RADIOIMMUNOCONJUGATES AND CHECKPOINT INHIBITOR COMBINATION THERAPY

Abstract

Combination therapies comprising administering radioimmunoconjugates and one or more checkpoint inhibitors.

Claims

1. A method of inducing an immune response to a tumor in a mammal, said method comprising: (i) administering to the mammal a radioimmunoconjugate, wherein the mammal has received or is receiving one or more checkpoint inhibitors; (ii) administering to the mammal one or more checkpoint inhibitors, wherein the mammal has received or is receiving a radioimmunoconjugate; or (iii) administering the mammal one or more checkpoint inhibitors at the same time as administering the mammal a radioimmunoconjugate, wherein: the radioimmnoconjugate has the structure of Formula I-b-1, or a pharmaceutically acceptable salt thereof: ##STR00007## wherein A is a metal complex of a chelating moiety, wherein said chelating moiety is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α′′ a‴-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-l,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-D03A (hydroxypropyltetraazacyclododecanetriacetic acid), wherein the metal of said metal complex is a radionuclide selected from the group consisting of .sup.47Sc, .sup.55Co, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.82Rb, .sup.86Y, .sup.87Y, .sup.89Zr, .sup.90Y, .sup.97Ru, .sup.99Tc, .sup.99mTc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.117mSn, .sup.149Pm, .sup.149Tb, .sup.153Sm, .sup.166Ho, .sup.177Lu.sub.,.sup.186Re, .sup.188Re, .sup.198Au, .sup.199Au, .sup.201Tl, .sup.203Pb, .sup.211At, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.225Ac, .sup.227Th, and .sup.229Th; L.sub.1 is optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl; L.sup.2 has the structure of Formula II: ##STR00008## wherein X.sup.1 is C═.sub.O(NR.sup.1) or NR.sup.1, in which R.sup.1 is H or optionally substituted C.sub.1— C.sub.6alkylor optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted aryl or heteroaryl; L.sup.3is optionally substituted C.sub.1-C.sub.50 alkyl or optionally substituted C.sub.1-C.sub.50 heteroakyl; and Z.sup.1 is CH.sub.2, C═O, C═S, OC═O,NR.sup.1C═O, or NR.sup.1, in which R.sup.1 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or pyrrolide-2,5-dione, and B is a human or humanized IgG antibody or an antigen-binding fragment thereof.

2. The method of claim 1, said method comprising administering to a mammal one or more checkpoint inhibitors, wherein the mammal has received or is receiving the radioimmunoconjugate.

3. The method of claim 1, wherein the one or more checkpoint inhibitors or the radioimmunoconjugate is administered in a lower effective dose.

4-5. (canceled)

6. The method of claim 1, wherein the human or humanized IgG antibody or antigen-binding fragment thereof is capable of binding to a tumor-associated antigen.

7-8. (canceled)

9. The method of claim 8, wherein the human or humanized IgG antibody or antigen-binding fragment thereof is an IGF-1R antibody or an antigen-binding fragment thereof.

10-12. (canceled)

13. The method of claim 1, wherein the radionuclide is an alpha emitter.

14. The method of claim 13, wherein the radionuclide is an alpha emitter selected from the group consisting of Astatine-211 (.sup.211At), Bismuth-212 (.sup.212Bi), Bismuth-213 (.sup.213Bi), Actinium-225 (.sup.225Ac), Radium-223 (.sup.223Ra), Lead-212 (.sup.212Pb), Thorium-227 (.sup.227Th), and Terbium-149 (.sup.149Tb).

15. The method of claim 14, wherein the radionuclide is .sup.225Ac.

16. The method of claim 1, wherein the radioimmunoconjugate comprises the following structure: ##STR00009## wherein B is the targeting moiety.

17. The method of claim 1,wherein the one or more checkpoint inhibitors comprise a PD-1 inhibitor.

18. (canceled)

19. The method of claim 1,wherein the one or more checkpoint inhibitors comprise an CTLA-4 inhibitor.

20. (canceled)

21. The method of claim 1, wherein the one or more checkpoint inhibitors comprises both a PD-1 inhibitor and a CTLA-4 inhibitor.

22. The method of claim 1, wherein the mammal is a human.

23. The method of claim 1, wherein the mammal is diagnosed with cancer.

24. The method of claim 23, wherein the cancer is selected from the group comprising: breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing’s Sarcoma, multiple myeloma, or acute myeloid leukemia.

