Use of radiopharmaceuticals for treatment of cancer

Abstract

Disclosed are methods of co-administration of a radioimmunoconjugate with a cold targeting molecule (e.g., an antibody) at a therapeutic dose for treatment of cancer.

Description

USE OF RADIOPHARMACEUTICALS FOR TREATMENT OF CANCERRELATED APPLICATIONS

[0001] The present application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 501,372, filed May 10, 2023, the entire contents of which are hereby incorporated by reference for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on April 25, 2024, is named FPI_049.xml and is 13.2 kilobytes in size.BACKGROUND

[0003] Radiopharmaceuticals have seen great advancement in the last several years as both diagnostic and therapeutic approaches for cancer.

[0004] Radioactive decay can cause direct physical damage (such as single or double- strandedDNA breaks) or indirect damage (such as by-stander or crossfire effects) to the biomolecules that constitute a cell. Drugs that deliver radionuclides to cancer cells, e.g., radiopharmaceuticals or radioimmunoconjugates, provide a mechanism to generate DNA damage with anti-cancer therapeutic effect and hold significant promise for improved patient outcomes.

[0005] Delivery of therapeutically effective doses of radiopharmaceuticals or radioimmunoconjugates have been limited by efficacy, toxicities, and side effects. Therefore, there is a need for improved methods of delivering radionuclides that would allow tolerable and effective treatment of cancer patients.SUMMARY

[0006] The present disclosure encompasses the insight that co-administration of a radioimmunoconjugate with a cold targeting molecule (e.g., an antibody) at a therapeutic dose may be tolerable and effective for treatment of cancer. The therapeutic dose of the cold targeting molecule can be determined based on its pharmacokinetic (PK) profile.

[0007] Specifically, the present disclosure provides a method of treating a patient having cancer, wherein the method comprises administering to a patient in need thereof an effective amount of a radioimmunoconjugate or a pharmaceutically acceptable salt thereof, wherein the radioimmunoconjugate comprises a structure of Formula I:A-L-B(I) wherein A is a chelating moiety comprising a radionuclide, B is antibody or an antigen-binding fragment thereof, and L is a linker; wherein the patient is being co- administered a therapeutic dose (DT) of a cold targeting molecule, wherein the DT of said cold targeting molecule is determined by:(a) administering the cold targeting molecule to the patient at various dose levels to determine a pharmacokinetic (PK) profile of the cold targeting molecule; and(b) formulating a DT of said cold targeting molecule based on the PK profile.

[0008] In some embodiments, provided are methods of treating a patient having cancer, wherein the method comprises administering to a patient in need thereof an effective amount of a radioimmunoconjugate or a pharmaceutically acceptable salt thereof, wherein the radioimmunoconjugate comprises a structure of Formula I:A-L-B(I) whereinA is a chelating moiety comprising a radionuclide, B is antibody or an antigen-binding fragment thereof, and L is a linker, wherein the patient is being co-administered a therapeutic dose (DT) of a cold targeting molecule, wherein the DT is equal to Cmaxx Vc, wherein Cmaxis the maximal concentration after bolus injection and Vcis the distribution volume of the central compartment where mixed linear and nonlinear clearance pathways are present and the bolus injection administration is occurred.

[0009] In some embodiments, the radioimmunoconjugate comprises225Ac.

[0010] In some embodiments, the radioimmunoconjugate comprises a structure of Formula II:A-LJ-(L2)n-B(II)whereinA is a chelating moiety comprising a radionuclide;B is an antibody or an antigen-binding fragment thereof;L1is a bond, C=O, C=S, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, or optionally substituted aryl or heteroaryl; n is an integer of 1 to 5 (inclusive); and each L2, independently, has a structure of -X'-L ’-Z1-. whereinX1is -C(O)NR1-*, -NR1C(O)-*, -C(S)NR1-*, -NR1C(S)-*, -OC(O)NR1-*, - NR1C(O)O-*, -NR1C(O)NR1-, -CH2-Ph-C(O)NR1-*, -NR1COH’h-CIB-*, -CH2-Ph- NH-C(S)NR1-*, -NR1C(S)-NH-Ph-CH2-*, -O-, or -NR1-, wherein “*” indicates the attachment point to L3, and each R1is independently hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, or optionally substituted aryl or heteroaryl;L3is optionally substituted C1-C50alkyl or optionally substituted C1-C50heteroalkyl; andZ1is -CH2-, -C(O)-, -C(S)-, -OC(O)-#, -C(O)O-#, -NR2C(O)-#, -C(O)NR2-#, or - NR2- , wherein indicates the attachment point to B, and each R2is independently hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0011] In some embodiments, the radioimmunoconjugate comprises the following structure chelated with a radionuclide:wherein B is an antibody or an antigen-binding fragment thereof. The antibody can be a monoclonal antibody or a bispecific antibody.

[0012] In some embodiments, the cold targeting molecule is a cold antibody, wherein the cold antibody refers to a non-radioactive drug directed to the same target antigen as the radioimmunoconj ugate.

[0013] In some embodiments, the PK profile of the cold targeting molecule is characterized by a one- or two-compartment PK model having either a nonlinear only clearance pathway or mixed linear and nonlinear clearance pathways.

[0014] In some embodiments, to formulate a DT of the cold targeting molecule, the following steps are performed: a. obtaining parameters of the PK profile, wherein the parameters comprise Michaelis-Menten constant (KM), maximal velocity (VM), first-order / linear clearance rate (Cl), and central volume of distribution (Vc); b. determining a critical concentration ( Ccrit) based on the parameters, wherein the PK profile of the cold targeting molecule is linear before Ccrit; and c. formulating a DT of the cold targeting molecule based on Ccritto reach a desired clinical endpoint.

[0015] In some embodiments, to formulate a DT of the cold targeting molecule, the following steps are performed: d. obtaining parameters of the PK profile, wherein the parameters comprise Michaelis-Menten constant ( KM), maximal velocity (VM), first-order / linear clearance rate (Cl), and central volume of distribution (Vc); e. determining a critical concentration (Ccrit) and a transitional concentration (Ctrans) based on the parameters, wherein the PK profile of the cold targeting molecule is linear both before Ccritand after KM / 3, and exhibits a transition at Ctransbetween Ccritand KM / 3; and f. formulating a DT of the cold targeting molecule based on Ctransto reach a desired clinical endpoint.

[0016] In some embodiments, the co-administration of the cold targeting molecule at the DT results in an improvement of the therapeutic index of the radioimmunoconjugate.

[0017] In some embodiments, the improvement of the therapeutic index of the radioimmunoconjugate is increase of tumor uptake of the radioimmunoconjugate, increase of tumor-to-background ratio, decrease of radioimmunoconjugate uptake in non-target tissues, reduction of hematological toxicity, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 illustrates dimensionless elimination rates for dimensionless number R being 4. Here the transitional concentration is calculated as sqrt(R) = 2; at this transitional concentration, the total mass elimination rate is almost 1, relative to VM, refer to black dot.

[0019] FIG. 2A illustrates dimensionless PK profde in a semi-log scale with dimensionless slopes at a few key concentrations for dimensionless concentration Xmaxbeing 100 and dimensionless number R being 10. FIG. 2B illustrates dimensionless slope vs dimensionless concentration. Both FIGs 2A and 2B show that Ctranslies in the middle of the transition zone of the PK profde.

[0020] FIG. 3 illustrates dimensionless analytical solution and two approximate solutions and their convergence and divergence around the transition zone for dimensionless concentration Xmax being 100 and dimensionless number R being 10.

[0021] FIG. 4 illustrates dimensionless PK profdes based on a PK model having two compartments with mixed (or parallel) linear and nonlinear clearance pathways for dimensionless concentration Xmaxbeing 100 and 1000, respectively; and dimensionless number R being 10; and dimensionless number Q / Cl being 4.0 and dimensionless number Vp / Vc being 2.5.

[0022] FIG. 5 illustrates the PK profde of Panitumumab, together with some key concentrations.

[0023] FIG. 6A is a schematic depicting the general structure of bifunctional chelate comprising a chelate, a linker, and a cross-linking group.

[0024] FIG. 6B is a schematic depicting the general structure of a bifunctional conjugate comprising a chelate, a linker, and a targeting moiety.

[0025] FIGs. 6C-6D are schematics depicting the structures of |1 11In|-DOTA-anti-IGF- 1 R and [225Ac]-DOTA-anti-IGF-lR, two exemplary radioimmunoconjugates disclosed herein.

[0026] FIG. 7 is a schematic depicting the synthesis of the bifunctional chelate, 4- { [11-oxo- 11 -(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl} -2-[4,7, 10-tris(carboxymethyl)-1 ,4,7, 10- tetraazacyclododecan-l-yl]butanoic acid (Compound B).

[0027] FIG. 8 is a schematic depicting the synthesis of the bifunctional chelate, 4-{[2-(2- {2-[3-oxo-3-(2, 3,5, 6-tetr afluorophenoxy jpropoxy] ethoxy }ethoxy)ethyl] carbamoyl} -2-[4, 7,10- tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-l-yl]butanoic acid (Compound C).

[0028] FIG. 9 is a schematic depicting the synthesis of IGF-lR-targeting radioimmunoconjugate [225Ac]-DOTA-anti-IGF-lR.

[0029] FIG. 10 shows a plot of pharmacokinetics of [111In ]-DOTA-anti-IGF- 1 R conjugate with and without cold IGF-1R antibody.

[0030] FIGs. 11A-11E show a plot of uptake of radioimmunoconjugate in various organs (FIG. 11 A: kidney; FIG. 11B: liver; FIG. 11C: red marrow; FIG. 11D: spleen; FIG. HE: whole body) via imaging analysis.

[0031] FIGs. 12A-12E show a plot of radioimmunoconjugate tumor uptake relative to background in various organs (FIG. 12A: lesion / liver; FIG. 12B: lesion / muscle; FIG. 12C: lesion / red marrow; FIG. 12D: lesion / total body; FIG. 12E: lesion / muscle) via imaging analysis.

[0032] FIG. 13A shows the plasma exposures of co-administration of [225Ac]-DOTA-anti- IGF-1R conjugate at 15 kBq / kg with cold IGF-1R antibody as compared to [225Ac]-DOTA- anti-IGF-lR conjugate alone at 20 kBq / kg or 40 kBq / kg. FIG. 13B shows the platelet count following co-administration of [225Ac]-DOTA-anti-IGF-lR conjugate at 15 kBq / kg with cold IGF-1R antibody as compared to [225Ac]-DOTA-anti-IGF-lR conjugate alone at 20 kBq / kg or 40 kBq / kg.

[0033] FIG. 14 shows the bio-distribution of co-administration of [177Lu]-DOTA-anti- FGFR3 conjugate with cold vofatamab as compared to [177Lu]-DOTA-anti-FGFR3 conjugate alone.

[0034] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way whatsoever.DETAILED DESCRIPTION

[0035] The present disclosure relates to methods of co-administration of a radioimmunoconjugate with a cold targeting molecule (e.g., an antibody) at a therapeutic dose for treatment of cancer.

[0036] 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). Delivering a radioactive payload results in targeted alpha,beta, gamma particle or Auger electron emission 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.

[0037] Radioimmunoconjugates typically contain a biological targeting moiety (e.g., an antibody or antigen-binding fragment thereof), a radionuclide (e.g., an alpha or beta emitter), and a molecule that links the two. Conjugates are formed when a bifunctional chelate is appended to the biological targeting moiety so that structural alterations are minimal while maintaining target affinity. Once radiolabelled, the final radioimmunoconjugate is formed.

[0038] Bifunctional chelates structurally contain a chelate, a linker, and a cross-linking group (FIG. 6A). When developing new bifunctional chelates, most efforts focus on the chelating portion of the molecule. Several examples of bifunctional chelates have been described with various cyclic and acyclic structures conjugated to a targeted moiety. [Bioconjugate Chem. 2000, 11, 510-519; Bioconjugate Chem. 2012, 23, 1029-1039; Mol Imaging Biol. 2011, 13, 215-221, Bioconjugate Chem. 2002, 13, 110-115.]

[0039] One of 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 tenets 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 by an antibody drug conjugate (“ADC”), 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 therapeutic radioimmunoconjugates.

[0040] 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). One mechanism is to increase the rate of clearance of the biological targeting agent. Without being bound by theory, this approach may require identifying ways to shorten the half-life of the biological targeting agent, which is 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 totaldose and would not achieve the desired results of reducing total radioactive dose to normal tissue.

[0041] 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 chelate 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. 2013, 12(11), 2472-2482; Methods Mol Biol. 2009, 539, 191-211; Bioconjug Chem. 2003, 14(5), 927-33], 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 two agents.

[0042] The present disclosure provides methods of co-administration of a radioimmunoconjugate with a cold targeting molecule (e.g., an antibody) at a therapeutic dose for treatment of cancer, thereby saturating non-tumoral tissue uptake by the cold targeting molecule and maximizing tumoral tissue uptake by the radioimmunoconjugate to achieve greater therapeutic efficacy while limiting side effects. This unexpected superiority is achieved, at least in part, by co-administering a therapeutic dose (DT) (explained further herein) of the cold targeting molecule.Definitions

[0043] As used herein, the term “bind” or “binding” of a targeting moiety means an at least temporary interaction or association with or to a target molecule as described herein.

[0044] The terms “bifunctional chelate,” as used herein, refers to a compound that comprises a chelate, a linker, and a cross-linking group. See, e.g., FIG. 6A. A “cross-linking group” is a reactive group that is capable of joining two or more molecules, e.g., joining a bifunctional chelate and a targeting moiety, by a covalent bond.

[0045] The term “bifunctional conjugate,” as used herein, refers to a compound that comprises a chelate or metal complex thereof, a linker, and a targeting moiety, e.g., an antibody or antigen-binding fragment thereof. See, e.g., FIG. 6B.

[0046] The term “cancer,” as used herein, refers to any disease caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. In some embodiments, a cancer of the present disclosure comprises cells (e.g., tumor cells) expressing a specific target, such as, but not limited to, lung cancer, colorectal cancer, pancreatic cancer, or head and neck cancer.

[0047] As used herein, the phrase “co-administer” or “co-administration” means that two or more agents are administered to a subject (e.g., a patient) at the same time or within an interval such that there may be an overlap of an effect of each agent on the subject. Thus, two or more agents that are administered in combination need not be administered together, although they may be. For example, one agent may be pre-administered before the other agent. In some embodiments, the two or more agents are administered within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour(s) of one another, or within about 60, 30, 15, 10, 5, or 1 minute(s) of one another. In some embodiments, the two or more agents are administered together, e.g.., in the same formulation or, e.g., in different formulations but at the same time.

[0048] As used herein, the term “cold,” when used to describe an agent (e.g., a targeting molecule or a targeting moiety, such as an antibody or antigen-binding fragment thereof) means that the agent is not radioactive, e.g., not labeled with a radionuclide. A “cold” agent may or may not be conjugated to another moiety or modified in some way, so long as the cold agent is not radioactive. A cold targeting molecule may be the same as or different from the antibody or antigen-binding fragment thereof contained in a radioimmunoconjugate as described herein.

[0049] The term “chelate,” as used herein, refers to an organic compound or portion thereof that can be complexed with a central metal or radiometal atom at two or more points.

