Residualizing linkers and uses thereof

Abstract

The present invention relates to conjugates including a residualizing linker, methods for their production, and uses thereof.

Claims

1. A conjugate comprising a polypeptide linked to a detection agent, the conjugate having a structure of Formula I: ##STR00063## or a salt thereof, wherein A-NH— is a polypeptide; L.sup.1 and L.sup.2 are independently absent, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted aryl, or optionally substituted C2-C100 polyethylene glycol; L.sup.3 is absent or optionally substituted C1-C6 alkyl; m is 0 or 1; R.sup.1 is hydrogen and R.sup.2 is —CH.sub.2CO.sub.2H or —CH.sub.2CH.sub.2CO.sub.2H, or R.sup.1 is —CH.sub.2CO.sub.2H or —CH.sub.2CH.sub.2CO.sub.2H and R.sup.2 is hydrogen; each R.sup.3 and R.sup.4 is hydrogen or R.sup.3 and R.sup.4 combine to form C═O; n is 0 or 1; o is 3; R.sup.5 is hydrogen or L.sup.4-B; R.sup.6 and R.sup.7 are independently hydrogen, optionally substituted C1-C6 heteroalkyl, or L.sup.4-B; L.sup.4 is optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl; B is an organic moiety comprising a detection agent; at least one of L.sup.1 and L.sup.2 is present and when L.sup.1 or L.sup.2 is absent, m is 0; and one and only one of R.sup.5, R.sup.6, and R.sup.7 is L.sup.4-B, wherein B has a structure: ##STR00064## to which a metallic radionuclide is chelated.

2. The conjugate of claim 1, wherein L.sup.4 is optionally substituted C1-C6 alkyl.

3. The conjugate of claim 1, wherein R.sup.5 is L.sup.4-B and R.sup.6 and R.sup.7 are both hydrogen.

4. The conjugate of claim 1, wherein R.sup.6 is L.sup.4-B and R.sup.5 and R.sup.7 are both hydrogen.

5. The conjugate of claim 1, wherein L.sup.2 is selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted aryl, and optionally substituted C2-C100 polyethylene glycol.

6. The conjugate of claim 5, wherein L.sup.2 is optionally substituted C1-C6 alkyl.

7. The conjugate of claim 6, wherein said optionally substituted C1-C6 alkyl is n-butyl.

8. The conjugate of claim 5, wherein L.sup.2 is optionally substituted C1-C6 heteroalkyl.

9. The conjugate of claim 8, wherein said optionally substituted C1-C6 heteroalkyl has the structure: ##STR00065##

10. The conjugate of claim 5, wherein L.sup.2 is optionally substituted C3-C10 cycloalkyl.

11. The conjugate of claim 10, wherein said optionally substituted C3-C10 cycloalkyl is cyclohexyl.

12. The conjugate of claim 5, wherein L.sup.2 is optionally substituted aryl.

13. The conjugate of claim 12, wherein said optionally substituted aryl is phenyl.

14. The conjugate of claim 5, wherein L.sup.2 is optionally substituted C2-C100 polyethylene glycol.

15. The conjugate of claim 14, wherein said optionally substituted C2-C100 polyethylene glycol is: ##STR00066##

16. The conjugate of claim 1, wherein L.sup.1 is absent or optionally substituted C3-C10 cycloalkyl.

17. The conjugate of claim 1, wherein R.sup.1 is hydrogen.

18. The conjugate of claim 1, wherein R.sup.1 is —CH.sub.2CO.sub.2H.

19. The conjugate of claim 1, wherein L.sup.3 is absent.

20. The conjugate of claim 1, wherein L.sup.3 is optionally substituted C1-C6 alkyl.

21. The conjugate of claim 1, wherein said polypeptide is an antibody, or an antigen-binding fragment thereof.

22. The conjugate of claim 21, wherein said antibody, or antigen-binding fragment thereof, binds to insulin-like growth factor-1 receptor (IGF1R).

23. A pharmaceutical composition comprising a conjugate of claim 1 and a pharmaceutically acceptable excipient.

24. The conjugate of claim 1, wherein the metallic radionuclide is a radionuclide selected from the group consisting of .sup.64Cu, .sup.67Cu, .sup.89Zr, .sup.90Y, .sup.97Ru, .sup.99Tc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.149Pm, .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, and .sup.229Th.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph illustrating binding affinities for select conjugates.

(2) FIG. 2 is a graph illustrating residualization of select conjugates.

(3) FIG. 3 is the amino acid sequence of the CDRs of figitumumab.

(4) FIG. 4 is the amino acid sequence of the variable domains of figitumumab.

(5) FIG. 5 is the amino acid sequence of the heavy chain of figitumumab.

(6) FIG. 6 is the amino acid sequence of the light chain of figitumumab.

DETAILED DESCRIPTION

(7) The present invention relates to linkers that enhance residualization or retention of detection agents in cells after internalization of polypeptide-linker-detection agent conjugate. Typically cell internalizing labeled polypeptides are limited by loss of the label after lysosomal processing. This leads to significant loss of signal or payload at the desired site and potential toxicity in normal tissues. To overcome this limitation a platform of residualizing linkers capable of carrying a wide variety of detection agents has been developed and demonstrated improved radioactivity retention (residualization) in target cells when linked to polypeptides.

