106 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. 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): (Compound A)-HuMIGF-1R found m/z 151759; HuMIGF-1R found m/z 149835.
[0278] As a typical reaction, the Ac-225 (1.1 mCi, 14 μL) is 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)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [.sup.225Ac]-Compound A-HuMIGF-1R, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
Example 19. Synthesis of [.SUP.225.Ac]-Compound B-HuMIGF-1R
[0279] Compound B (0.7 moles) was dissolved in sodium acetate buffer (69 μL, pH 6.5). An aliquot of Compound B solution (4 μL, 40 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-HuMIGF-1R: found m/z 152988 [M+H].sup.+; HuMIGF-1R: found m/z 149835 [M+H].sup.+.
[0280] As a typical reaction, the Ac-225 (1.15 mCi, 14 μL) is added to a solution of Compound B-HuMIGF-1R (75 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [.sup.225Ac]-Compound C-HuMIGF-1R, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
Example 20. Synthesis of [.SUP.225.Ac]-Compound C-HuMIGF-1R
[0281] The Compound C (17.5 moles) was dissolved in sodium acetate buffer (1.32 mL, pH 6.5). An aliquot of Compound C solution (8 μL, 91 nmoles) was added to a solution containing the antibody HuMIGF-1R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-HuMIGF-1R found m/z 152166 [M+H].sup.+; HuMIGF-1R found m/z 149724 [M+H].sup.+.
[0282] As a typical reaction, the Ac-225 (1.6 mCi, 16 μL) is added to a solution of Compound C-HuMIGF-1R (150 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. [.sup.225Ac]-Compound C-HuMIGF-1R is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
OTHER EMBODIMENTS
[0283] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.