[0050] The term “conjugate,” as used herein, refers to a molecule that contains a chelating group or metal complex thereof, a linker group, and which optionally contains a targeting moiety, e.g., an antibody or antigen-binding fragment thereof.

[0051] “Antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies). As disclosed herein, an antibody can be either a human antibody or a humanized antibody. The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Al Lazikani et al., (1997)J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5thedit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:90 1-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mai. Biol. 273:927-948. In preferred embodiments, variable domains, e.g., CDRs, are defined according to the Kabat numbering scheme.

[0052] A “humanized” antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from human frameworks (FWs). A humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FWs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0053] “Antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay includes a homogeneous time-resolved fluorescence (HTRF) assay.

[0054] “Antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab’, F(ab’)2, Fab’-SH, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen-binding fragments. For a review of certain antigen-binding fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antigen-binding fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Single-domainantibodies are antigen-binding fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. Antigen- binding fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage).

[0055] As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and / or salts thereof of the structures disclosed herein.

[0056] The compounds recited or described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds discussed in the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.

[0057] As used herein, “detection agent” refers to a molecule or atom which is useful in diagnosing a disease by locating the cells containing the antigen. Various methods of labeling polypeptides with detection agents are known in the art. Examples of detection agents include, but are not limited to, radioisotopes and radionuclides, dyes (such as with the biotin- streptavidin complex), contrast agents, luminescent agents (e.g., fluorescein isothiocyanate or FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.

[0058] As used herein, the term “radionuclide,” refers to an atom capable of undergoing radioactive decay (e.g.,3H,14C,15N,18F,35S,44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,75Br,76Br ,77Br ,82Rb,89Zr,86Y,87Y,90Y,97RU,99TC, "mTc,105Rh,109Pd,111In,117mSn,123I,124I,125I,1311,149Pm,149Tb,153Sm,166Ho,177Lu,186Re,188Re,198Au,199Au,2O1T1,203Pb,211At,212Pb ,212Bi,213Bi,223Ra,225Ac,227Th, or229Th). The terms radioactive nuclide, radioisotope, or radioactive isotope may also be used to describe a radionuclide. Radionuclides may be used as detection agents, as described herein. In some embodiments, the radionuclide may be used as therapeutic agents, e.g., an alpha-emitting radionuclide.

[0059] The term an “effective amount” of an agent (e.g., any of the foregoing conjugates such as a radioimmunoconjugate), as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in therapeutic applications, an “effective amount” may be an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications, to substantially improve at least one symptomassociated with the disease or a medical condition, to slow the progression of symptoms of the disorder and its complications, and / or to slow the progression of at least one symptom associated with the disease or a medical condition. Typically, an “effective amount” in the context of the present disclosure is an amount of a radioimmunoconjugate disclosed herein, e.g., an Ac-225-radioimmunoconjugate, that produces at least some measurable therapeutic response or desired effect in some fraction of the patient to whom it is administered. 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 may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed. For example, the disease or condition may become less severe and / or recovery is accelerated in an individual. An effective amount may be administered by administering a single dose or multiple (e.g., at least two, at least three, at least four, at least five, or at least six) doses.

[0060] The term “immunoconjugate,” as used herein, refers to a conjugate that includes a targeting moiety, such as an antibody (or antigen-binding fragment thereof), nanobody, affibody, or a consensus sequence from Fibronectin type III domain. In some embodiments, the immunoconjugate comprises an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8 conjugates per targeting moiety). In some embodiments, the immunoconjugate comprises an average of at least or about 0.20 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.30 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.40 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.50 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.60 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.70 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.80 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.90 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 1 conjugate per targeting moiety.

[0061] The term “radioconjugate,” as used herein, refers to any conjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

[0062] The term “radioimmunoconjugate,” as used herein, refers to any immunoconjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein. A radioimmunoconjugate provided in the present disclosure typically refers to a bifunctional conjugate that comprises a metal complex formed from a radioisotope or radionuclide.

[0063] The term “radioimmunotherapy,” as used herein, refers a method of using a radioimmunoconjugate to produce a therapeutic effect. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate produces a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a cell, wherein administration of the radioimmunoconjugate kills the cell. Wherein radioimmunotherapy involves the selective killing of a cell, in some embodiments the cell is a cancer cell in a subject having cancer.

[0064] The term “pharmaceutical composition” as used herein, represents a composition containing a radioimmunoconjugate described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein.

[0065] A “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non- inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotectants, sorbents, suspending or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calciumcarbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0066] The term “pharmaceutically acceptable salt,” as use herein, represents those salts of the compounds described here that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and m ' Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

[0067] The compounds of the disclosure may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the disclosure be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

[0068] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, among others. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0069] The term “polypeptide,” as used herein, refers to a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides can include one or more “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may comprise two or more individual polypeptide chains, which may in some cases be linked to one another, for example by one or more disulfide bonds or other means.

[0070] As used herein, and as well understood in the art, “to treat” a condition or “treatment” of the condition (e.g., the conditions described herein such as cancer) is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

[0071] As used herein, the term “about” or “approximately,” when used in reference to a quantitative value, includes the recited quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” or “approximately” refers to a ±10% variation from the recited quantitative value unless otherwise indicated or inferred from the context.

[0072] As used herein, the term “targeting moiety” refers to any molecule or any part of a molecule that is capable of binding to a given biological target. As used herein, a targetingmoiety can be an antibody or antigen binding fragment thereof that is capable of specifically binding to a biological target, e.g., insulin-like growth factor 1 receptor (IGF-1R).

[0073] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0074] Unless specifically stated or obvious from context, as used herein the term “or” is understood to be inclusive and covers both “or” and “and”.

[0075] The term “and / or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other.

[0076] The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. The term “consisting of’ is to be construed as close-ended.Methods of treatment

[0077] In one aspect, provided are methods of treating cancer that comprise a step of administering to a subject (e.g., a patient) in need thereof a pharmaceutical composition comprising an effective amount of a radioimmunoconjugate as described further herein (e.g., a radioimmunoconjugate comprising a targeting moiety as disclosed herein), and wherein the subject is being co-administered a cold targeting molecule (e.g., a cold targeting molecule or a cold antibody as disclosed herein).

[0078] By “cold targeting molecule” it means that the targeting molecule is not radioactive, e.g., not labeled with a radionuclide. For example, in some embodiments, the cold targeting molecule is an antibody or antigen-binding fragment thereof that is capable of binding to a target. In some embodiments, the radioimmunoconjugate and the cold targeting molecule are capable of binding the same epitope on a biological target.

[0079] For example, an “IGF-lR-targeting molecule” as used herein refers to a molecule comprising an IGF-lR-targeting moiety, e.g., any IGF-lR-targeting moiety as described herein. By “cold IGF-lR-targeting molecule” it means that the IGF-lR-targeting molecule is not radioactive, e.g., not labeled with a radionuclide. For example, in some embodiments, the cold IGF-lR-targeting molecule is an antibody or antigen-binding fragment thereof that is capable of binding to IGF-1R. In some embodiments, the radioimmunoconjugate and the cold targeting molecule are capable of binding the same epitope on a biological target (e.g., IGF- 1R).Administration

[0080] By “co-administered,” it means that the radioimmunoconjugate and the cold targeting molecule are administered to a subject (e.g., patient) at the same time or within an interval such that there may be an overlap of an effect of each agent in the subject. The radioimmunoconjugate and the cold targeting molecule need not be administered together, although they may be. For example, one agent may be pre-administered before the other agent. For example, in the context of the presently disclosed methods, the cold targeting molecule may be pre-administered before the radioimmunoconjugate. For example, in some embodiments, a radioimmunoconjugate and a cold targeting moiety are administered within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour(s) of one another, or within about 60, 30, 15, 10, 5, or 1 minute(s) of one another. In some embodiments, the IGF-lR-targeting moiety together, e.g., in the same formulation or, e.g., in different formulations but at the same time.

[0081] By “pre-administered,” it means that the cold targeting molecule is administered before the radioimmunoconjugate is administered. For example, in some embodiments, the cold targeting molecule is administered less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours before, less than 1 hour before, or less than 30 minutes before the radioimmunoconjugate is administered.

[0082] In some embodiments, the cold targeting molecule is pre-administered prior to the administration of the radioimmunoconjugate.

[0083] Radioimmunoconjugates and pharmaceutical compositions thereof disclosed herein may be administered by any of a variety of routes of administration, including systemic and local routes of administration.

[0084] Systemic routes of administration include parenteral routes and enteral routes. In some embodiments, radioimmunoconjugates or pharmaceutical compositions thereof are administered by a parenteral route, for example, intravenously, intraarterially, intraperitoneally, subcutaneously, or intradermally. In some embodiments, radioimmunoconjugates or pharmaceutical compositions thereof are administered intravenously. In some embodiments, radioimmunoconjugates or pharmaceutical compositions thereof are administered by an enteral route of administration, for example, trans- gastrointestinal, or orally.

[0085] Local routes of administration include, but are not limited to, peritumoral injections and intratumoral injections.Dosages

[0086] Radioimmunoconjugates or pharmaceutical compositions comprising the same can be administered for radiation treatment planning, diagnostic, and / or therapeutic treatments. When administered for radiation treatment planning or diagnostic purposes, the radioimmunoconjugate may be administered to a subject in a diagnostically effective dose and / or an amount effective to determine the therapeutically effective dose. In therapeutic applications, pharmaceutical compositions may be administered to a subject (e.g., a patient) 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 may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed. For example, the disease or condition may become less severe and / or recovery is accelerated in an individual. In some embodiments, a subject is administered a first dose of a radioimmunoconjugate or composition in an amount effective for radiation treatment planning, then administered a second dose or set of doses of the radioimmunoconjugate or composition in a therapeutically effective amount.

[0087] Effective amounts may depend on the severity of the disease or condition and other characteristics of the subject (e.g., weight). Therapeutically effective amounts of disclosed radioimmunoconjugates and compositions for subjects (e.g, mammals such as humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences (e.g, differences in age, weight, and the condition of the subject.

[0088] In some embodiments, the subject (e.g., a patient) is pre-administered or co- administered a dosage of 0.1 to 10 mg / kg (e.g., 0.2 to 8 mg / kg, 0.3 to 7 mg / kg, 0.4 to 6 mg / kg, 0.5 to 5 mg / kg, 0.5 to 4 mg / kg, 0.5 to 3 mg / kg, 0.5 to 2 mg / kg, or 0.5 to 1 mg / kg) of a cold targeting molecule (e.g., an IGF-1R antibody). In some embodiments, the patient is pre- administered or co-administered about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, about 8.0 mg / kg, about 9.0 mg / kg, or about 10 mg / kg of a cold targeting molecule. In some embodiments, the patient is pre- administered a dosage of 0.5 to 3 mg / kg (e.g., 0.5 mg / kg or 1.5 mg / kg) of a cold targeting molecule (e.g., an IGF-1R antibody).

[0089] In some embodiments, the radioimmunoconjugate is administered at a dosage of 10 kBq to 100 kBq / kg (e.g., 15 kBq to 80 kBq / kg, 20 kBq to 60 kBq / kg, 25 kBq to 50 kBq / kg, 30 kBq to 40 kBq / kg, 25 kBq to 40 kBq / kg, 20 kBq to 40 kBq / kg, or 15 kBq to 40 kBq / kg) of body weight of said patient. In some embodiments, the radioimmunoconjugate is administered at a dosage of 15 kBq to 40 kBq / kg (e.g., about 15 kBq / kg, about 20 kBq / kg, about 25 kBq / kg, about 30 kBq / kg, about 35 kBq / kg, about 40 kBq / kg) of body weight of said patient.

[0090] In some embodiments, the radioimmunoconjugate is administered with a cumulative exposure of 20 kBq to 300 kBq / kg (e.g., 20 kBq to 200 kBq / kg, 20 kBq to 150 kBq / kg, 20 kBq to 100 kBq / kg, 25 kBq to 50 kBq / kg, 30 kBq to 60 kBq / kg, 35 kBq to 70 kBq / kg, or 35 kBq to 80 kBq / kg) of body weight of said patient.

[0091] To practice the methods of this disclosure, either the radioimmunoconjugate or the cold targeting molecule can be administered in a single dose or multiple doses (e.g., twice, three times, or four times within any given treatment).

[0092] Single or multiple administrations of radioimmunoconjugates disclosed herein including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. 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. With respect to radioactive doses, by way of non-limiting example, in some embodiments, the subject (e.g., a patient) is pre-administered about 0.5 to 3 mg / kg (e.g., 0.5 mg / kg or 1.5 mg / kg) of a cold IGF-1R antibody, followed by administration of an IGF-lR-targeting radioimmunoconjugate at a dosage of about 15 kBq to 40 kBq / kg (e.g., 15 kBq / kg or 30 kBq / kg).Functional outputs

[0093] In some embodiments, a subject who has been treated with a method disclosed herein exhibits one or more improved characteristics as measured relative to a reference level. As used herein, the term “reference level” is a level as determined by the use of a controlmethod in an experimental animal model or clinical trial. In some embodiments, the reference level refers to a level observed in a subject administered the same radioimmunoconjugate (and in some embodiments, with the same dosing protocol, including radioactive dose) but without co-administration of a cold targeting molecule.

[0094] In some embodiments, a subject who has been treated with a method disclosed herein exhibits increased tumor uptake of the radioimmunoconjugate relative to a reference level, e.g., at least 1.2 times greater, at least 1.5 times greater, at least 2.0 times greater, at least 2.5 times greater, or at least 3 times greater levels in a tumor than a reference level at 24 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits at least 1.2 times greater, at least 1.5 times greater, at least 2.0 times greater, at least 2.5 times greater, or at least 3 times greater levels of uptake in a tumor than a reference level at 48 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits at least 1.2 times greater, at least 1.5 times greater, at least 2.0 times greater, at least 2.5 times greater, or at least 3 times greater levels of uptake in a tumor than a reference level at 96 h after administration of the radioimmunoconjugate.

[0095] In some embodiments, the subject exhibits a %ID / g of greater than 10%, greater than 15%, or greater than 20% in a tumor at 24 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, or greater than 45% in a tumor at 96 h after administration of the radioimmunoconj ugate.

[0096] In some embodiments, a subject who has been treated with a method disclosed herein exhibits reduced uptake of the radioimmunoconjugate in one or more normal (non- tumor) tissues relative to a reference level, e.g., 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 65% or less, or 50% or less of a reference level in one or more normal tissues at 24 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 65% or less, or 50% or less of a reference level in one or more normal tissues at 48 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 65% or less, or 50% or less of a reference level in one or more normal tissues at 96 h after administration of the radioimmunoconjugate.

[0097] In some embodiments, the subject exhibits a %ID / g of less than 10% in an internal organ (e.g., intestines, kidneys, adrenals, liver, gall bladder, lungs, spleen, skin, and / or bladder) at 4 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of less than 10% in an internal organ (e.g., intestines, kidneys, adrenals, liver, gall bladder, lungs, spleen, skin, and / or bladder) at 24 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of less than 10% in an internal organ (e.g., intestines, kidneys, adrenals, liver, gall bladder, lungs, spleen, skin, and / or bladder) at 48 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of less than 10% in an internal organ (e.g., intestines, kidneys, adrenals, liver, gall bladder, lungs, spleen, skin, and / or bladder) at 96 h after administration of the radioimmunoconjugate.