(8) Polypeptides

(9) 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.

(10) 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, Mac1, 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 chemookine 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.

(11) Antibodies

(12) 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).

(13) Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized 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.

(14) 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.

(15) 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.

(16) 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).

(17) Insulin-Like Growth Factor 1 (IGF-1R) Antibodies

(18) 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). IGF-1R is implicated in several cancers including breast cancer, non-small cell lung cancer, prostate cancer, colon cancer, sarcoma, and adrenocortical carcinoma, increased levels of IGF-1R are expressed on the surface of tumor cells of these cancers.

(19) Several IGF-1R antibodies have been developed and investigated for the treatment of various types of cancers including figitumumab, cixutumumab, AMG479, BIIB002, SCH717454, and R1507. After binding to IGF-1R, these antibodies are internalized into the cell and degraded by ysosomal 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.

(20) Modified Polypeptides

(21) 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).

(22) 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.

(23) Modifications include those by natural processes, such as posttranslational 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 posttranslational 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 fiavin, 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.

(24) 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).

(25) 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.

(26) 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.

(27) TABLE-US-00001 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

(28) 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.

(29) Detection Agents

(30) 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-strepavidin 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.

(31) Radioisotopes and Radionuclides

(32) 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.64Cu, .sup.67Cu, .sup.75Br, .sup.76Br, .sup.77Br, .sup.89Zr, .sup.90Y, .sup.97Ru, .sup.99Tc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.149Pm, .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, and .sup.229Th.

(33) Metal Chelates

(34) Chelating groups known in the art for their utility as detection agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriamiepentaacetic acid (DTPA), porphyrins, polyamines (such as diaminodioximes, diaminodithiols (N.sub.2S.sub.2 chelates), and triaminothiol (N.sub.3S)), crown ethers, bis-thiosemicarbazones, 1,4,7-triazacyclononane-N,N′,N,″-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), triethylenetetramime (TETA), poly-oximes, and Re/Tc(I) difunctional chelates. Chelates are coupled to the linker using standard chemistries. 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.62Cu, .sup.64Cu, .sup.67Ga, .sup.90Y, .sup.94mTc, .sup.99mTc, .sup.111In, .sup.177Lu, .sup.213Bi, .sup.225Ac, .sup.227Th, and .sup.229Th.

(35) Luminescent and Fluorescent Agents

(36) Luminescent and fluorescent agents known in the art for their utility as detection agents include, but are not limited to, indocyanines (eg. Cy3 or Cy5), coumarins, fluorescein (eg. FITC), rhodamines, carbopyronins, oxazines, and luciferins (bioluminescent).

(37) Residualizing Linkers

(38) Residualizing linkers are designed to retain the label intracellularly after lysosomal degradation of the internalized polypeptide conjugate.

(39) Linkers of the invention have the structure of Formula II:

(40) ##STR00027## or a salt thereof, L.sup.1 and L.sup.2 are independently absent, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C10 cycloalkynyl, optionally substituted oxime, optionally substituted hydrazone, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted C2-C100 polyethylene glycol, or L.sup.4-B; L.sup.3 is absent or optionally substituted C1-C6 alkyl; m is 0 or 1; each R.sup.1 and R.sup.2 are independently hydrogen, —CH.sub.2CO.sub.2H, or —CH.sub.2CH.sub.2CO.sub.2H; each R.sup.3 and R.sup.4 is independently hydrogen or R.sup.3 and R.sup.4 combine to form C═O; n is 0 or 1; is an integer between 1 and 10; R.sup.5 is hydrogen or L.sup.4-B; R.sup.6 and R.sup.7 are independently hydrogen, optionally substituted C1-C6 heteroalkyl, or L.sup.4-B; R.sup.9 is —CO.sub.2H, —N═C═O, —N═C═S,

(41) ##STR00028## L.sup.4 is independently absent, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C4-C10 cycloalkenyl, optionally substituted C4-C10 cycloalkynyl, optionally substituted oxime, optionally substituted hydrazone, optionally substituted aryl, or optionally substituted heterocyclic; B is an organic moiety including a detection agent; wherein at least one R.sup.1 or R.sup.2 is —CH.sub.2CO.sub.2H or CH.sub.2CH.sub.2CO.sub.2H; at least one of L.sup.1 and L.sup.2 are present and when L.sup.1 or L.sup.2 are absent, m is 0;
and one and only one of L, L.sup.2, R.sup.5, R.sup.6, and R.sup.7 is L.sup.4-B.

(42) The linkers of the invention comprise three distinct modules that together result in their increased effectiveness compared to those known in the art.

(43) 1. Amine Reactive Linker Module:

(44) The R.sup.9 groups of the linkers of the invention react with free amine groups of polypeptides and facilitate one-step conjugation reactions without additional modification to the antibody structure. The linker portion of the module may be any length or lipophilicity to allow for enhanced physiochemical properties and biological activity.

(45) 2. Negatively Charged or Zwitterionic Residualizer Module:

(46) The negatively charged or zwitterionic backbone is advantageous as it displays increased residualization compared to positively charged or neutral backbones and is not taken up by the kidney non-specifically after release from the polypeptide in vivo.