[0098] In some embodiments, a subject who has been treated with a method disclosed herein exhibits reduced clearance of the radioimmunoconjugate from the blood relative to a reference level, e.g., as evidenced by a higher %ID / g in the blood.

[0099] In some embodiments, a subject who has been treated with a method disclosed herein exhibits at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold greater levels of radioactivity in the blood than a reference level at 24 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold greater levels of radioactivity in the blood than a reference level at 48 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits at least 5-fold, at least 10-fold, at least 20-fold, at least 30- fold greater levels of radioactivity in the blood than a reference level at 96 h after administration of the radioimmunoconjugate.

[0100] In some embodiments, the subject exhibits a %ID / g of greater than 10%, greater than 15%, greater than 20%, or greater than 25% in the blood at 24 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of greater than 10%, greater than 12.5%, greater than 15%, or greater than 17.5% in the blood at 48 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of greater than 10%, greater than 12.5%, or greater than 15% in the blood at 96 h after administration of the radioimmunoconjugate.

[0101] In some embodiments, a subject who has been treated with a method disclosed herein exhibits reduced excretion of the radioimmunoconjugate in urine relative to a reference level, e.g., as evidenced by a lower %ID / g in the urine.

[0102] In some embodiments, a subject who has been treated with a method disclosed herein exhibits less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of levels of radioactivity in the urine as compared to a reference level at 24 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of levels of radioactivity in the urine as compared to a reference level at 48 h after administration of the radioimmunoconjugate. In some embodiments, a subject who has been treated with a method disclosed herein exhibits less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% of levels of radioactivity in the urine as compared to a reference level at 96 h after administration of the radioimmunoconjugate.

[0103] In some embodiments, the subject exhibits a %ID / g of less than 10%, less than 8%, or less than 6% in urine at 24 h after administration of the radioimmunoconjugate. In some embodiments, the subject exhibits a %ID / g of less than 10% in urine at 96 h after administration of the radioimmunoconjugate.

[0104] In some embodiments, a subject who has been treated with a method disclosed herein exhibits reduced toxicity as compared to a reference level. In some embodiments, toxicity is assessed based on one or more of clinical observations (e.g., severity and / or frequency of side effects), food consumption, body weight, ophthalmologic examination, hematology, clinical chemistry, urinalysis, and examination of biopsy tissue.

[0105] In some embodiments, the co-administration of a cold targeting molecule at the DT as described herein results in an improvement of the therapeutic index of the radioimmunoconj ugate.

[0106] In some embodiments, the improvement of the therapeutic index is increase of tumor uptake of the radioimmunoconjugate, increase of tumor-to-background ratio, decrease of radioimmunoconjugate uptake in non-target tissues, reduction of hematological toxicity, or a combination thereof.

[0107] In some embodiments, use of a method as disclosed herein allows a subject to tolerate a higher radioactive dose than a method in which the subject is not pre-administered or co-administered a cold targeting molecule.

[0108] In some embodiments, the method of the present disclosure can be used for treating cancer including, but not limited to, solid tumor cancer.

[0109] In certain embodiments, the solid tumor cancer is selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing’s sarcoma, gallbladder carcinoma, glioma, head and neck cancer, liver cancer, lung cancer, neuroblastoma, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary adenoid cystic cancer, spermatocytic seminoma, and uveal melanoma.Radiopharmaceuticals or radioimmunoconjugates

[0110] In this disclosure, the radiopharmaceuticals or radioimmunoconjugates comprise the structure of Formula I, or pharmaceutically acceptable salts thereof:(I) wherein A is a chelating moiety comprising a radionuclide, B is antibody or an antigen- binding fragment thereof, and L is a linker.

[0111] In some embodiments, the radiopharmaceuticals or radioimmunoconjugates comprise the structure of Formula II, or pharmaceutically acceptable salts thereof:(II) whereinA is a chelating moiety or a metal complex thereof, B is an antibody or antigen-binding fragment thereof;L1is a bond, C=O, C=S, optionally substituted C1-C6alkyl, optionally substituted C1- C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer between 1 and 5 (inclusive); andL2each independently has the structure of -X'-L ’-Z1- whereinX1is -C(O)NR'-*. -NR1C(O)-*, -C(S)NR1-*, -NR1C(S)-*, -OC(O)NR1-*, -NR1C(O)O-*. -NR1C(O)NR'-*. -CH2-Ph-C(O)NR1-*. -NR1(O)-Ph-CH2-*, -CH2-Ph- NH-C(S)NR1-*, -NR1C(S)-NH-Ph-CH2-*, -O-*, or -NR1-*; wherein “*” indicates the attachment point to L3, and R1is hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, or optionally substituted aryl or heteroaryl;L3is optionally substituted C1-C50alkyl or optionally substituted C1-C50heteroalkyl;Z1is-CH2-#, -C(0)- #, -C(S)- #, -0C(0)-#, -C(0)0-#, -NR2C(O)-#, -C(O)NR2-#, or -NR2-#, whereinindicates the attachment point to B, and R2is hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0112] Typical substituents of alkyl, heteroalkyl, aryl, or heteroaryl include, but are not limited to halo (e.g., F, Cl, Br, I), OH, CN, nitro, amino, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, C1-6heteroalkyl, C1-6heterocycloalkyl, haloalkyl (e.g., CF3), alkoxy (e.g., OCH3), alkylamino (e.g., NH2CH3), sulfonyl, aryl, and heteroaryl.

[0113] In some embodiments, referring to Formula I, A-L- is a metal complex of a moiety selected from the group consisting of:(Moiety 2),

[0114] In some embodiments, the radioimmunoconjugate or pharmaceutically acceptable salt thereof (e.g immunoconjugate or radioimmunoconjugate) has or comprises:wherein B is an antibody or antigen-binding fragment thereof as disclosed herein.[01151 In some embodiments, the radioimmunoconjugate or pharmaceutically acceptable salt thereof comprises an alpha emitter. In certain embodiments, the alpha emitter is selected from the group consisting of Astatine-211 (211 At), Bismuth-212 '(21261), Bismuth-213 (213Bi), Actinium-225 (225 Ac), Radium -223 (223 Ra), Lead-212 (212Pb), Thorium-227 (227Th), and T erbium-149 (149Tb), or a progeny thereof. In certain embodiments, the metal complex comprises 225 Ac or a progeny thereof.

[0116] In some embodiments, the radioimmunoconjugate comprises the following structure:wherein is an antibody or antigen-binding fragment thereof, as disclosed herein. In some embodiments, the antibody or antigen-bindi ng fragmetn thereof is an IGF-1R antibody, e.g., AVE1642:

[0117] In some embodiments, the method of the present disclosure features that the cold targeting molecule is an antibody or antigen-binding fragment thereof targeting IGF-1R. In certain embodiments, the cold IGF- IR-taigeting molecule is AVE 1642 or an IGF-lR-binding fragment thereof.Chelating moiety or metal complex thereofChelating moieties

[0118] Examples of suitable chelating moieties include, but are not limited to, DOTA (1,4,7, 10-tetraazacyclododecane-1, l,7, 10-tetraacetic acid), DOTMA (lR,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(carbamoylm ethyl )- 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-l-yl)acetic acid), DOTA-GA anhydride (2, 2’, 2”-( 10-(2, 6-dioxotetrahydro-2H-pyran-3:-yl)’l, 4, 7, 10-tetraazacyclododecane-l ,4, 7- triyljtriacetic acid, DOTP ( 1,4,7, 10-tetraazacyclododecane-l, 4,7, 10-tetra(m ethylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, D0TA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido- methylenephosphonic acid), CB-TE2A (1,4,8,l l-tetraazabicyclo[6.6.2]hexadecane-4,l l- 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- tetraazacy cl otetradecane- 1,4, 8, 11 -tetrapropionic acid), TETA (1,4,8,11- tetraazacyclotetradecane-1,4,8,ll-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), EUoctapa (N,N’-bis(6- carboxy-2-pyridylmethyl)-ethylenediamine-N,N’-diacetic acid), H2dedpa (1,2-[[6-(carboxy)- pyridin-2-yl] -methylamino] ethane), H6phospa (N,N’-(methylenephosphonate)-N,N’-[6- (methoxy carbonyl)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), octadentate-HOPO (octadentate hydroxypyridinones), or porphyrins.

[0119] Preferably, the chelating moiety is selected from DOTA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (lR,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), DO3 AM-acetic acid (2-(4,7,10- tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-l-yl)acetic acid), DOTP (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTA-4AMP (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), NOTA (1,4,7- triazacyclononane-1,4,7-triacetic acid), and HP-DO3A (10-(2 -hydroxypropyl)-1, 4, 7- tetraazacyclododecane-1,4,7-triacetic acid).

[0120] In some embodiments, the chelating moiety is DOTA.

[0121] In some embodiments, compounds comprise a metal complex of a chelating moiety. For example, 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 10,000 keV), such as any of the radioisotopes and radionuclides discussed herein.

[0122] In some embodiments, chelating moieties are useful as detection agents, and compounds comprising such detectable chelating moieties can therefore be used as diagnostic or theranostic agents.Radioisotopes and Radionuclides

[0123] In some embodiments, the metal complex comprises a radionuclide. Examples of suitable radioisotopes and radionuclides include, but are not limited to,3H,14C,15N,18F,35S, 44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,66Ga,67Ga,67Cu,68Ga,75Br,76Br,77Br ,82Rb,89Zr, 86Y,87Y,90Y,97RU,99TC,99mTc,105Rh,109Pd,111In,1231,1241,125I,1311,149Pm,149Tb,153Sm, 166HO,177LU,117mSn,186Re,188Re,198Au,199Au,2O1T1,203Pb,211At,212Pb ,212Bi,213Bi,223Ra, 225Ac,227Th, and229Th.

[0124] In some embodiments, the metal complex comprises a radionuclide selected from 44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,89Zr,90Y,97Ru, 99Tc,99mTc,105Rh,109Pd,111In,117mSn,149Pm,149Tb,153Sm,166Ho,177Lu,186Re,188Re,198Au, 199Au,2O1T1,203Pb,211At,212Pb,212Bi,213Bi,223Ra,225Ac,227Th, and229Th.

[0125] In certain embodiments, the metal complex comprises a radionuclide selected from 68Ga,89Zr,90Y,111In,177Lu, and225Ac. In certain embodiments, the metal complex comprises a radionuclide of177Lu or225Ac.

[0126] In some embodiments, the radionuclide is an alpha emitter, e.g., Astatine-211 (211At), Bismuth-212 (212Bi), Bismuth-213 (213Bi), Actinium-225 (225Ac), Radium-223 (223Ra), Lead-212 (212Pb), Thorium-227 (227Th), or Terbium-149 (149Tb), or a progeny thereof. In some embodiments, the alpha-emitter is Actinium-225 (225Ac), or a progeny thereof.

[0127] In certain embodiments, the metal complex comprises an alpha emitter of225Ac or a progeny thereof.Linker

[0128] The compounds of this disclosure comprise the structure of Formula I below:Formula I wherein each of the variables is defined in the SUMMARY section above.Each of the compounds of Formula I comprises a linker moiety as -L1-(L2)n-, wherein:L1is a bond, C=O, C=S, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer between 1 and 5 (inclusive); and each L2, independently, has the structure:Formula II wherein:X1is-C(O)NR1-*, -NR1C(O)-*, -C(S)NR1-*, -NR1C(S)-*, -OC(O)NR1-*, - NR1C(O)O-*, -NR1C(O)NR1-*, -CH2-Ph-C(O)NR1-*, -NR1-NR1C(O)- *, -CH2-Ph- NH-C(S)NR1-*, -NR1C(S)-NH-Ph-CH2-*, -O-*, or -NR1-*; wherein “*” indicates the attachment point to L3, and R1is hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, or optionally substituted aryl or heteroaryl;L3is optionally substituted C1-C50alkyl or optionally substituted C1-C50heteroalkyl (e.g., (CH2CH20)2.20); andZ1is -CH2-#, -C(O)- #, -C(S)- #, -OC(O)-#, -C(O)O-#, -NR2C(O)-#, -C(O)NR2-#, or -NR2-#, whereinindicates the attachment point to B, and R2is hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0129] As described herein, the phrase “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein one or more of the carbons of said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g., alkyl) per se is optional. Without limitation, a substituent can be alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, each of which has the same meaning as commonly used in the field. For example, the term “optionally substituted aryl” means an aryl that may be optionally substituted with one, two, three, or four substituents independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heterocycloalkyl, haloalkyl, aryl, and heteroaryl.

[0130] In some embodiments, L1is optionally substituted C1-C6alkyl or optionally substituted C1-C6heteroalkyl. In certain embodiments, L1is substituted C1-C6alkyl or substituted C1-C6heteroalkyl, the substituent comprising a heteroaryl group (e.g., six- membered nitrogen-containing heteroaryl). In some embodiments, L1is C1-C6alky. For example, L1is -CH2CH2- In some embodiments, L1is a bond. In some embodiments, L1is, wherein RLis hydrogen or -CO2H.

[0131] In some embodiments, X1is-C(O)NR1-*, -NR1C(O)-*, or -NR1-, “*” indicating the attachment point to L3, and R1is hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, X1is -C(O)NR1-*, “*” indicating the attachment point to L3, and R1is hydrogen.

[0132] In some embodiments, L3is optionally substituted C1-C50alkyl (e.g., C3-C30alkyl, C3-C25alkyl, C3-C20alkyl, C3-C15alkyl, C3-C10alkyl, C5-C30alkyl, C5-C25alkyl, C5-C20alkyl, C5-C15alkyl, and C5-C10alkyl) or optionally substituted C1-C50heteroalkyl (e.g., C3-C30heteroalkyl, C3-C25heteroalkyl, C3-C20heteroalkyl, C3-C15heteroalkyl, C3-C10heteroalkyl, C5-C30heteroalkyl, C5-C25heteroalkyl, C5-C20heteroalkyl, C5-C15heteroalkyl, and C5-C10heteroalkyl). An exemplary C1-C50heteroalkyl is C5-C30polyethylene glycol (e.g., C5-C25polyethylene glycol, C5-C20polyethylene glycol, C5-C15polyethylene glycol). In certain embodiments, L3is C5-C25polyethylene glycol, C5-C20polyethylene glycol, or C5-C15polyethylene glycol.

[0133] In some embodiments, L3is optionally substituted C1-C50heteroalkyl (e.g., C1-C40heteroalkyl, C1-C30heteroalkyl, C1-C20heteroalkyl, C2-C18heteroalkyl, C3- C16heteroalkyl, C4-C14heteroalkyl, C5-C12heteroalkyl, C6-C10heteroalkyl, C8-C10heteroalkyl, C4heteroalkyl, C6heteroalkyl, C8heteroalkyl, C10heteroalkyl, C12heteroalkyl, C16heteroalkyl, C20heteroalkyl, or C24heteroalkyl).