(47) 3. Detection Agent Module:

(48) The linkers of the invention have an independent module for incorporation of the detection agent allowing for installation of the detection agent without modification to the residualizing backbone.

(49) Administration and Dosage

(50) 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).

(51) 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.

(52) 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 7 and 8, such as 7 to 7.5. 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.

(53) 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.

(54) 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.

(55) 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.

(56) 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.

(57) 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.

(58) By “antiproliferative” 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.

(59) TABLE-US-00002 TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazine ifosfamide altretamine hexamethylmelamine estramustine phosphate thiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomide cyclophosphamide Semustine Platinum agents spiroplatin lobaplatin (Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464 (Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo) ZD-0473 (AnorMED) AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecan mesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) J-107088 (Merck & Co) rebeccamycin analogue (Exelixis) BNP-1350 (BioNumerik) BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang) rubitecan (SuperGen) KW-2170 (Kyowa Hakko) irinotecan (CPT-11) hydroxycamptothecin (SN-38) topotecan Antitumor antibiotics valrubicin azonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycin MEN-10755 (Menarini) porfiromycin GPX-100 (Gem Pharmaceuticals) mitoxantrone (novantrone) Epirubicin amonafide mitoxantrone doxorubicin Antimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma) auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075 (GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin A paclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi) atamestane (BioMedicines) formestane letrozole exemestane anastrazole Thymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagonists trabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxter International) mafosfamide (Baxter International) albumin + 32P (Isotope Solutions) apaziquone (Spectrum thymectacin (NewBiotics) Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferase arglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex) inhibitors marimastat (British Biotech) BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox (Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene) agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoic acid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptor agonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy (Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK (Progenies) Technology) adenocarcinoma vaccine (Biomira) ISF-154 (Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2 (Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira) synchrovax vaccines (CTL Immuno) MGV (Progenies) melanoma vaccine (CTL Immuno) ß-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy (Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCam Biotherapeutics) abatacept (BMS) MPDL3280A (Genentech) Hormonal and estrogens dexamethasone antihormonal agents conjugated estrogens prednisone ethinyl estradiol methylprednisolone chlortrianisen prednisolone idenestrol aminoglutethimide hydroxyprogesterone caproate leuprolide medroxyprogesterone octreotide testosterone mitotane testosterone propionate; P-04 (Novogen) fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosterone arzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofine bicalutamide goserelin flutamide Leuporelin nilutamide bicalutamide Photodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda) agents Theralux (Theratechnologies) lutetium texaphyrin (Pharmacyclics) motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (lmClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (lmClone) trastuzumab (Genentech) Tyrphostins OS1-774 (Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B-Raf enzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BOX-1777 (PNP inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent, SRI CapCell ™ (CYP450 stimulant, Bavarian Nordic) International) GCS-100 (gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent, Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen (NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, Allos Therapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88 (heparanase inhibitor, Progen) seocalcitol (vitamin D receptor agonist, Leo) tesmilifene (histamine antagonist, YM 131-I-TM-601 (DNA antagonist, BioSciences) TransMolecular) histamine (histamine H2 receptor agonist, Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDH inhibitor, Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide (integrin antagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinase inhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE V inhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst) CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer, AG-2037 (GART inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogen activator inhibitor, Wilex) Immunol ™ (triclosan oral rinse, Endo) PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine (uridine prodrug, Wellstat) bortezomib (proteasome inhibitor, Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant, SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor, TransMID-107 ™ (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosis promotor, Procyon) PT-100 (growth factor agonist, Point doranidazole (apoptosis promotor, Pola) Therapeutics) CHS-828 (cytotoxic agent, Leo) midostaurin (PKC inhibitor, Novartis) trans-retinoic acid (differentiator, NIH) bryostatin-1 (PKC stimulant, GPC Biotech) MX6 (apoptosis promotor, MAXIA) CDA-II (apoptosis promotor, Everlife) apomine (apoptosis promotor, ILEX Oncology) SDX-101 (apoptosis promotor, Salmedix) urocidin (apoptosis promotor, Bioniche) rituximab (CD20 antibody, Genentech Ro-31-7453 (apoptosis promotor, La Roche) carmustine brostallicin (apoptosis promotor, Pharmacia) Mitoxantrone β-dapachone Bleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesium oxides caffeic acid BRAF inhibitors, Tyrphostin AG PDL1 inhibitors PD-1 inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenib angiogenesis inhibitors dabrafenib