[0134] In some embodiments, L3is optionally substituted C1-C50heteroalkyl comprising a polyethylene glycol (PEG) moiety comprising 1-20 oxy ethylene (-O-CH2-CH2-) units, e.g., 2 oxyethylene units (PEG2), 3 oxyethylene units (PEG3), 4 oxyethylene units (PEG4), 5 oxyethylene units (PEG5), 6 oxyethylene units (PEG6), 7 oxyethylene units (PEG7), 8 oxyethylene units (PEG8), 9 oxyethylene units (PEG9), 10 oxyethylene units (PEG10), 12 oxy ethylene units (PEG12), 14 oxy ethylene units (PEG14), 16 oxy ethylene units (PEG16), or 18 oxy ethylene units (PEG18).

[0135] In certain embodiments, L3is optionally substituted C1-50heteroalkyl comprising a polyethylene glycol (PEG) moiety comprising 1-20 oxy ethylene (-O-CH2-CH2-) units or portions thereof. For example, L3comprises PEG3 as shown below:

[0136] In some embodiments, L3is (CH2CH2O)m(CH2)w, and m and w are each independently an integer between 0 and 10 (inclusive), and at least one of m and w is not 0.

[0137] In some embodiments, L3is substituted C1-C50alkyl or substituted C1-C50heteroalkyl, the substituent comprising a heteroaryl group (e.g., six-membered nitrogen- containing heteroaryl).

[0138] In some embodiments, Z1is CH2, C=O, or NR1; wherein R1is H, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0139] In certain embodiments, A-L1-(L2)n-B can be represented by the following structure:wherein Y1is -CH2OCH2(L2)n-B, C=O(L2)n-B, or C=S(L2)n-B and Y2is -CH2CO2H; or wherein Y1is H and Y2is L1-(L2)n-B.Targeting moieties and cold targeting molecules

[0140] Targeting moieties, as well as cold targeting molecules, include any molecule or any part of a molecule that is capable of binding to a given target, e.g., IGF-1R. In some embodiments, the targeting moiety or cold targeting molecule comprises a protein or polypeptide. In some embodiments, the targeting moiety or cold targeting molecule is selected from the group consisting of antibodies or antigen binding fragments thereof, nanobodies, affibodies, and consensus sequences from Fibronectin type III domains (e.g., Centyrins or Adnectins). In some embodiments, a moiety is both a targeting and a therapeutic moiety, i.e., the moiety is capable of binding to a given target and also confers a therapeutic benefit.Antibodies and antigen-binding moieties

[0141] Antibodies typically comprise 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 of 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. Light chains typically comprise one variable region (VL) and one constant region (CL). An IgG heavy chain includes a variable region (VH), a first constant region (CHI), 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).

[0142] Antibodies suitable for use with the present disclosure 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. In some embodiments, the antibody or antigen-binding fragment thereof is humanized. In some embodiments, the antibody or antigen-binding fragment thereof is chimeric. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0143] 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')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). In some embodiments, an “antigen binding fragment” comprises a heavy chain variable region and a light chain variable region. 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.

[0144] Antibodies or antigen-binding 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.

[0145] 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), or any of the molecules described herein.

[0146] “Avimer” relates to a multimeric binding protein or peptide engineered using, for example, in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in greater affinity and specificity compared to single epitope immunoglobin domains.

[0147] “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 are capable of binding 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, yeastdisplay, bacterial display, mRNA display, ribosome display). “Affibodies” are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may be considered to mimic certain functions of antibodies.

[0148] 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).

[0149] Affibody molecules showing specific binding to a variety of different proteins (e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gpl20, CD28, human serum albumin, IgA, IgE, IgM, HER2 and EGFR) have been generated, demonstrating affinities (Kd) in the pM to pM range. “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See for example Hudson et al., (2003). Single-chain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all, or a portion of the light chain variable domain of an antibody. Antibody fragments can be made by various techniques including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant hosts (e.g., E. coli or phage) as described herein.

[0150] In certain embodiments, the antibody or antigen-binding fragment thereof is a multispecific, e.g. bispecific. Multispecific antibodies (or antigen-binding fragments thereof) include monoclonal antibodies (or antigen-binding fragments thereof) that have binding specificities for at least two different sites.

[0151] In certain embodiments, amino acid sequence variants of antibodies or antigen- binding fragments thereof are contemplated; e.g., variants that are capable of binding to a target antigen . For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or antigen-binding fragment thereof. Amino acid sequence variants of an antibody or antigen-binding fragment thereof may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody or antigen-binding fragment thereof. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct possesses desired characteristics, e.g. antigen binding.

[0152] In some embodiments, the antibody or antigen binding fragment thereof is an inhibitory antibody (also called “antagonistic antibody”) or antigen-binding fragment thereof, e.g., the antibody or antigen binding fragment thereof at least partially inhibits one or more functions of the target molecule as explained further herein.

[0153] In some embodiments, the antibody or antigen binding fragment thereof is an agonistic antibody (also known as stimulatory antibody).

[0154] In some embodiments, the antibody is selected from the group consisting of an IGF- 1R antibody, a fibroblast growth factor receptor 3 (FGFR3) antibody, a tumor epithelial marker-1 (TEM-1) antibody, a Claudin 18.2 antibody, and a carbonic anhydrase 9 (CA9) antibody.

[0155] Examples of antibodies, or antigen-binding fragments thereof capable of binding to IGF-1R include, but are not limited to, human or humanized monoclonal antibodies such as figitumumab, cixutumumab, dalotuzumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab, and antigen-binding fragments of any of the foregoing. In some embodiments, the antibody, or an antigen binding fragment thereof is AVE 1642 or an IGF-lR-binding fragment thereof.

[0156] In certain embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises specific heavy chain complementarity determining regions CDR- Hl, CDR-H2 and / or CDR-H3 as described herein. In some embodiments, the complementarity determining regions (CDRs) of the antibody or antigen-binding fragment thereof are flanked by framework regions. A heavy or light chain of an antibody or antigen- binding fragment thereof containing three CDRs typically contains four framework regions.

[0157] In some embodiments, the CDRs of the light chain variable region of AVE1642 comprises the sequences:SEQ ID NO:SEQ ID NO:

[0158] In some embodiments, the light chain variable region of AVE1642 comprises the sequence:SEQ ID NO: 9

[0159] In some embodiments, the light chain of AVE1642 comprises the sequence: SEQ ID NO: 4

[0160] In some embodiments, the CDRs of the heavy chain variable region of AVE1642 comprises the sequences:

[0161] In some embodiments, the heavy chain variable region of AVE1642 comprises the sequence:SEQ ID NO: 10

[0162] In some embodiments, the heavy chain of AVE1642 comprises the sequence:SEQ ID NO: 8

[0163] In some embodiments, the antibody, or antibody-binding fragment thereof includes a light chain variable domain including at least one, two, or all three complementarity determining regions (CDRs) selected from:(a) CDR-L1 including the amino acid sequence of SEQ ID NO: 1;(b) CDR-L2 including the amino acid sequence of SEQ ID NO: 2; and(c) CDR-L3 including the amino acid sequence of SEQ ID NO: 3.

[0164] In some embodiments, the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain including at least one, two, or all three CDRs selected from:(a) CDR-H1 including the amino acid sequence of SEQ ID NO: 5;(b) CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and(c) CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0165] In certain embodiments, the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain including at least one, two, three, four, five, or all six CDRs selected from:(a) CDR-L1 including the amino acid sequence of SEQ ID NO: 1;(b) CDR-L2 including the amino acid sequence of SEQ ID NO: 2;(c) CDR-L3 including the amino acid sequence of SEQ ID NO: 3;(d) CDR-H1 including the amino acid sequence of SEQ ID NO: 5;(e) CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and(f) CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0166] In some embodiments, the antibody, or antibody-binding fragment thereof features that the heavy chain variable domain includes the amino acid sequence of SEQ ID NO: 10.

[0167] In some embodiments, the antibody, or antibody-binding fragment thereof features that the light chain variable domain includes the amino acid sequence of SEQ ID NO: 9.

[0168] The antibody or antigen-binding fragment thereof may be any antibody or antigen- binding fragment thereof of natural and / or synthetic origin, e.g., an antibody of mammalian origin. In some embodiments, the constant domain, if present, is a human constant domain. In some embodiments, the variable domain is a mammalian variable domain, e.g., a humanized or a human variable domain.

[0169] In some embodiments, antibodies used in accordance with this disclosure are monoclonal antibodies. In some embodiments, antibodies are recombinant murine antibodies, chimeric, humanized or fully human antibodies, multispecific antibodies (e.g., bispecific antibodies), or antigen-binding fragments thereof.

[0170] Examples of antibodies, or antigen-binding fragments thereof capable of binding to FGFR3 include, but are not limited to, human or humanized monoclonal antibodies such as MFGR1877S (CAS No. 1312305-12-6; Genentech) (a human monoclonal antibody also known as vofatamab, and whose lyophilized form is also known as B-701 or R3Mab); PRO- 001 (Prochon); PRO-007 (Fibron); IMC-D11 (Imclone); and AV-370 (Aveo Pharmaceuticals), and antigen-binding fragments of any of the foregoing. (See, e.g., U.S. Pat. No. 8,410,250; US 10,208,120; and International Patent Publication Nos. W02002102972A2, W02002102973A2, W02007144893 A2, W02010002862A2, and W02010048026A2.)

[0171] Examples of antibodies, or antigen-binding fragments thereof capable of binding to TEM-1 include, but are not limited to, human or humanized monoclonal antibodies such as hMP-E-8.3 (Mediapharma), ontuxizumab (also known as MORAb-004) (Morphotek), anti- TEM-1 antibodies from Kirin Brewery, and antigen-binding fragments of any of the foregoing. (See, e.g., U.S. Pat. No. 8,895,000, and International Patent Publication Nos. WO 2017 / 134234 Al and WO 2006 / 017759 A2.)

[0172] Additional known TEM-1 antibodies and antigen-binding fragments thereof include, for example, human or humanized antibodies such as humanized FB5; mouse monoclonalantibodies such as, for example, mMP-E-8.3 (Mediapharma) (see, e.g., WO 2017 / 134234), FB5, 1F9B4 (Proteintech Group), G-9 (sc-377221) (Santa Cruz Biotechnology), and Bl / 35 (MilliporeSigma); rat monoclonal antibodies such as, e.g., 9G5, 16D2, 7H12, 15F8, 9A5, 11D1, 15D10, 7D3, or 16D2 from Morphotek (see, e.g., WO 2013 / 148250), clone 458606 (R&D systems), CBFYE-0326 (Creative Biolabs); rabbit monoclonal antibodies such as EPR17081 (Abeam); chimeric antibodies such as cMP-E-8.3 (Mediapharma) (see, e.g., WO 2017 / 134234); and antigen-binding fragments of any of the foregoing.

[0173] Examples of antibodies, or antigen-binding fragments thereof capable of binding to Claudin 18.2 include, but are not limited to, human or humanized monoclonal antibodies such as osemitamab (TST001) zolbetuximab (IMAB362; also known as claudiximab), Claudin 18.2 antibodies disclosed in International Patent Publication WO 2023 / 070201 (e.g., clones YU574-D03, YU574-D11, YU574-E07, YU574-H09, YU575-H03, YU577-B01, YU577- Bl l, YU577-D10, YU577-E08, YU577-E12, YU577-H07, YU578-A02, YU578-D01, and YU578-F02 disclosed therein) and antigen-binding fragments of any of the foregoing.

[0174] Examples of antibodies, or antigen-binding fragments thereof capable of binding to CA9 include, but are not limited to, human or humanized monoclonal antibodies such as MSC3 (as disclosed, e.g., in International Patent Publication WO 2011 / 139375), and antigen- binding fragments thereof.Polypeptides

[0175] 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.

[0176] A reference polypeptide described herein can include a target-binding domain that is capable of binding to a target of interest (e.g., is capable of binding to an antigen, e.g., IGF- 1R). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor).Modified polypeptides

[0177] Polypeptides suitable for use with compositions and methods of the present disclosure may have a modified amino acid sequence. Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acidsequence 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.

[0178] 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.

[0179] 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 theamino or carboxy-terminus of a polypeptide herein 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).

[0180] 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 NH2(CH2)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.

[0181] 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.

[0182] Table 1. Amino acid substitutions

[0183] 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, and / or (c) the bulk of the side chain.Therapeutic dose (DT) of cold targeting molecule

[0184] Determination of the appropriate dose level, e.g., a therapeutic dose (DT), of a cold targeting molecule (e.g., an antibody) can be made based upon the pharmacokinetics (PK) of the targeting moiety. To practice the methods in this disclosure, provided below are the details as to how to determine the unique DT of a cold targeting molecule (e.g., a cold antibody).

[0185] The pharmacokinetic properties of a targeting moiety, or a cold targeting molecule, depend upon attributes of the targeting moiety itself and also attributes of the target (such as expression levels among various normal and tumoral tissues, turnover rate, cellular trafficking, etc.) and the binding kinetics of the interaction. If the pharmacokinetic properties of a targeting moiety are understood and described quantitatively, they can be utilized to determinea mass dose level suitable to optimize tumor targeting and tumormormal organ uptake of targeted radiopharmaceuticals. Conceptually, a mass dose of targeting moiety should be identified which is sufficient to saturate the non-tumoral tissue uptake (or clearance) for a certain time period post-administration and to maximize tumoral tissue uptake during this time. Dose levels higher than this threshold value are not desirable because they may begin to saturate tumor targets in addition to normal tissue and thus negatively impact tumormormal tissue uptake. Antibodies are used as examples to demonstrate the practice of the method disclosed herein to determine the DT of the cold targeting molecule (or a total mass dose level).

[0186] In some embodiments, the PK profile is characterized by a one- or two-compartment PK model having either a nonlinear only clearance pathway or mixed linear and nonlinear clearance pathways.

[0187] Therapeutic agents like monoclonal antibodies (e.g., cetuximab) and some small molecules (e.g., phenytoin) exhibit nonlinear pharmacokinetics (PK) at a certain range of doses. Nonlinearity typically results from nonlinear kinetics in any step of key pharmacokinetic processes, such as absorption, distribution, metabolism, and elimination. Two major factors leading to nonlinear PK are the capacity -limited metabolism and target- mediated drug disposition (TMDD).

[0188] Many antibodies targeted to cell surface receptors exhibit nonlinear plasma PK, which can be described by a full target-mediated drug disposition (TMDD) model or approximated by models with a nonlinear clearance pathway described by the Michaelis- Menten equation or other model. In either case, the saturation of the nonlinear antibody clearance pathway can be described by a parameter known as the critical concentration denoted as Ccrit[Stein & Peletier, 2018], When antibody plasma concentration is above Ccrit, the target-mediated pathway of antibody plasma clearance is saturated.

[0189] It is known in the field that nonlinear PK are frequently described by compartmental models comprising mixed linear and nonlinear clearance pathways. The linear clearance pathway can be described by first order kinetics with a rate constant of Cl, which can be used to determine the mass clearance rate as ClxCp(t), where Cp(t) is the plasma concentration of the compound with respect to time. The nonlinear clearance pathway can be described by a TMDD model (whose governing equations are listed in Equations 11A / 11B / 11C below) or Michaelis-Menten equation, which has the following formula (1):where VM (unit of mass / time) and KM(unit of concentration) are M-M equation coefficients.

[0190] In some embodiments, to formulate a DT of the cold targeting molecule, the following steps are performed: i. obtaining parameters of the PK profile, wherein the parameters comprise Michaelis-Menten constant (KM), maximal velocity (VM), first-order / linear clearance rate (Cl), and central volume of distribution (Vc); ii. determining a critical concentration (Ccrit) based on the parameters, wherein the PK profile of the cold targeting molecule is linear before Ccrit; and iii. formulating a DT of the cold targeting molecule based on Ccritto reach a desired clinical endpoint.