(60) 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

(61) General Methods

(62) All reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions, unless otherwise specified. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light and an ethanolic solution of phosphomolybdic acid and cerium sulfate followed by heating as visualizing strategies. E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash column chromatography. 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 electrosparay 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. Analysis and purification of radioactive materials were performed using similar HPLC systems equipped with an additional Bioscan Flow Count radiodetector (FC-3300). SEC was performed on a Phenomenex BioSep s2000, 7.8×300 mm, 1 mL/min, λ220/280 nm under isocratic conditions with mobile phase of 100 mM PO4- at pH 6.8. HPLC elution method 1: Waters Acquity BEH C18 2.1×50 (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; 0.fwdarw.3 min, 90.fwdarw.0% A; 3.fwdarw.4 min, 0% A. HPLC elution method 2: Waters XBridge Prep Phenyl 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.5% v/v AcOH); mobile phase B: Acetonitrile (0.5% v/v AcOH); flow rate: 10 mL/min; 0.fwdarw.10 minutes, 50%.fwdarw.30% A; 10.fwdarw.12 minutes, 30% A. HPLC elution method 3: 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: 11 mL/min; 0.fwdarw.11.5 minutes, 75%.fwdarw.56% A. HPLC elution method 4: Waters Acquity BEH C18 2.1×50 (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; 0.fwdarw.8 min, 90.fwdarw.0% A. 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; 0.fwdarw.10 minutes, 80%.fwdarw.50% A. HPLC elution method 6: Waters XBridge Prep Phenyl 19×100 mm (5 μm) column; mobile phase A: H.sub.2O (0.5% v/v AcOH); mobile phase B: Acetonitrile (0.5% v/v AcOH); flow rate: 10 mL/min; 0.fwdarw.2 minutes, 46% A; 2.fwdarw.13 minutes, 46%.fwdarw.30% A.

Example 1: Synthesis of CPD-1022 and CPD-1023

(63) Reaction Scheme

(64) ##STR00029##
Amine Intermediate:

(65) ##STR00030##

(66) The tetrapeptide backbone of CPD-1022 (Fmoc-D-Glu-D-Glu-D-Glu-D-Lys-OH) was prepared using standard solid phase peptide synthesis techniques. To a solution of the backbone tetrapeptide (100 mg, 0.132 mmol) in 1.5 mL of DMF were added 2,3,5,6-tetrafluorophenyl 3-tributylstannylbenzoate (191 mg, 0.341 mmol) and diisopropylethylamine (40 μL, 0.229 mmol) at room temperature. The reaction solution was stirred for 1 hour then diluted with a mixture of cold diethyl ether and hexanes (45 mL, 4:1 v/v). The resultant suspension was centrifuged at 4000 rpm for 15 minutes and the white precipitate (138 mg, 0.120 mmol, 91% yield) was collected and washed with 20 mL of cold diethyl ether. To this white solid (90 mg, 0.078 mmol) was added 1.0 mL of 20% piperidine/DMF and the mixture was stirred at room temperature for 20 minutes before dilution with cold diethyl ether (45 mL). The resultant white precipitate (collected after centrifuging at 4000 rpm for 15 minutes) was taken up in acetonitrile/water/AcOH (4.0 mL, 45:45:10 v/v/v) and loaded on a C-18 plus Seppak SPE cartridge. The cartridge was flushed with acetonitrile/water (20 mL, 1:2 v/v) first to remove residual piperidine and the desired product was eluted off the cartridge with acetonitrile/water (200 mL, 3:1 v/v). The final product was obtained as white amorphous powder after evaporation of solvents (65 mg, 0.07 mmol, 90% yield).

(67) HPLC-MS elution method 1; retention time: 3.03 min; MS (positive ESI): found m/z 928.0 [M+H].sup.+; C.sub.40H.sub.66N.sub.5O.sub.12Sn (calc. 928.3).

(68) Synthesis of CPD-1022:

(69) ##STR00031##

(70) To a solution of the above free amine (24 mg, 0.026 mmol) in DMF (0.5 mL) were added an excess of isophthalic acid NHS ester (100 mg, 0.278 mmol, 10.7 equiv.) and diisopropylethylamine (6.0 μL, 0.035 mmol) at room temperature. LC-MS indicated the reaction was complete within 10 minutes. Cold ether was added to precipitate the crude product, which was collected after centrifuging at 4000 rpm for 15 minutes. Purification was accomplished by preparative HPLC (method 2, retention time: 9.9 min) to afford CPD-1022 in 62% yield (19 mg, 0.016 mmol).

(71) HPLC-MS elution method 1; retention time: 3.30 min; MS (positive ESI): found m/z 1173.1 [M+H].sup.+; C.sub.52H.sub.73N.sub.6O.sub.17Sn (calc. 1173.4).

(72) .sup.1H NMR (700 MHz, DMSO-d.sub.6) δ 8.92 (1H, d, J=7.4 Hz), 8.60 (1H, s), 8.44 (1H, t, J=5.5 Hz), 8.32 (1H, d, J=7.8 Hz), 8.26 (1H, d, J=7.8 Hz), 8.16 (1H, d, J=7.6 Hz), 8.14 (1H, m), 7.99 (1H, d, J=7.4 Hz), 7.89 (1H, s), 7.77 (1H, t, J=7.8 Hz), 7.74 (1H, d, J=7.8 Hz), 7.56 (1H, d, J=7.1 Hz), 7.40 (1H, dd, J=7.8, 7.1 Hz), 4.48 (1H, m), 4.32-4.26 (2H, m), 4.13 (1H, m), 3.24 (2H, dt, J=5.5, 6.8 Hz), 2.92 (4H, s), 2.40-2.20 (6H, m), 2.05 (1H, m), 1.97-1.86 (3H, m), 1.82-1.69 (3H, m), 1.63 (1H, m), 1.58-1.43 (8H, m), 1.41-1.33 (2H, m), 1.30 (6H, m), 1.08 (6H, m), 0.86 (9H, t, J=7.3 Hz) (Sn—H couplings are not presented for simplicity);

(73) .sup.13C NMR (175 MHz, DMSO-d.sub.6) δ 174.00, 173.44, 171.17, 171.08, 170.90, 170.22, 166.48, 165.19, 161.44, 141.37, 138.75, 135.12, 134.75, 134.46, 133.99, 132.58, 129.69, 128.96, 127.65, 126.78, 124.58, 53.11, 52.01, 51.89, 51.58, 39.01, 30.61, 30.52, 30.14, 29.98, 28.90, 28.56, 27.48, 27.28, 26.79, 26.65, 25.56, 22.99, 13.54, 9.18 (two carboxyls missing due to signal overlapping; Sn—C couplings are not presented for simplicity).

(74) CPD-1023:

(75) ##STR00032##

(76) To a solution of CPD-1022 (5.0 mg, 4.3 μmol) in acetonitrile/water (0.5 mL, 3:1 v/v) was added iodine (2.0 mg, 7.9 μmol). Excess of iodine was quenched by the addition of Na.sub.2S.sub.2O.sub.5 (0.1 mL, 0.1 M aq.) and the reaction mixture purified by preparative HPLC (method 3, retention time: 10.4 min) to give the desired product in 85% yield (3.7 mg, 3.7 μmol).

(77) HPLC-MS elution method 1; retention time: 1.79 min; MS (positive ESI): found m/z 1009.4 [M+H].sup.+; C.sub.40H.sub.46IN.sub.6O.sub.17 (calc. 1009.2).

(78) .sup.1H NMR (700 MHz, DMSO-d.sub.6) δ 8.92 (1H, d, J=7.5 Hz), 8.60 (1H, s), 8.55 (1H, t, J=5.5 Hz), 8.32 (1H, d, J=7.9 Hz), 8.26 (1H, d, J=7.8 Hz), 8.18 (1H, s), 8.17-8.12 (2H, m), 7.98 (1H, d, J=7.8 Hz), 7.88 (1H, d, J=7.7 Hz), 7.85 (1H, d, J=7.9 Hz), 7.78 (1H, dd, J=7.9, 7.8 Hz), 7.27 (1H, dd, J=7.9, 7.7 Hz), 4.47 (1H, m), 4.32-4.25 (2H, m), 4.13 (1H, m), 3.23 (2H, dt, J=5.5, 6.8 Hz), 2.92 (4H, s), 2.41-2.22 (6H, m), 2.05 (1H, m), 1.97-1.88 (3H, m), 1.81-1.70 (3H, m), 1.63 (1H, m), 1.55-1.48 (2H, m), 1.40-1.33 (2H, m);

(79) .sup.13C NMR (175 MHz, DMSO-d.sub.6) δ 174.01, 173.99, 173.98, 173.44, 171.19, 171.10, 170.91, 170.23, 165.20, 164.53, 161.45, 139.53, 136.63, 135.56, 135.12, 134.47, 132.59, 130.44, 129.70, 128.96, 126.63, 124.57, 94.62, 53.13, 51.96, 51.89, 51.55, 39.09, 30.54, 30.51, 30.12, 29.98, 28.65, 27.47, 27.27, 26.78, 25.56, 22.93.

Example 2: Synthesis of CPD-1052

(80) CPD-1051:

(81) ##STR00033##

(82) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(83) HPLC-MS elution method 1; retention time: 3.60 min; MS (positive ESI): found m/z 1224.7 [M+H].sup.+; C.sub.54H.sub.70F.sub.4N.sub.5O.sub.15Sn (calc. 1224.4).

(84) CPD-1052:

(85) ##STR00034##

(86) The title compound was prepared from CPD-1051 in a similar manner to the synthesis of CPD-1023.

(87) HPLC-MS elution method 1; retention time: 2.19 min; MS (positive ESI): found m/z 1060.4 [M+H].sup.+; C.sub.42H.sub.43F.sub.41IN.sub.5O.sub.15 (calc. 1060.2).

Example 3: Synthesis of CPD-1055

(88) CPD-1054:

(89) ##STR00035##

(90) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(91) HPLC-MS elution method 1; retention time: 3.27 min; MS (positive ESI): found m/z 1179.6 [M+H].sup.+; C.sub.52H.sub.79N.sub.6O.sub.17Sn (calc. 1179.4).

(92) CPD-1055:

(93) ##STR00036##

(94) The title compound was prepared from CPD-1054 in a similar manner to the synthesis of CPD-1023.

(95) HPLC-MS elution method 1; retention time: 1.75 min; MS (positive ESI): found m/z 1014.9 [M+H].sup.+; C.sub.40H.sub.52IN.sub.6O.sub.17 (calc. 1015.2).

Example 4: Synthesis of CPD-1065

(96) CPD-1064:

(97) ##STR00037##

(98) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(99) HPLC-MS elution method 1; retention time: 2.43 min; MS (positive ESI): found m/z 1174.6 [M+H].sup.+; C.sub.51H.sub.72NO.sub.17Sn (calc. 1174.4).

(100) CPD-1065:

(101) ##STR00038##

(102) The title compound was prepared from CPD-1064 in a similar manner to the synthesis of CPD-1023.

(103) HPLC-MS elution method 1; retention time: 1.54 min; MS (positive ESI): found m/z 1010.4 [M+H].sup.+; C.sub.39H.sub.45IN.sub.7O.sub.17 (calc. 1010.2).