[0191] In some embodiments, for a PK model for which a nonlinear clearance pathway is described by the Michaelis-Menten equation, Ccritis equal to VM / C1, where VM is the maximal velocity of Michaelis-Menten equation and Cl is the linear clearance rate constant of the antibody.

[0192] In some embodiments, for a PK model described by the full TMDD model, Ccritis determined according to formula Ksyn / (C1 / Vo) wherein Ksynis the synthesis rate of the target and Vo is the distribution of the central compartment.

[0193] In some embodiments, for a PK model described by the full TMDD model, Ccritis determined according to formula Kint×Ttot / (Cl / Vc), wherein Kintis the rate constant of internalization of drug-target complex with a unit of 1 / time, and Ttotis the total receptor concentration.

[0194] In some embodiments, the desired clinical endpoint includes a time to reach Ccrit, denoted as Tcrit, that is determined according to the following equation:wherein Vcand Cl are parameters of a one-compartment PK model.

[0195] In some embodiments, the desired clinical endpoint includes a time to reach Ccrit, denoted as Tcritthat is determined according to the following equation:when Ccritis three times or more greater than KM, wherein Vcand Cl are parameters determined by a one-compartment PK model.

[0196] In some embodiments, the desired clinical endpoint includes a time to reach Ccrit,denoted as Tcrit, that is determined according to the following equation:when Cent is three times or more greater than KM, wherein A, B, α, β, and kelare parameters determined by a two-compartment PK model.

[0197] In addition to the critical concentration Ccrit, the time interval for which the systemic concentration of the antibody should be equal to or greater than Ccritmust be determined. For radiopharmaceuticals, this time interval is a function of both the physical half-life of the therapeutic radionuclide and the biological clearance rate of the radiopharmaceutical from the body. Consider Ac-225 as an example, which has a radioactive half-life of 9.92 days, and a typical antibody with a terminal half-life of approximately 14 days. The effective half-life of the radioimmunoconjugate in vivo will therefore be about 5.8 days, which is determined according to the following equation: In such a case. a reasonabletime period for saturation of the normal tissue sink can be chosen to be about 10 days, or approximately 2 effective half-lives, post-administration of hot dose administration, during which time the majority of the radiation emitted in vivo will be deposited.

[0198] In some embodiments, to formulate a DT of the cold targeting molecule, the following steps are performed: iv. obtaining parameters of the PK profde, wherein the parameters comprise Michaelis-Menten constant (KM), maximal velocity (VM), first-order / linear clearance rate (Cl), central volume of distribution (Vc); v. determining a critical concentration (Ccrit) and a transitional concentration (Ctrans) based on the parameters, wherein the PK profde of the cold targeting molecule is linear both before Ccritand after KM / 3, and exhibits a transition at Ctransbetween Ccritand KM / 3; and vi. formulating a DT of the cold targeting molecule based on Ctransto reach a desired clinical endpoint.

[0199] In some embodiments, the DT is formulated based on a transitional concentration (Ctrans), which is applicable for any therapeutic agent (e.g., drug) whose pharmacokinetics (PK) is described by nonlinear PK models comprising a few compartments (1, 2, or 3) and also including parallel linear and nonlinear clearance pathways; the nonlinear pathwayincludes but is not limited to TMDD and empirical Michaelis-Menten (M-M) equation. Typically, PK of such drugs exhibits sharp transitions at some concentrations; such transition can be captured by the defined Ctrans.

[0200] For PK models comprising mixed linear and nonlinear M-M clearance pathways, a transitional concentration, denoted herein as Ctrans, is defined as:(5)In Equation (5), sqrt(KMxVM / Cl) can also be expressed asare interchangeably used in this invention.

[0201] If only nonlinear M-M equation exits (i.e., Cl is zero), the transitional concentration is defined as:(6) Ctransis used to predict and locate the transition zone of the PK profiles. Determination of Ctranscan be done in a few approaches: either directly calculated from the PK model parameters or estimated or visually determined from the observed PK profiles.

[0202] The definition of Ctransis demonstrated with one example; the PK of one drug is described by a one-compartment open model with parallel linear (first order with rate constant Cl) and nonlinear clearance pathways; nonlinear pathway is described by M-M equation (with parameters of VM and KM). In this case, one dimensionless number can be defined as R = VM / (C1 x KM).

[0203] The transitional concentration Ctransis defined as follows. Factors to be considered include the mass elimination rates (i.e., Elimination in FIG. 1 for R=4) of the first order clearance, the M-M equation, and the total rate (the sum of the first order clearance and the M- M equation). Ctransis defined as the concentration to let the total mass elimination rate equal to VM. It is found that when R is greater than or equal to 1 / 3, dimensionless number sqrt(R) is the best choice for determining the transitional concentration, referring to FIG. 1; in dimensional form (relative to KM), the transitional concentration is determined by formula KMx sqrt(R), which is KMX sqrt(VM / (Cl x KM)), i.e., sqrt(KMx VM / CI). When R is less than 1 / 3, drug PK profiles are almost linear no matter how much dose is administered via bolus injection as if the nonlinear clearance term described by M-M equation (1) is dropped.

[0204] In some embodiments, a PK profile shows a nonlinear clearance pathway featuring target mediated drug disposition (TMDD), Ctranscan be determined according to formula sqrt(KMx VM / CI), wherein KM= Koff / Konand VM Kint×Ttotx Vc, where Konis the forward rate constant of drug-target binding with a unit of l / concentration / time; Koffis the reverse rateconstant of drug-target binding with a unit of 1 / time; Kintis the rate constant of internalization of drug-target complex with a unit of 1 / time, and Ttotis the total receptor concentration.

[0205] In some embodiments, a PK profde shows a nonlinear clearance pathway featuring target mediated drug disposition (TMDD), and Ctranscan be determined according to formula sqrt(KMx VM / C1), wherein KM= ( Koff+Kint)Konand VM = Ksynx Vc, where Konis the forward rate constant of drug-target binding with a unit of l / concentration / time; Koffis the reverse rate constant of drug-target binding with a unit of l / time; Kintis the rate constant of internalization of drug-target complex with a unit of 1 / time, and Ksynis the target synthesis rate constant. In this disclosure, two formulations are used: one in a dimensional form and the other in a dimensionless form. The latter has an advantage to reduce model parameters by at least 2 and to neatly summarize the results for various combinations of dimensional model parameters. In the dimensionless form, all concentrations are scaled relative to KMwhile all slopes are scaled relative to Cl / Vc. Dimensionless number R, i.e., VM / (C1 X KM), can be referred to a critical concentration (Ccrit); in dimensional form, Ccritis determined by formula R x KM= VM / C1.The dimensionless transitional concentration is sqrt(R); in dimensional form, Ctransis determined by formula sqrt(KM×VM / C1). The maximal concentration after bolus injection is Cmax= DT / VCand its dimensionless form is denoted as Xmax, which is determined by formula DT / ( Vcx KM).

[0206] The use of Ctransto predict the transition zone is analyzed by two approaches. Firstly, considering the slope of the PK curve at a few key concentrations; it is found that the slope at Ctranslies in the middle part of the slopes of the PK curve, referring to FIG. 2 for R=10, where drug PK is described by one compartment model with mixed linear and nonlinear clearance pathways.

[0207] Secondly, two approximate solutions are obtained for two scenarios: (i) Cp far greater than Ctrans, which is greater than KM; and (ii) Cp far less than KM. It is found that Ctranslies in the central region of the transition zone where those two approximate solutions diverge from the analytical solution at the boundaries of the transition zone. See FIG. 3 for R=10 and Xmax=100, where drug PK is described by one compartment model with mixed linear and nonlinear clearance pathways.

[0208] Further, it is found that the definition of Ctransis also applicable for PK models comprising two compartments with mixed (or parallel) linear and nonlinear (M-M) clearance pathways. See FIG. 4.

[0209] Referring to FIG. 4, Ctranscan be defined in the same way as in the one compartment model shown in FIGs. 2-3, i.e., Ctrans= sqrt(KMx VM / C1); also, Ctranslies in the central region of the transition zone where two approximate solutions diverge from the analytical solution at the boundaries of the transition zone.

[0210] As demonstrated in FIGs. 2-4 above, Ctransas disclosed herein can be used to predict the transition zone of PK profdes for various therapeutic agents such as antibodies and small molecule drugs. The existence of the transition zone in the PK profde is dependent on the therapeutic dose (DT) and PK model parameters. In certain embodiments, the DT of a cold targeting molecule is formulated to reach Ctransat a pre-determined time point, wherein DT is determined according to the following equation:where Vcis the distribution volume of the central compartment where the linear and nonlinear clearance pathways are present and the bolus injection administration is occurred. If Cmaxis greater than Ctrans, the time to reach Ctrans, denoted herein as Ttrans, is dependent on Cmaxand PK model parameters; if Cmaxis equal to Ctrans, Ttrans= 0, i.e., the administration time; if Cmaxis less than Ctrans, Ttransis not defined.

[0211] For a PK model comprising only one compartment (whose distribution volume is Vc) and including mixed linear and nonlinear M-M clearance pathways, the desired clinical endpoint includes a time to reach Ctrans, denoted as Ttrans, that can be determined according to the following equation:where Vc is the distribution volume of the compartment, Cl is the clearance rate constant for the linear / first order clearance, KMand VM are M-M equation coefficients, Cmaxand Ctransare defined in Equations (7) and (5), respectively. With known values for PK model parameters (Vc, Cl, KM, VM) and Ctransdefined, Ttransand Cmaxare related to each other according to Equation (8): if one is known, the other is uniquely determined. Equation (8) can be used to determine Cmaxand the therapeutical dose based on equation Dose = CmaxxVcfor a given Ttrans; or to determine Ttranswith the known therapeutic dose and Cmax.

[0212] Equation (8) is a special application at the transitional concentration Ctransof the analytical solution for the aforementioned PK model, where the analytical solution can be referenced to Wagner (1988) and Wagner (1993). The analytical solution was obtained for small molecule drugs before the advent of protein therapeutics (such as mAbs); it was used tocalculate major PK parameters such as AUC (area under the curve) of drug products, the trough concentration, and accumulation ratio for multiple administrations. To our best knowledge, the analytical solution has not been used to predict dosage forms for protein therapeutics and / or monoclonal antibodies. This would be the first time to use the analytical solution for calculating the time to reach the transitional concentration Ctrans, thereby formulating a therapeutic dose of the drug (e.g., antibody) based on Ctransto reach a desired clinical endpoint.

[0213] In some embodiments, the pre-determined time point or Ttransdescribed above can be 7, 14, 21, or 28 days or longer.

[0214] In some embodiments, the DT of a cold targeting molecule is formulated to reach a plasma concentration (CP) that is greater than Ctransfor a pre-determined duration of time (Tdur), wherein DT is determined according to the following equation: DT = CmaxX Vc.

[0215] In some embodiments, the desired clinical endpoint includes a time to reach Ctrans, denoted as Ttrans, that is determined according to the following equation:wherein Ttransis greater than Tdur.

[0216] For other PK models, such as a PK model comprising two compartments and including mixed linear and nonlinear M-M clearance pathways, Ctransis still defined by equation (5); Ttranscan be obtained via numerically solving the governing equations for the PK model. The governing equations for such a model can be defined as below:where Vc and Vp are distribution volumes of the central and peripheral compartments, Q is the exchange rate between two compartments, C2 is the drug concentration in the peripheral compartment defined as C2 = Mp / Vp. where Mp is drug mass in the peripheral compartment. The initial conditions are:

[0217] Equations 10A / 10B / 10C shown above can be solved with any ordinary differential equation (ODE) solver such as Berkeley Madonna®, package deSolve of R, or various ODE solvers in MATLAB®. Once the numerical solution for Cp(t) is obtained, Ttrans, which is achieved when Cp(t) is equal to Ctrans, can be read out and recorded.

[0218] In TMDD models, drug PK can be obtained via solving mechanistic equations describing the key processes of drug PK, including distribution, clearance, drug-target binding / unbinding and internalization. Assuming one TMDD model has one compartment with a distribution volume Vc and a first-order clearance rate Cl, the governing equations for such a TMDD model are defined as below:where Cp(t) is drug plasma concentration, T is free target concentration, and CT is the concentration of drug-target complex. Model parameters are defined as below:Kei = Cl / V c, the elimination rate constant with a unit of / time; Ksyn, the rate constant of target synthesis with a unit of concentration / time;Kieg, the rate constant of degradation of free target with a unit of 1 / time; Kon, the forward rate constant of drug-target binding with a unit of1 / concentration / time; Koff, the reverse rate constant of drug-target binding with a unit of 1 / time; Kint, the rate constant of internalization of drug-target complex with a unit of 1 / time.The initial conditions can be defined as below:Cp(0) = Dose / Vc, T(t=O) = Ksyn / Kdeg, and CT(t=0) = 0 (1 ID)

[0219] Equations 11 A / l 1B / 11C with the initial conditions as defined in 1 ID can be solved with any ODE solver such as Berkeley Madonna®, package deSolve of R, or various ODE solvers in MATLAB®. Hence, their numerical solutions can be obtained accordingly. Once the numerical solution is obtained, Ttransto achieve Ctransfor any Cmaxvalue (where Cmaxis greater than Ctrans) can be determined.

[0220] Under certain conditions such as quasi-equilibrium (QE) and quasi-steady state (QSS) conditions, a TMDD model is equivalent to a M-M model for a certain range of plasma concentrations. As a result, VM and KMof M-M equation can be expressed by the TMDD model parameters.

[0221] More specifically, under the QE condition, a TMDD model is equivalent to M-M model; see e.g., Yan et al., 2010; Mager & Krzyzanski, 2005. This equivalence is valid when the following conditions are satisfied:Kint=Kdegso that Ttot= Ksyn / Kdegis a constant (where Ttotis total receptor concentration); rapid binding, i.e, KonX Cp(t) x T = Koffx CT is valid, for any time t;Cp2>(Kdx Ttot) where Kdis the dissociation constant: Ka= Koff / Kon. (Yan et al., 2021).

[0222] Under the QE condition, VM and KMcan be determined as below:where Ctransis also defined by Equation (5) with VM and KMdefined in Equation (12) and (13). Expressed in an explicit form by TMDD model parameters, Ctransis determined as below:

[0223] Alternatively, under the QSS condition, a TMDD model is also equivalent to M-M model; see e.g., Gibiansky et al., 2008. The equivalence is valid for rapid binding and for Cp2>(KQSSx Ttot) where K.QSS ( Koff+Kint / )Kon. It is demonstrated that QSS approximation is preferred over QE when Kintis not negligible compared to Koff.

[0224] Under the QSS condition, VM and KMcan be determined as below:KMwhere Ctransis also defined by Equation (5) with VM and KMdefined in Equation (15) and (16). Expressed in an explicit form by TMDD model parameters, Ctransis determined as below:

[0225] For typical TMDD model described by Equations 11 A / l 1B / 11 C / 1 ID, its analytical solution is generally hard to be obtained while its numerical solution can be obtained with any ODE solver. Once its numerical solution is obtained, Ttransto achieve Ctransfor any Cmaxvalue (where Cmaxis greater than Ctrans) can be found.

[0226] Based on the descriptions set forth above, the therapeutic dose of a cold targeting molecule can be determined for any given Ttranswith known PK model parameters. The following examples are merely illustrative for demonstrating how Ctransis determined and how it is used to determine the therapeutic dosage of a drug to reach a desired clinical endpoint.EXAMPLESExample 1. Determination of Ctransof an antibody whose PK in humans is described by a two- compartment model with nonlinear only clearance pathway

[0227] This example demonstrates how Ctransis determined for two antibodies: cetuximab and alemtuzumab, where their PK are described by a two-compartment model with nonlinear only clearance pathway described by M-M equation. For this kind of PK model, Ctransis determined according to Equation (6):Table 2 PK parameters and calculated Ctransfor cetuximab and alemtuzumab

[0228] PK parameters listed in Table 2, i.e., Vc, Vp, Q, VM and KM, were obtained based on the protocols disclosed in Dirks et al., 2008 and Mould et al., 2007, with modification to be consistent with the current model description provided herein.Example 2. Determination of Ctransof a small molecule drug whose PK in rats is described by a two-compartment model with nonlinear only clearance pathway

[0229] This example demonstrates how Ctransis determined for small molecule phenytoin. Its PK in rats is described by a two-compartment model with nonlinear only clearance described by M-M equation. For this kind of PK model, Ctransis determined according to Equation (6):Table 3 PK parameters for rats and calculated Ctransfor phenytoin

[0230] PK parameters listed in Table 3, i.e., Vc, Vp, K12, K21, Q, VM and KM, were obtained based on the protocols disclosed in Della Paschoa et al., 1998, with modification to be consistent with the current model description provided herein.Example 3. Determination of Ctransof an antibody whose PK is described by a two- compartment model with mixed linear and nonlinear clearance pathways (M-M equation)

[0231] This example demonstrates how Ctransis determined for four antibodies: tocilizumab, sibrotuzumab, panitumumab, and vedolizumab, where their PKs are described by a two- compartment model with mixed linear and nonlinear clearance pathways (described by M-M equation). For this kind of PK model, Ctransis determined according to Equation (5):Table 4 PK parameters and calculated Ctransfor four antibodies

[0232] PK parameters listed in Table 4, i.e., Vc, Cl, Vp, Q, VM and KM, were obtained based on the protocols disclosed in Frey et al., 2010; KI oft et al., 2004; Ma et al., 2009; and Okamoto et al., 2021, with modification to be consistent with the current model description provided here.Example 4. Determination of Ctransof an antibody whose PK in humans and / or monkeys is described by TMDD model under the quasi-equilibrium (QE) condition

[0233] This example demonstrates how Ctransis determined for two antibodies: TRX1 and mAb-7, where their PKs are described by a two-compartment model with TMDD model under rapid binding and QE conditions. For this kind of PK model, Ctransis determined according to Equation (5), specified by Equation (14):where VM and KMare calculated from TMDD model parameters according to Equations (12) and (13):Table 5 PK (TMDD) model parameters and calculated Ctransfor two antibodiesAssuming a human subject of 70 kg weight.#From Yan et al., 2010.

[0234] PK parameters listed in Table 5 were obtained based on the protocols disclosed in Ng et al., 2006, Yan et al., 2010, and Singh et al., 2015, with modification to be consistent with the model description listed in Equations (11 A)-(l ID).Example 5. Determination of Ctransof an antibody whose PK is described by TMDD model under the quasi-steady state (QSS) condition

[0235] This example demonstrates how Ctransis determined for two antibodies: mavrilimumab and efalizumab, where their PKs in humans are described by a two- compartment model with TMDD model under the QSS condition. For this kind of PK model, Ctransis determined according to Equation (5), specified by Equation (17):where VM and KMare calculated from TMDD model parameters according to Equations (15) and (16).Table 6 PK (TMDD) model parameters and calculated Ctransfor two antibodies

[0236] PK parameters listed in Table 6 were obtained based on the protocols disclosed in Stein & Peletier, 2018 and Rodriguez-Vera et al., 2015, with modification to be consistent with the current model description listed in Equations (11 A)-(l ID).Example 6. Determination of a therapeutic dose of one compound to reach Ctransat a certain time point

[0237] This example demonstrates how Ctransis used to determine a therapeutic dose of evolocumab in humans, where its PK is described by a one-compartment model with mixed linear and nonlinear clearance pathways. Ttransto reach the transitional concentration Ctransforany dose level Cmaxis determined according to Equation (8). When Ttransand Ctransare known, Cmaxand the therapeutic dose can be determined as follows.Evolocumab PK model parameters such as Vc, Cl, KMand VM are provided in Table 7 below. According to Equation (5), Ctransis determined to be 115.18 nM or 17.28 mg / L assuming the molecular weight of 150 kDa or 150 kg / mol for a typical antibody. Ttransis pre- determined to be 28 days. Ttransand Ctranscan be used to determine Cmaxbased on Equation (8). Cmaxcan be obtained from Equation (8) via any root solver as provided in MATLAB®, R, MAPLE®.With R (version x64 4.0.5), Cmaxis determined to be 536.4 nM. which is converted into 80.46 mg / L as shown below assuming the molecular weight of 150 kDa or 150 kg / mol for a typical antibody. Cmax= 536.4 nM, which is equivalent to 536.4 nmol / Lx 150,000 g / mol, which is equivalent to 536.4x0.15 mg / L, i.e., 80.46 mg / LThe therapeutic dose (Dose or DT) is determined as shown below:DT = CmaxxVc = 80.46 mg / Lx5.18 L = 416.8 mg.Hence, the desired DT is 416.8 mg to reach Ctransat Ttrans= 28 day.Table 7 PK model parameters and calculated Ctransfor Evolocumab

[0238] PK parameters listed in Table 7 were obtained based on the protocols disclosed in Kuchimanchi et al., 2018, with modification to be consistent with the current model description listed in Equations (5) and (7).Example 7. Determination of the minimal dose level to have plasma concentration Cp(t) above Ctransfor a certain time duration

[0239] This example demonstrates how Ctransis used to determine a therapeutic dose of tocilizumab, where its PK in humans is described by a two-compartment model with mixed linear and nonlinear pathways.This example requires that Ttransfor the desired therapeutic dose be greater than the pre-determined duration time point post-administration, which is denoted herein as Tdur.PK model parameters for tocilizumab such as Vc, Cl, Vp, Q, KMand VM are provided in Example 3. Ctransis determined to be 8.22 pg / mL or 8.22 mg / L; Tdur is pre-determined as 28 days, which is equal to or less than Ttrans. Cmaxfor Ttrans= 28 day can be obtained via solving equations (10A / 10B / 10C) via any ODE solver. With package deSolve of R (version x644.0.5), Cmaxis determined to be 237.2 mg / L.The therapeutic dose (DT) is calculated as shown below:DT = CmaxxVc = 237.2 mg / Lx3.5 L = 830.2 mgIt is found that Ttranscorrelates with Cmaxas the greater the Cmax, the greater the Ttrans. Having Ttransgreater than Tdur, the minimal Cmaxvalue is 237.2 mg / L; and the minimal dose to have tocilizumab reach a Cp(t) above Ctransfor at least 28 days is 830.2 mg.Example 8. Determination of a therapeutic dose (DT) of PanitumumabPanitumumab PK

[0240] Panitumumab is an epidermal growth factor receptor (EGFR) antagonist monoclonal antibody indicated for the treatment of wild-type RAS (defined as wild-type in both KRAS and NRAS as determined by an FDA-approved test for this use) metastatic colorectal cancer (Vectibux label). Reference is made to the PK profile of panitumumab in a male subject of 80 kg weight and age of 60 years, who was dosed at 6 mg / kg or 480 mg of panitumumab.

[0241] Panitumumab PK is described by a two-compartment model with mixed (or parallel) linear and nonlinear (Michaelis-Menten) clearance pathways. PK parameters are available in Ma et al., (2009). Model parameters for this subject are assumed to be identical to the values listed in the abstract.

[0242] The simulated PK profile, together with some key concentrations, is plotted as shown in FIG. 5, which can be used to demonstrate the various parameters.First, the PK profile (solid blackcurve) is divided into three phases: the initial linear phase, the transition phase, and the terminal linear phase.Second, the initial linear phase is bounded by Ccrit. Above Ccrit, PK profile is linear in log-linear scale.Third, the terminal linear phase is bounded by KM / 3. Below that, PK profile is linear in log-linear scale.Fourth, the initial linear phase in this case is extended to day 14.Fifth, Ctransis used to locate the transition phase, which is bounded by the initial linear and the terminal linear phases.Sixth, the initial linear phase and its extension to day 14 can be described by the PK profile (dotted blackcurve) predicted by a linear model where the M-M clearance rate is assumed to be constant VM. This is a very good approximation since Cp>Ccritin the initial linear phase and Ccrit=39.4 mcg / mL is about 100 times greater than KMvalue (0.401 mcg / mL).The initial linear phase can be extended to Day 14 since the approximation (CP>>KM) is still valid during 14 days post-administration. Further, this means that the nonlinear (M-M) clearance pathways is still saturated at day 14 or earlier. Refer to below for approximation.Determination of Ccrit

[0243] The plasma PK of Panitumumab is described by a two-compartment model with a mixed linear (Cl) and nonlinear clearance pathways; the nonlinear part is described by the Michaelis-Menten equation (VM and KM) [Ma et al., 2009], Cl = 0.269 L / day, VM = 10.6 mg / day. Hence Ccrit= VM / C1 = 39.41 mg / L. Note also that KM= 0.401 mg / L. The transition phase between the initial linear phase to the terminal linear phase varies from 0.401 mg / L to 39.41 mg / L, a range of two orders of magnitude.Determination of mass dose level to saturate Ccrit

[0244] Consider Panitumumab for which Ccrit= 39.4 mg / L. The post-administration time period to saturate clearance is can be considered to be 7 days, i.e. at 7 day after dose administration, the plasma concentration is required to be greater than 39.4 mg / L. Based on a published PK model and their model parameter estimates obtained via population PK analysis [Ma et al., 2009], numerical computation with an ODE solver such as Berkeley Madonna was conducted to solve the nonlinear PK problem and to predict the plasma concentration at day 7 for various dose levels. The minimal dose level or therapeutic dose (DT) of Panitumumab to reach the critical concentration of 39.4 mg / L at day 7 was determined to be about 6 mg / kg.Note: due to the wide range between KM(0.401 mg / L) and Ccrit(39.4 mg / L), the mean PK profde for a dose level of 6 mg / kg appears linear through day 14 post-administration (when the plasma concentration is 16 mg / L on average). The approved dose level of Panitumumab was 6 mg / kg every 14 days as an intravenous infusion over 60 minutes.Example 9. Determination of a therapeutic dose (DT) of AVE1642 Determination of Ccrit

[0245] AVE 1642 is a monoclonal antibody targeting IGR-1R. Its plasma PK is described by a two-compartment model with mixed linear (Cl) and nonlinear clearance pathways; the latter is described by the Michaelis-Menten equation. Cl=0.55 L / day, Vm = 1.0 mg / day. Hence Ccrit=Vm / Cl = 1.82 mg / L. Clinical PK data shows that a mass dose level of 0.5 mg / kg leads to almost linear pharmacokinetics and maintains drug concentrations greater than Ccritfor at least 7 days following IV administration.Determination of mass dose level to saturate Ccrit

[0246] Consider a radioimmunoconjugate comprising AVE-1642 conjugated to a bifunctional chelator and labeled with the alpha emitting radioisotope Ac-225. A time period of 10 days is estimated as a clinically relevant time interval based upon the effective half-life of the radioimmunoconjugate (derived from the decay rate of Ac-225 and the biological clearance rate of FPI-1434). Based on a nonlinear PK model and estimates of model parameters, numerical computation with an ODE solver was conducted to solve the nonlinear PK equations and to predict the plasma concentration at day 10 for various dose levels. For a subject of 80 kg weight, a cold dose level of 50 mg is sufficient to saturate target mediated clearance and to make plasma concentration at 10 days post-dose just exceed Ccrit. Hence, the target cold dose level, i.e., DT, is determined to be about 0.6 mg / kg. The actual cold dose level chosen in a clinical trial, which considers variation of PK parameters among subjects and balances the tumoral tissue uptake, was chosen to be 0.5 mg / kg.Example 10. General materials and methods

[0247] Lutetium- 177 can be obtained from Perkin Elmer as lutetium trichloride in a 0.05 N hydrochloric acid solution; indium-il l, as a trichloride salt, can be obtained from Nordi on; and actinium-225 can be obtained as actinium-225 trinitrate from Oak Ridge National Laboratories or actinium-225 trichloride from Canadian Nuclear Laboratories.

[0248] Analytical HPLC-MS can be 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, 2.1x50 (1.7 μm) column. Preparative HPLC can be 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 19x100 mm (5 μm) column.

[0249] HPLC elution method 1: Waters Acquity BEH C18 2.1x50 mm (1.7 μm) column; mobile phase A: H2O (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.

[0250] HPLC elution method 2: Waters XBridge Prep Phenyl 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0251] HPLC elution method 3: Waters Acquity BEH C18 2.1x50 mm (1.7 μm) column; mobile phase A: H2O (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.

[0252] HPLC elution method 4: Waters XBridge Prep Cl 8 OBD 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0253] HPLC elution method 5: Waters XBridge Prep Cl 8 OBD 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0254] HPLC elution method 6: Waters XBridge Prep Cl 8 OBD 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0255] HPLC elution method 7: Waters XBridge Prep Cl 8 OBD 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0256] HPLC elution method 8: Waters XBridge Prep C18 OBD 19x100 mm (5 μm) column; mobile phase A: H2O (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.

[0257] Analytical Size Exclusion Chromatography (SEC) can be 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, 7.8x300 mm column. The isocratic SEC method can have a flow rate of, e.g., mL / min, with a mobile phase of 0.1 M phosphate, 0.6 M NaCl, 0.025% sodium azide, pH = 7.

[0258] MALDI-MS (positive ion) can be performed using a MALDI Bruker Ultraflextreme Spectrometer.

[0259] Radio thin-layer chromatography (radioTLC) can be performed with Bioscan AR- 2000 Imaging Scanner, and can be carried out on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plates using citrate buffer (0.1 M, pH 5.5).Example 11. Synthesis of 4-{[ll-oxo-ll-(2,3,5,6-tetrafhrorophenoxy)undecyl]carbamoyl}-2- [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-l-yl]butanoic acid (Compound B)

[0260] A bifunctional chelate, 4-{[l l-oxo-ll-(2,3,5,6- tetrafluorophenoxy)undecyl]carbamoyl } -2-[4,7, 10-tris(carboxymethyl)-1 ,4,7, 10- tetraazacyclododecan-l-yl]butanoic acid (Compound B), can be synthesized according to the scheme provided in FIG. 7. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert- butoxy)-2-oxoethyl)- 1 ,4,7, 10-tetraazacy clododecan- 1 -yl)pentanoic acid (DOTA-GA-(tBu)4, 50 mg, 0.07 mmol) in ACN (2.0 mL), DSC (50 mg, 0.21 mmol) is added, followed by pyridine, (0.20 mL, 2.48 mmol). The reaction is stirred at room temperature for 1 hour. To the reaction mixture is added 11-aminoundecanoic acid, (70 mg, 0.36 mmol) followed by PBS solution (1.0 mL) at room temperature. The reaction is stirred for 72 hours at room temperature. The reaction mixture is filtered with a syringe filter and purified directly by Prep-HPLC using method 6 to yield Intermediate 2-A.