Example 5: Synthesis of CPD-1067

(104) CPD-1066:

(105) ##STR00039##

(106) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(107) HPLC-MS elution method 4; retention time: 5.97 min; MS (positive ESI): found m/z 1173.7 [M+H].sup.+; C.sub.52H.sub.73N.sub.6O.sub.17Sn (calc. 1173.4).

(108) CPD-1067:

(109) ##STR00040##

(110) The title compound was prepared from CPD-1066 in a similar manner to the synthesis of CPD-1023.

(111) HPLC-MS elution method 4; retention time: 3.05 min; MS (positive ESI): found m/z 1009.4 [M+H].sup.+; C.sub.40H.sub.46IN.sub.6O.sub.17 (calc. 1009.2).

Example 6: Synthesis of CPD-1048

(112) CPD-1047:

(113) ##STR00041##

(114) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(115) HPLC-MS elution method 4; retention time: 2.31 min; MS (positive ESI): found m/z 927.5 [M+H].sup.+; C.sub.42H.sub.51N.sub.6O.sub.18 (calc. 927.3).

(116) ##STR00042##

(117) The title compound was prepare in a similar manner to the synthesis of CPD-1022.

(118) HPLC-MS elution method 4; retention time: 2.76 min; MS (positive ESI): found m/z 1053.4 [M+H].sup.+; C.sub.42H.sub.50IN.sub.6O.sub.18 (calc. 1053.2).

Example 7: Synthesis of CPD-1107

(119) CPD-1107:

(120) ##STR00043##

(121) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(122) HPLC-MS elution method 4; retention time: 1.63 min; MS (positive ESI): found m/z 1165.7 [M+H].sup.+; C.sub.49H.sub.69N.sub.10O.sub.23 (calc. 1165.4).

Example 8: Synthesis of CPD-1072

(123) CPD-1071:

(124) ##STR00044##

(125) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(126) HPLC-MS elution method 1; retention time: 3.27 min; MS (positive ESI): found m/z 1153.7 [M+H].sup.+; C.sub.50H.sub.77N.sub.6O.sub.17Sn (calc. 1153.4).

(127) CPD-1072:

(128) ##STR00045##

(129) The title compound was prepared from CPD-1071 in a similar manner to the synthesis of CPD-1023.

(130) HPLC-MS elution method 1; retention time: 1.71 min; MS (positive ESI): found m/z 989.4 [M+H].sup.+; C.sub.38H.sub.50IN.sub.6O.sub.17 (calc. 989.2).

Example 9: Synthesis of CPD-1081

(131) CPD-1080:

(132) ##STR00046##

(133) The title compound was prepared in a similar manner to the synthesis of CPD-1022.

(134) HPLC-MS elution method 1; retention time: 3.23 min; MS (positive ESI): found m/z 1213.2 [M+H].sup.+; C.sub.52H.sub.81N.sub.6O.sub.19Sn (calc. 1213.5).

(135) CPD-1081:

(136) ##STR00047##

(137) The title compound was prepared from CPD-1080 in a similar manner to the synthesis of CPD-1023.

(138) HPLC-MS elution method 1; retention time: 1.68 min; MS (positive ESI): found m/z 1049.1 [M+H].sup.+; C.sub.40H.sub.53IN.sub.6O.sub.19 (calc. 1049.2).

Example 10: Synthesis of CPD-1085

(139) CPD-1084:

(140) ##STR00048##

(141) Starting from a β-peptide backbone (Fmoc-β-Glu-β-Glu-β-Glu-D-Lys-OH), which was prepared using standard solid phase peptide synthesis techniques, the title compound was prepared in a similar manner to the synthesis of CPD-1022.

(142) HPLC-MS elution method 1; retention time: 3.21 min; MS (positive ESI): found m/z 1179.7 [M+H].sup.+; C.sub.52H.sub.79N.sub.6O.sub.17Sn (calc. 1179.4).

(143) CPD-1085:

(144) ##STR00049##

(145) The title compound was prepared from CPD-1084 in a similar manner to the synthesis of CPD-1023.

(146) HPLC-MS elution method 1; retention time: 1.68 min; MS (positive ESI): found m/z 1015.6 [M+H].sup.+; C.sub.40H.sub.52N.sub.6O.sub.17 (calc. 1015.2).

Example 11: Synthesis of CPD-1092

(147) CPD-1091:

(148) ##STR00050##

(149) Starting from the hybrid backbone Fmoc-Nglu-Nglu-Nglu-D-Lys-OH, the title compound was prepared in a similar manner to the synthesis of CPD-1022.

(150) HPLC-MS elution method 4; retention time: 6.60 min; MS (positive ESI): found m/z 1173.5 [M+H].sup.+; C.sub.52H.sub.73N.sub.6O.sub.17Sn (calc. 1173.4).

(151) CPD-1092:

(152) ##STR00051##

(153) The title compound was prepared in a similar manner to the synthesis of CPD-1023.

(154) HPLC-MS elution method 1; retention time: 1.74 min; MS (positive ESI): found m/z 1009.4 [M+H].sup.+; C.sub.40H.sub.46IN.sub.6O.sub.17 (calc. 1009.2).

Example 12: Synthesis of CPD-1098

(155) CPD-1097:

(156) ##STR00052##

(157) Starting from the hybrid backbone Fmoc-β-Glu-β-Glu-β-Glu-Nlys-OH, the title compound was prepared in a similar manner to the synthesis of CPD-1022.

(158) HPLC-MS elution method 1; retention time: 3.23 min; MS (positive ESI): found m/z 1173.5 [M+H].sup.+; C.sub.52H.sub.73N.sub.6O.sub.17Sn (calc. 1173.4).

(159) CPD-1098:

(160) ##STR00053##

(161) The title compound was prepared from CPD-1097 in a similar manner to the synthesis of CPD-1023.

(162) HPLC-MS elution method 1; retention time: 1.70 min; MS (positive ESI): found m/z 1009.4 [M+H].sup.+; C.sub.40H.sub.46IN.sub.6O.sub.17 (calc. 1009.2).

Example 13: Synthesis of CPD-1102

(163) CPD-1101:

(164) ##STR00054##

(165) Starting from the peptoid backbone Fmoc-Nglu-Nglu-Nglu-Nlys-OH, the title compound was prepared in a similar manner to the synthesis of CPD-1022.

(166) HPLC-MS elution method 1; retention time: 3.29 min; MS (positive ESI): found m/z 1174.0 [M+H].sup.+; C.sub.52H.sub.73N.sub.6O.sub.17Sn (calc. 1173.4).

(167) CPD-1102:

(168) ##STR00055##

(169) The title compound was prepared from CPD-1101 in a similar manner to the synthesis of CPD-1023.

(170) HPLC-MS elution method 1; retention time: 1.75 min; MS (positive ESI): found m/z 1009.1 [M+H].sup.+; C.sub.40H.sub.46IN.sub.6O.sub.17 (calc. 1009.2).

Example 14: Synthesis of CPD-1094

(171) ##STR00056## ##STR00057##
Polyamine Backbone (Free Amine):

(172) ##STR00058##

(173) To a solution of DTPA-anhydride (0.150 g, 0.419 mmol) in 2 mL of DMF was added H-Nlys(Boc)-Ot-Bu (0.152 g, 0.502 mmol). The reaction mixture was stirred at room temperature for 1 hour before the addition of N-Fmoc-1,4-butanediamine hydrochloride salt (0.146 g, 0.419 mmol). After an additional 1 hour of stirring the mixture was diluted with diethyl ether (25 mL) and the suspension centrifuged at 4000 rpm for 15 minutes. The white precipitate was then collected and treated with a mixture of TFA/water/TES (95:2.5:2.5 v/v/v, 2 mL) at room temperature for 2 hours. Volatiles were removed under vacuum and the residue purified by preparative-HPLC (method 5, retention time: 8.9 min) to produce the desired product as an amorphous white solid (0.123 g, 0.151 mmol, 36% yield).

(174) HPLC-MS elution method 4; retention time: 2.97 min; MS (positive ESI): found m/z 814.5 [M+H].sup.+; C.sub.39H.sub.56N.sub.7O.sub.12 (calc. 814.4).

(175) Polyamine Backbone (Stannane):

(176) ##STR00059##

(177) To a solution of the polyamine backbone (62 mg, 0.076 mmol) in 1.0 mL of DMF were added 2,3,5,6-tetrafluorophenyl 3-tributylstannylbenzoate (84 mg, 0.151 mmol) and diisopropylethylamine (30 μL, 0.172 mmol) at room temperature. The reaction solution was stirred for 1 hour and then diluted with a mixture of cold diethyl ether (25 mL). The resultant suspension was centrifuged at 4000 rpm for 15 minutes and the white precipitate collected and washed with 20 mL of cold diethyl ether. This intermediate (91 mg, 0.075 mmol, 99% yield) was used in the next step without further purification. HPLC-MS elution method 1; retention time: 3.42 min; MS (positive ESI): found m/z 1208.5 [M+H].sup.+; C.sub.58H.sub.86N.sub.7O.sub.13Sn (calc. 1208.5).

(178) CPD-1094:

(179) ##STR00060##

(180) To a solution of the above polyamine-stannane (45 mg, 0.037 mmol) and adipic acid bis-NHS active ester (0.127 g, 0.372 mmol) in 1 mL of DMF was added triethylamine (0.20 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours before dilution with diethyl ether (25 mL). The resultant suspension was centrifuged at 4000 rpm for 15 minutes and the solid collected. Purification was accomplished by preparative-HPLC (method 6, retention time: 7.7 min) to yield the final product as an amorphous white solid (6.5 mg, 5.4 μmol, 12% yield).

(181) HPLC-MS elution method 1; retention time: 3.00 min; MS (positive ESI): found m/z 1211.6 [M+H].sup.+; C.sub.53H.sub.87N.sub.8O.sub.16Sn (calc. 1211.5).

(182) CPD-1095:

(183) ##STR00061##

(184) The title compound was prepared from CPD-1094 in a similar manner to the synthesis of CPD-1023.

(185) HPLC-MS elution method 1; retention time: 1.61 min; MS (positive ESI): found m/z 1047.5 [M+H].sup.+; C.sub.41H.sub.60IN.sub.8O.sub.16 (calc. 1047.3).