[0261] 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) is added pyridine (0.05 mL, 50 mg, 0.62 mmol) at room temperature. The solution is stirred at room temperature for 24 hours. The reaction is purified directly by Prep-HPLC using method 7 to provide Intermediate 2-B as a wax after concentration using a Biotage V10 Rapid Evaporator.

[0262] Intermediate 2-B is dissolved in DCM / TFA (1.0 mL / 2.0 mL) and allowed to stir at room temperature for 24 hours. The reaction is concentrated by air stream and purified directly by Prep-HPLC using method 8 to yield Compound B as a clear wax after concentration. An aliquot is analyzed by HPLC-MS elution method 3.

[0263] 1H NMR (600 MHz, DMSO-d6) δ 7.99 - 7.88 (m, 1H), 7.82 (t, J= 5.5 Hz, 1H), 3.78 (broad s, 4H), 3.43 (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 12. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy] ethoxy } ethoxy)ethy 1] carbamoyl } -2- [4,7, 10-tris(carboxymethy 1)- 1,4,7,10- tetraazacyclododecan-l-yl]butanoic acid (Compound C)

[0264] A bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy] ethoxy } ethoxy)ethy 1] carbamoyl } -2- [4,7, 10-tris(carboxymethy 1)- 1,4,7,10- tetraazacyclododecan-l-yl]butanoic acid (Compound C), is synthesized according to the scheme provided in FIG. 8.

[0265] To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)- l,4,7,10-tetraazacyclododecan-l-yl)pentanoic acid (DOTA-GA(tBu)4, 100 mg, 0.143 mmol) in ACN (8.0 mL) is added DSC (73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol). The reaction mixture is stirred for 90 min at ambient temperature. This solution is added to a semi- solution of amino-PEG3-acid (63 mg, 0.285 mmol in 1.2 mL of DMF) in a 100 mL round bottom flask. After 4 hours at ambient temperature, the reaction is worked up by concentrating to dryness under a stream of air. The crude material is purified by HPLC elution method 2 (dissolved the crude in 6 mL of 20% ACN / H2O). The fractions containing product are pooled and concentrated under vacuum and then co-evaporated with ACN (3 x 2 mL).

[0266] To a vial containing Intermediate 1-A (82 mg, 60 pmol) is added ACN (2 mL), NEts (50 μL, 360 pmol, 6 equiv.), HBTU (23 mg, 60 pmol, 1 equiv) and a TFP solution (50 mg, 300 pmol, 5 equiv., dissolved in 250 μL of ACN). The resulting clear solution is stirred at ambient temperature for 3 hours. The reaction is worked up by concentrating the solution to dryness under an air stream and is then diluted with ACN / H2O (1:1, 3 mL total) and purified on preparative HPLC using elution method 4. Fractions containing product are pooled and concentrated under vacuum and then co-evaporated with ACN (3 x 2 mL). Intermediate 1-B is obtained as a clear residue.

[0267] To a vial containing Intermediate 1-B (67 mg, 64 pmol) is added DCM (2 mL) and TFA (2 mL). The resulting solution is stirred at ambient temperature for 16 hour. Additional, TFA (2 mL) is added, and the reaction is stirred at ambient temperature for 6 hours. The reaction is concentrated to dryness under an air stream, with the crude product being finally dissolved in ACN / H2O (1 mL of 10% ACN / H2O). The crude reaction solution is then purifiedby preparative HPLC using elution method 5. The fractions containing product are pooled, frozen and lyophilized. Compound C is obtained as a white solid . An aliquot is analyzed by HPLC-MS elution method 3.

[0268] 1H NMR ( DMSO-d6, 600 MHz) 87.97-7.91 (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 13. Synthesis of radioimmunoconjugates targeting IGF-1R

[0269] FIG. 9 shows the steps for synthesis of two radioimmunoconjugates targeting IGF- 1R, namely, [111In]-DOTA-anti-IGF-l R conjugate and [225Ac]-DOTA-anti-IGF-lR conjugate. The synthesis followed the protocols below.[111In] -Compound C-cmti-IGF-lR conjugate

[0270] Compound C (17.5 pmoles) 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 IGF-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-anti-IGF-lR was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-anti- IGF-1R found m / z 152166 [M+H]+; IGF-1R found m / z 149724 [M+H]+.

[0271] As a typical reaction, In-111 (60 mCi, 215 μL) was added to a solution of Compound C-anti-IGF-lR (7 mg, 1.6 mL in acetate buffer (pH 6.5)) and ascorbic acid (100 μL, 0.2 Min acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37°C for 30 minutes. [111In]-Compound C-anti-IGF-lR conjugate was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).[225Ac] -Compound C-anti-IGF-lR conjugate

[0272] Compound C (1 pmole) is dissolved in a hydrochloric acid solution (0.001 M). An aliquot of Compound C solution (5 μL, 70 nmole) is added to a solution containing anti-IGF- 1R antibody (1.8 nmoles) in a phosphate buffer (pH 8). After 3 hours at ambient temperature, the resulting immunoconjugate is purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-anti-IGF-lR is eluted from the column with acetate buffer (pH 6.5). Identities of eluates can be confirmed by, e.g., MALDI-TOF.

[0273] Ac-225 (15 pCi, 10 μL) is added to a solution of Compound C-anti-IGF-lR (300 pg in acetate buffer (pH 6.5). The radiolabeling reaction is incubated at 30° C for 1 hour. Thecrude product, [225Ac] -Compound C-anti-IGF-lR, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer.Example 14. Imaging and pharmacokinetics of IGF-lR-targeting radioimmunoconjugates involving cold IGF-1R antibody pre-administration

[0274] A study was performed to evaluate the imaging and pharmacokinetics in patients of receiving cold IGF-1R antibody pre-administration prior to administration of [111In]-DOTA- anti-IGF-lR conjugate. The study followed the protocols below.

[0275] Five patients were evaluated in the cold antibody pre-administration study at two different dose levels of cold antibody. Patients were first imaged and samples were taken for pharmacokinetic analysis after receiving a fixed dose of 5 mCi [111In]-DOTA-anti-IGF-l R conjugate (the “no cold” regimen) and subsequently patients received the same 5 mCi dose of the imaging agent [111In]-DOTA-anti-IGF-l R conjugate and either 0.5 or 1.5 mg / kg of non- radioactive IGF-1R antibody AVE1642 (“cold antibody” dosing regimen) followed by imaging and pharmacokinetic assessment. Four patients had sufficient data with and without pre-administration of cold antibody to estimate radiation doses of [225Ac]-DOTA-anti-IGF-lR conjugate to the whole body, liver, spleen, red bone marrow, and kidneys. One patient was unable to complete all imaging time points needed for dosimetry. All five patients were evaluated for the comparison of lesion uptake where up to three lesions and backgrounds consisting of liver, muscle, red bone marrow, and total body were sampled and compared in each patient. All five patients were also evaluated for the determination of [111In]-DOTA-anti- IGF-1R conjugate plasma pharmacokinetics as measured by total radioactivity.

[0276] It was observed that the pre-administration of cold antibody prior to the [111In]- DOTA-anti-IGF-lR conjugate dosing regimen had a significant effect on the plasma pharmacokinetics of [111In]-DOTA-anti-IGF- 1 R conjugate in all five patients. See Fig. 10. In each patient, the clearance of the [111In]-DOTA-anti-IGF-l R conjugate was markedly reduced in the cold antibody dosing regimen at both the 0.5 and 1.5 mg / kg dose levels. The pharmacokinetics of [111In]-DOTA-anti-IGF- I R conjugate in each dose level of IGF-1R antibody AVE1642 (i.e., 0.5 or 1.5 mg / kg) were comparable, suggesting that doses equal to or greater than 0.5 mg / kg are sufficient to saturate the endogenous antigen sink and achieve linear pharmacokinetics. On average for the five evaluable patients, the pre-administration of IGF-1R antibody AVE1642 to the dosing regimen increased the total exposure (AUCinf) of [111In]-DOTA-anti-IGF- 1 R conjugate by 6-fold relative to [111In]-DOTA-anti-IGF- 1 R conjugate only. See Table 8 below.Table 8. Pharmacokinetics of IGF-lR-targeting radioimmunoconjugates with / without coldIGF-1R antibody pre-administration

[0277] Among the five patients, imaging study shows that, at the cold antibody dose level of 0.5 mg / kg, radiation dose to the whole body, kidneys, and red marrow increased with the addition of cold antibody, while radiation dose to the liver was decreased in one patient, stayed about the same in another patient. Radiation dose to spleen decreased in both patients. At the cold antibody dose level of 1.5 mg / kg, radiation dose to the whole body, kidneys, liver, and red marrow increased. There was a mixed result in the spleen at this dose level with one patient showing a slight increase while the other showed a decrease in radiation dose with the addition of cold antibody. See FIGs. 11A-11E.

[0278] Further, ratios of tumor uptake relative to background increased for each patient at the cold antibody dose level of 0.5 mg / kg and 1.5 mg / kg. It was noted that the relative gain in tumor uptake was greater in patients treated with 0.5 mg / kg (n=2) compared to those treated in the 1.5 mg / kg group (n=3). This study demonstrated that pre-administration of cold IGF-1R antibody increased tumor lesion uptake of radioimmunoconjugate targeting IGF-1R in all patients. See FIGs. 12A-12E.Example 15. Efficacy of IGF-lR-targeting radioimmunoconjugates involving cold IGF-1R antibody pre-administration

[0279] The above findings indicate that the pre-administration of cold IGF-1R antibody AVE1642 at 0.5 mg / kg prior to treatment with [225Ac]-DOTA-anti-IGF-lR conjugate shall improve uptake of the compound leading to a greater deposition of radiation to the tumorrelative to the normal tissues, thereby likely increasing efficacy and decreasing off-target toxicities.

[0280] In addition, based on the imaging and pharmacokinetic data above, the predicted therapeutic efficacious range of [225Ac]-DOTA-anti-IGF-lR conjugate can be extrapolated from non-clinical mouse xenograft efficacy data as below: without cold antibody, the [225Ac]- DOTA-anti-IGF-lR conjugate is expected to provide radioactivity in the range of 80-160 kBq / kg cumulative exposure, while with pre-admini strati on of cold IGF-1R antibody, the [225Ac]-DOTA-anti-IGF-lR conjugate is expected to provide radioactivity in 35-70 kBq / kg cumulative exposure. Accordingly, when pre-administered with cold IGF-1R antibody (AVE1642) at 0.5 mg / kg, [225Ac]-DOTA-anti-IGF-lR conjugate can be administered at a dosage of 15 kBq / kg, 20 kBq / kg, 25 kBq / kg, 30 kBq / kg, or 35 kBq / kg to achieve therapeutic efficacy.Example 16. Reduced hematological toxicity of IGF-lR-targeting radioimmunoconjugates involving cold IGF-1R antibody pre-administration

[0281] The above findings suggest that the pre-administration of cold IGF-1R antibody AVE1642 at 0.5 mg / kg prior to treatment with [225Ac]-DOTA-anti-IGF-lR conjugate shall reduce deposition of radiation to normal tissues, thereby potentially decreasing toxicities.

[0282] The hematology data collected from N=3 advanced cancer patients following administration of 0.5 mg / kg cold IGF-1R antibody (AVE1642) followed by 15 kBq / kg of [225Ac]-DOTA-anti-IGF-lR conjugate is suggestive of an improved hematological safety profile with cold antibody as a part of the dosing regimen. Due to the reduced systemic clearance of the [225Ac]-DOTA-anti-IGF-lR conjugate with cold antibody pre-dosing, the systemic exposure (area under the curve [AUC]) of 15 kBq / kg [225Ac]-DOTA-anti-IGF-lR conjugate with 0.5 mg / kg AVE1642 is several times greater than the AUC of 20 kBq / kg [225Ac]-DOTA-anti-IGF-lR conjugate and is comparable to the AUC of 40 kBq / kg [225Ac]- DOTA-anti-IGF-lR conjugate without cold antibody. Although the systemic exposure to [225Ac]-DOTA-anti-IGF-lR conjugate is comparable between the 15 kBq / kg [225Ac]-DOTA- anti-IGF-lR conjugate with 0.5 mg / kg cold antibody and the 40 kBq / kg [225Ac]-DOTA-anti- IGF-1R conjugate (no cold) dosing regimens, the hematological toxicity as determined by platelet counts over time was markedly improved in the dosing regimen which includes cold antibody pre-dosing (FIGs. 13A-13B).Example 17. Improved tumor uptake and reduced normal tissue uptake of [225Ac]-DOTA- anti-FGFR3 conjugate with cold antibody dosing in a mouse model.

[0283] The fibroblast growth factor 3 (FGFR3) targeted monoclonal antibody vofatamab is known to be cross-reactive to both human and murine FGFR3. Biodistribution studies in xenograft tumor-bearing mice were conducted to evaluate the effect of adding cold vofatamab to the dosing regimen of [177Lu]-DOTA-anti-FGFR3 conjugate.