Example 15: Radiochemistry

(186) All compounds were prepared using the following general procedure described for the radiolabeling of CPD1028 with Na.sup.131I. Table 3 describes representative labeling results for some of the different residualizing linkers described herein.

(187) Radiolabeling Precursor CPD1022 with Na.sup.131I to Prepare CPD1028

(188) ##STR00062##

(189) Precursor CPD 1022 (80 μg) was dissolved in 50 μL of acetonitrile and added to a 20 mL scintillation vial. Sodium iodide (Na.sup.131I, 20 μL) was premixed with 5 uL of acetic acid in a 1.5 mL plastic vial and subsequently added to the scintillation vial containing the precursor. Iodogen (5 μL of a 1 mg/mL solution in acetonitrile) was added and the resultant solution allowed to stand for 5 minutes at room temperature after which the reaction was quenched with 25 μL of sodium thiosulfate. Intermediate radiolabeled linker CPD1023 was then purified via HPLC.

(190) Purified HPLC fractions of CPD1023 were combined and dried under vacuum in a 60° C. water bath. The vial was further dried with 2×200 μL of EtOH to remove any residual TFA. The resulting residue was dissolved in 20% DMSO in PBS (containing 0.01% Tween80), vortexed and centrifuged. To this vial was added 10 μL of figitumumab followed by 5 μL of borate buffer (0.1M, pH=8.5) and allowed to sit at room temperature for 2 hours. The crude conjugated product was subsequently purified via a Sephadex packed column containing 35 mm G50 resin in a 1 mL housing. The final radiolabeled product, CPD 1028 was eluted from the column with PBS containing 0.01% Tween80 in high purity as analyzed by SEC and iTLC for purity.

(191) TABLE-US-00003 TABLE 3 Residualizing radiolabeled antibodies Radiochemical Pre- Yield (%) Radio- Specific Final Pre- cursor La- Conju- Over- chemical Activity Product cursor Standard beling gation all Purity (□Ci/□g) 1028 1022 1023 80 43 33 98 14 1028 1051 1052 55 75 20 98 15.1 1049 1054 1055 62 18 6 95 7 1068 1066 1067 56 36 20 99 4.4 1073 1071 1072 82 50 20 96 12.5 1074 1080 1081 74 46 33 96 14.2 1079 1084 1085 34 56 18 96 13.7 1093 1091 1092 80 33 23 99 14.6 1096 1094 1095 43 40 17 99 9.4 1100 1097 1098 81 41 32 98 16.4 1104 1101 1102 77 41 30 99 11 1106 1022 1023 72 8.5 6 99 5.6 Note: Most compounds were labeled with antibody Figitumumab with the exception of 1106 which used an anti-EGFR clone 528 monoclonal antibody

Example 16: In Vitro Evaluation

(192) Saturation Binding Experiments

(193) Saturation binding experiments measure the specific binding at equilibrium of a radioligand at various concentrations in order to determine the Kd (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 radioligand binding in the presence of a fixed concentration of an unlabeled compound that binds to essentially all the receptors. Since all the receptors are occupied by the unlabeled drug, the radioligand only binds nonspecifically. Kd and Bmax values are calculated by nonlinear regression analysis and computerized curve fitting.

(194) The purpose of this assay was to ensure that these new radiolabelled conjugates 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 labeled conjugate 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 h. 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 h with mild shaking. Following the blocking step, 100 μl of labeled conjugate was added to each well. The plates were then incubated at 4° C. for 2 h. 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 labeled conjugate 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 FIG. 1. The K.sub.d and B.sub.max were derived by curve fitting of the specific binding data to a single-site hyperbola model (GraphPad Prism Software).

(195) Residualization

(196) 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 radioligand 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 radioligand. Fresh medium is reapplied and the cells are again incubated for a pre-determined amount of time. It is during this period that cell processes degrade the radioligand 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.

(197) 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. Labeled conjugate was diluted in serum-free DMEM to a concentration of 2 nM. 500 uL of radioligand 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, 2 and 24 h. 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 (FIG. 2) was determined as CPM (lysate)/CPM (efflux+recycled+Iysate).

Example 17: Diagnostic and Radiation Treatment Planning Use

(198) Antibody-linker conjugates prepared by these means can used for diagnostic purposes including but not limited to detection of antigen expressing tumors and/or for measuring antigen biomarker levels to obtain biochemical or pathological information about course of disease, severity of disease, change in disease phenotype or indicate treatment planning. For example, high levels of antigen target expression in tumors measured by imaging antibody-linker conjugates in patients may be indicative of developing resistance to an ongoing chemotherapeutic treatment and provide information to enable change of a current course of treatment. High levels of target antigen in tumors measured by antibody-linker conjugate imaging may indicate aggressive, proliferative or metastatic disease and dictate subsequent treatment. Another example is that detection of antigen on target expressing tumors by imaging with an antibody-linker conjugate may indicate therapeutic efficacy of the same conjugate labeled with a therapeutic radioisotope or any other suitable chemotherapeutic in the form of an antibody drug conjugate. Follow up diagnostic imaging of an appropriate biomarker with an antibody-linker conjugate is also useful for tracking patient response to an ongoing treatment plan.

Other Embodiments

(199) All patents, patent applications, and publications mentioned in this specification are herein incorporated by reference, to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.