[0284] These studies demonstrated a marked effect of the addition of cold vofatamab to the tumor uptake, the rate of blood clearance, and the normal organ biodistribution and kinetics of [177Lu]-DOTA-anti-FGFR3. In the absence of cold vofatamab, blood clearance is rapid and tumor uptake is generally low with a peak uptake of <10% ID / g. When 5 mg / kg of cold vofatamab was pre-dosed prior to [177Lu]-DOTA-anti-FGFR3, the blood kinetics were slowed substantially and the tumor uptake increased several fold with a maximum of about 40% / g at 96 h (FIG. 14) With cold vofatamab, normal organ uptake was also reduced relative to the no-cold dosing regimen. Collectively, these results suggest that the therapeutic index is substantially improved when cold vofatamab is added to the dosing regimen of [177Lu]- DOTA-anti-FGFR3.References:1. WJ Jusko. Pharmacokinetics of capacity -limited systems. J Clin Pharmacol. 29:488- 493, 1989.2. TM Ludden. Nonlinear pharmacokinetics. Clinical Pharmacokinetics. 20: 429-446, 1991.3. G Levy. Pharmacologic target-mediated drug disposition. Clin Pharmacol Ther 56:248-252, 1994.4. GH An. Small-molecule compounds exhibiting target-mediated drug disposition (TMDD): a minireview. The Journal of Clinical Pharmacology 57: 137-150, 2017.5. John G. Wagner. Direct Method of Simulating Concentration-Time Data for Michaelis-Menten Elimination. Drug Intelligence & Clinical Pharmacy, 22: 381-386, 1988.6. John G. Wagner. 1993. Pharmacokinetics for the pharmaceutical scientist. Technomic Publishing Company, Inc. Lancaster.7. XY Yan, DE Mager and W Krzyzanski. Selection between Michaelis-Menten and target-mediated drug disposition pharmacokinetic models. J Pharmacokinet Pharmacodyn. 37: 25-47, 2010.Mager, D.E. & Krzyzanski, W. Quasi-equilibrium pharmacokinetic model for drugs exhibiting target-mediated drug disposition. Pharm. Res. 22, 1589-1596 (2005). doi: 10.1007 / s 11095-005-6650-0. Gibiansky, L., Gibiansky, E., Kakkar, T. & Ma, P. Approximations of the target mediated drug disposition model and identifiability of model parameters. J. Pharmacokinet Pharmacodyn. 35, 573-591 (2008). doi:10.1007 / sl0928-008-9102-8. Dirks NL, Nolting A, Kovar A, Meibohm B. Population pharmacokinetics of cetuximab in patients with squamous cell carcinoma of the head and neck. J Clin Pharmacol. 2008;48(3): 267-78. Mould DR, Baumann A, Kuhlmann J, Keating MJ, Weitman S, Hillmen P, et al. Population pharmacokinetics-pharmacodynamics of alemtuzumab (Campath) in patients with chronic lymphocytic leukaemia and its link to treatment response. Br J Clin Pharmacol. 2007;64(3):278-91. OE Della Paschoa, JW Mandema, RA Voskuyl and M Danhof. Pharmacokinetic- Pharmacodynamic Modeling of the Anticonvulsant and Electroencephalogram Effects of Phenytoin in Rats. Journal of Pharmacology and Experimental Therapeutics February 1998, 284 (2) 460-466. Frey N, Grange S, Woodworth T. Population pharmacokinetic analysis of tocilizumab in patients with rheumatoid arthritis. J Clin Pharmacol. 2010;50(7):754-66. C. Kloft, E-U. Graefe, P. Tanswell, A. M. Scott, R. Hofheinz, A. Amelsberg & M. O. Karlsson. Population Pharmacokinetics of Sibrotuzumab, A Novel Therapeutic Monoclonal Antibody, in Cancer Patients. Investigational New Drugs 22:39-52, 2004. Ma P, Yang BB, Wang YM, Peterson M, Narayanan A, Sutjandra L, et al. Population pharmacokinetic analysis of panitumumab in patients with advanced solid tumors. J Clin Pharmacol. 2009;49(10): 1142-56. H Okamoto, NL Dirks, M Rosario, T Hori, T Hibi. Population pharmacokinetics of vedolizumab in Asian and non-Asian patients with ulcerative colitis and Crohn’s disease. Intest Res 2021;19(l):95-105. Ng CM, Stefanich E, Anand BS, Fielder PJ, Vaickus L.Pharmacokinetics / pharmacodynamics of nondepleting anti-CD4 monoclonal antibody (TRX1) in healthy human volunteers. Pharm Res. 2006;23(l):95-103. Aman P. Singh, Wojciech Krzyzanski, Steven W. Martin, Gregory Weber, Alison Betts, Alaa Ahmad, Anson Abraham, Anup Zutshi, John Lin, & Pratap Singh.Quantitative Prediction of Human Pharmacokinetics for mAbs Exhibiting Target- Mediated Disposition. AAPSJ, 2015; 17:389-399.19. AM Stein & LA Peletier. Predicting the onset of nonlinear pharmacokinetics. CPT: Pharmacometrics Syst. Pharmacol. 2018. 7: 670-677.20. Leyanis Rodriguez-Vera, Mayra Ramos-Suzarte, Eduardo Fernandez-Sanchez, Jorge Luis Soriano, Concepcion Peraire Guitart, Gilberto Castaneda Hernandez, Carlos O. Jacobo-Cabral, Niurys de Castro Suarez. Semimechanistic model to characterize nonlinear pharmacokinetics of nimotuzumab in patients with advanced breast cancer. J Clinical Pharmacology. 55: 888-898, 2015.21. Mita Kuchimanchi, Anita Grover, Maurice G. Emery, Ransi Somaratne, Scott M. Wasserman, John P. Gibbs & Sameer Doshi. Population pharmacokinetics and exposure-response modeling and simulation for evolocumab in healthy volunteers and patients with hypercholesterolemia. Journal of Pharmacokinetics and Pharmacodynamics 45:505-522, 2018.22. P Ma, B Yang , TM Wang, M Peterson, A Narayanan, L Sutjandra, R Rodriguez, A Chow. Population Pharmacokinetic Analysis of Panitumumab in Patients With Advanced Solid Tumors. J Clinical Pharmacology. 49:1142-1156, 2009.OTHER EMBODIMENTS

[0285] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a patient having cancer, wherein the method comprises administering to a patient in need thereof an effective amount of a radioimmunoconjugate or a pharmaceutically acceptable salt thereof, wherein the radioimmunoconjugate comprises a structure of Formula I:A-L-B(I) whereinA is a chelating moiety comprising a radionuclide,B is antibody or an antigen-binding fragment thereof, andL is a linker, wherein the patient is being co-administered a therapeutic dose (DT) of a cold targeting molecule, wherein the DT of said cold targeting molecule is determined by performing steps of: a) administering the cold targeting molecule to the patient at various dose levels to determine a pharmacokinetic (PK) profile of the cold targeting molecule; and b) formulating a DT of said cold targeting molecule based on the PK profile.

2. A method of treating a patient having cancer, wherein the method comprises administering to a patient in need thereof an effective amount of a radioimmunoconjugate or a pharmaceutically acceptable salt thereof, wherein the radioimmunoconjugate comprises a structure of Formula I:A-L-B(I) whereinA is a chelating moiety comprising a radionuclide,B is antibody or an antigen-binding fragment thereof, andL is a linker, wherein the patient is being co-administered a therapeutic dose (DT) of a cold targeting molecule, wherein the DT is equal to Cmaxx Vo, wherein Cmaxis the maximal concentration after bolus injection and Vcis the distribution volume of the central compartment where mixed linearand nonlinear clearance pathways are present and the bolus injection administration is occurred.

3. The method of claim 1 or 2, wherein the radioimmunoconjugate comprises225Ac.

4. The method of any one of claims 1-3, wherein the radioimmunoconjugate comprises a structure of Formula II:A-LJ-(L2)n-B(II) whereinA is a chelating moiety comprising a radionuclide;B is an antibody or an antigen-binding fragment thereof;L1is a bond, C=O, C=S, optionally substituted C1-C6alkyl, optionally substituted C1- C6heteroalkyl, or optionally substituted aryl or heteroaryl; n is an integer of 1 to 5 (inclusive); and each L2, independently, has a structure of -X'-L’-Z1-. whereinX1is -C(O)NR1-*, -NR1C(O)-*, -C(S)NR1-*, -NR1C(S)-*, -OCC(O)NR-1*, - NR1C(O)O-*, -NR1C(O)NR-1, -CH2-Ph-C(O)NR1-*. -NR1C(O)-Ph-CH2-*, -CH2-Ph- NH-C(S)NR.'-*. -NR1C(S)-NH-Ph-CH2-*, -O-, or -NR1-, wherein “*” indicates the attachment point to L3, and each R1is independently hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, or optionally substituted aryl or heteroaryl;L3is optionally substituted C1-C50alkyl or optionally substituted C1-C50heteroalkyl; andZ1is -CH2-, -C(O)-, -C(S)-, -OC(O)-#, -C(O)O-#, -NR2C(O)-#, -C(O)NR2-#, or -NR2- , wherein indicates the attachment point to B, and each R2is independently hydrogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

5. The method of any one of claims 1-4, wherein L3comprises (CH2CH2O)2-20or (CH2CH2O)2-20-C1-C6alkyl.

6. The method of any one of claims 1-5, wherein the chelating moiety is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (lR,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), DO3 AM-acetic acid (2-(4, 7,10-tris(2-amino-2-oxoethyl)- 1,4, 7,10- tetraazacyclododecan-l-yl)acetic acid), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10- tetra(methylene phosphonic acid)), DOTA-4AMP (1,4,7, 10-tetraazacy clododecane-1, 4, 7, 10- tetrakis(acetamido-methylenephosphonic acid), NOTA (1,4,7-triazacyclononane-1,4,7- triacetic acid), and HP-DO3A (10-(2 -hy droxypropyl)- 1,4, 7-tetraazacy clododecane-1, 4,7- triacetic acid).

7. The method of any one of claims 1-6, wherein the radionuclide is selected from the group consisting of44Sc,47Sc,55Co,60Cu,61Cu,62Cu,64Cu,67Cu,66Ga,67Ga,68Ga,82Rb,86Y,87Y,89Zr,90Y,97RU,99TC,99mTc,105Rh,109Pd, '" in.117mSn,149Pm,149Tb,153Sm,166HO,177LU,186Re,188Re,198Au,199AU,201T1,203Pb,211At,212Pb,212BI,213BI,223Ra,225Ac,227Th, and229Th.

8. The method of claim 3, wherein:A is DOTA comprising a radionuclide;B is a human or humanized IgG antibody or an antigen-binding fragment thereof;L1is a bond or C1-C6alkyl; n is 1; andL2has the structure:wherein:X1is -C(O)NR1-*, “*” indicating the attachment point to L3, and R1is H or C1-C6alkyl;L3is (CH2CH2O)m(CH2)w, and m and w are independently an integral between 0 and 10 (inclusive) and at least one of m and w is not 0; andZ1is -C(O)-.

9. The method of any one of claims 1-8, wherein the radioimmunoconjugate comprises the following structure:

10. The method of any one of claims 1-9, wherein the antibody is a monoclonal antibody or a bispecific antibody.

11. The method of any one of claims 1-10, wherein the antibody is selected from the group consisting of an IGF-1R antibody, an FGFR3 antibody, a TEM-1 antibody, a Claudin 18.2 antibody, and a CA9 antibody.

12. The method of any one of claims 1-11, wherein the cold targeting molecule is a cold antibody.

13. The method of any one of claims 1 or 3-12, wherein the PK profde is characterized by a one- or two-compartment PK model having either a nonlinear only clearance pathway or mixed linear and nonlinear clearance pathways.

14. The method of any one of claims 1 or 3-12, wherein step (b) for formulating a DT of said cold targeting molecule comprises:(b)(i) obtaining parameters of the PK profde, wherein the parameters comprise Michaelis-Menten constant (KM), maximal velocity (VM), first- order / linear clearance rate (Cl), and central volume of distribution (Vc);(b)(ii) determining a critical concentration (Ccrit) based on the parameters, wherein the PK profde of the cold targeting molecule is linear before Ccrit; and (b)(iii) formulating a DT of the cold targeting molecule based on Ccritto reach a desired clinical endpoint.

15. The method of claim 14, wherein the PK profde shows a nonlinear clearance pathway featuring empirical Michaelis-Menten (M-M) equation, and Ccritis determined according to formula VM / CI.

16. The method of claim 15, wherein the PK profile shows a nonlinear clearance pathway which is characterized by the full target mediated drug disposition (TMDD) model, and Ccritis determined according to formula Ksyn / (C1 / Vo), wherein Ksynis the synthesis rate of a target that said cold targeting molecule bonds to.

17. The method of claim 14, wherein the PK profile shows a nonlinear clearance pathway which is characterized by the full target mediated drug disposition (TMDD) model, and Ccritis determined according to formula KintxTtot / (Cl / Vc), wherein Kintis the rate constant of internalization of drug-target complex with a unit of 1 / time, and Ttotis the total receptor concentration.

18. The method of claim 14, wherein the desired clinical endpoint includes a time to reach Ccrit, denoted as Tcrit, that is determined according to the following equation:wherein Vcand Cl are parameters of a one-compartment PK model.

19. The method of claim 14, wherein the desired clinical endpoint includes a time to reach Ccrit, denoted as Tcrit, that is determined according to the following equation:when Ccritis three times or more greater than KM, wherein Vcand Cl are parameters determined by a one-compartment PK model.

20. The method of claim 14, wherein the desired clinical endpoint includes a time to reach Ccrit, denoted as Tcrit, that is determined according to the following equation:when Ccritis three times or more greater than KM, wherein A, B, α, β, and kelare parameters determined by a two-compartment PK model.

21. The method of any one of claims 1 or 3-12, wherein step (b) for formulating a DT of said cold targeting molecule comprises:c) obtaining parameters of the PK profile, wherein the parameters comprise Michaelis-Menten constant (KM), maximal velocity (VM), first-order / linear clearance rate (Cl), central volume of distribution (Vc); d) determining a critical concentration (Ccrit) and a transitional concentration (Ctrans) based on the parameters, wherein the PK profile of the cold targeting molecule is linear both before Ccritand after KM / 3, and exhibits a transition at Ctransbetween Ccritand KM / 3; and e) formulating a DT of the cold targeting molecule based on Ctransto reach a desired clinical endpoint.

22. The method of claim 21, wherein the PK profile shows a nonlinear clearance pathway featuring empirical Michaelis-Menten (M-M) equation, and Ctransis determined according to formula sqrt(KMx VM / C1).

23. The method of claim 21, wherein the PK profile shows a nonlinear clearance pathway featuring target mediated drug disposition (TMDD), and Ctransis determined according to formula sqrt(KMx VM / C1), wherein KM= Koff / Konand VM x Vc, Kint×Ttotwhere Konis the forward rate constant of drug-target binding with a unit of l / concentration / time; Koffis the reverse rate constant of drug-target binding with a unit of I / time; Kintis the rate constant of internalization of drug-target complex with a unit of I / time, and Ttotis the total receptor concentration.

24. The method of claim 21, wherein the PK profile shows a nonlinear clearance pathway featuring target mediated drug disposition (TMDD), and Ctransis determined according to formula sqrt(KMx VM / C1), wherein KM= (KoffHCint) / Konand VM = Ksynx Vc, where Konis the forward rate constant of drug-target binding with a unit of l / concentration / time; Koffis the reverse rate constant of drug-target binding with a unit of I / time; Kintis the rate constant of internalization of drug-target complex with a unit of I / time, and Ksynis the target synthesis rate constant.

25. The method of claim 21, wherein the desired clinical endpoint includes a time to reach Ctrans, denoted as Ttrans, that is determined according to the following equation:

26. The method of claim 21, wherein the DT of the cold targeting molecule is formulated to reach Ctransat a pre-determined time point (Ttrans), wherein DT is determined according to the following equation: DT = Cmaxx Vc.

27. The method of claim 26, wherein the desired clinical endpoint includes a time to reach Ctrans, denoted as Ttrans, that is determined according to the following equation:wherein Ttransis equal to the pre-determined time point.

28. The method of any one of claims 25-27, wherein the pre-determined time point or Ttransis 7, 14, 21, or 28 days or longer.

29. The method of claim 21, wherein the DT of the cold targeting molecule is formulated to reach a plasma concentration (Cp) that is greater than Ctransfor a pre-determined duration of time (Tdur), wherein DT is determined according to the following equation: DT = CmaxX Vc-30. The method of claim 29, wherein the desired clinical endpoint includes a time to reach Ctrans, denoted as Ttrans, that is determined according to the following equation:wherein Ttransis greater than Tdur.

31. The method of any one of claims 1-30, wherein the co-administration of said cold targeting molecule at the DT results in an improvement of the therapeutic index of the radioimmunoconj ugate.

32. The method of claim 31, wherein the improvement of the therapeutic index comprises increase of tumor uptake of the radioimmunoconjugate, increase of tumor-to- background ratio, decrease of radioimmunoconjugate uptake in non-target tissues, reduction of hematological toxicity, or a combination thereof.

Images