25. (canceled)

26. The method of claim 1, wherein said administering results in a therapeutic effect.

27. The method of claim 26, wherein the targeting moiety is capable of binding to a tumor-associated antigen, and said therapeutic effect comprises an increase in T cells specific for the tumor-associated antigen.

28-29. (canceled)

30. The method of claim 27, wherein said administering results in at least 15% of the total T cell population in a sample from the mammal being specific for the tumor-associated antigen.

31-41. (canceled)

42. The method of claim 30, wherein the sample is a tumor sample.

43. The method of claim 26, wherein said therapeutic effect comprises (a) a decrease in tumor volume, a stable tumor volume, or a reduced rate of increase in tumor volume or (b) a decreased incidence of recurrence or metastasis.

44. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1 illustrates relative tumor volume in the CT26 syngeneic mouse tumor model after treatment with various checkpoint inhibitors.

[0106] FIG. 2 illustrates [.sup.177Lu]-FPI-1755 biodistribution in the CT-26 syngeneic mouse tumor model.

[0107] FIG. 3 illustrates the enhanced efficacy of [.sup.225Ac]-FPI-1792 in immunocompetent mice vs. immunodeficient mice.

[0108] FIG. 4 illustrates synergy between [.sup.225Ac]-FPI-1792 and α-CTLA-4/PD-1 treatment in the CT26 syngeneic mouse model.

[0109] FIG. 5 illustrates the development of protective immunity in [.sup.225Ac]-FPI-1792 (“[.sup.225Ac]-FPI-TAT” in the graph labels) treated mice upon CT26 re-challenge.

[0110] FIG. 6 illustrates cytokine response and T-cell recruitment after [.sup.225Ac]-FPI-1792 treatment.

[0111] FIG. 7 illustrates “humanized” IGF-1R model development.

[0112] 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 apparatuses, systems, and 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.

DETAILED DESCRIPTION

[0113] The present disclosure relates to combination therapies for inducing or improving an immune response to cancer using radioimmunoconjugates and checkpoint inhibitors. In some embodiments, use of methods disclosed herein results in treatment or amelioration of cancer.

[0114] In some embodiments, a lower effective dose of the radioimmunoconjugate and/or of the checkpoint inhibitor is used.

[0115] Radiolabelled targeting moieties (also known as radioimmunoconjugates) are designed to target a protein or receptor that is upregulated in a disease state and/or specific to diseased cells (e.g., tumor cells) to deliver a radioactive payload to damage and kill cells of interest. “Radioimmunotherapy” refers to this therapy when the targeting moiety comprises an antibody, typically a monoclonal antibody. Radioactive decay of the payload produces an alpha, beta, or gamma particle or Auger electron that can cause direct effects to DNA (such as single or double stranded DNA breaks) or indirect effects such as by-stander or crossfire effects.

[0116] Radioimmunoconjugates typically contain a biological targeting moiety (e.g., an antibody or antigen binding fragment thereof that specifically binds to a molecule expressed on or by a tumor, e.g., IGF-1R or TEM-1/endosialin), a chelating moiety or a metal complex of a chelating moiety (e.g., comprising a radioisotope), and a linker. Conjugates may be formed by appending a bifunctional chelate to the biological targeting molecule so that structural alterations are minimal while maintaining target affinity. A radioimmunoconjugate may be formed by radiolabelling such a conjugate.

[0117] Bifunctional chelates structurally contain a chelate, a linker, and a cross-linking group. When developing new bifunctional chelates, most efforts focus around 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].

Radioimmunoconjugates

[0118] Radioimmunoconjugates suitable for use in accordance with the present disclosure generally have the structure of Formula I-a:

##STR00003##

[0119] wherein A is a chelating moiety or metal complex thereof), [0120] wherein B is a targeting moiety, and [0121] wherein L is a linker.

[0122] In some embodiments, the radioimmunoconjugate comprises the following structure:

##STR00004##

wherein B is the targeting moiety.

Targeting Moieties

[0123] Targeting moieties include any molecule or any part of a molecule that is capable of binding to a given target. In some embodiments, the targeting moiety comprises a protein or polypeptide. In some embodiments, the targeting moiety 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

[0124] 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 (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).

[0125] Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. 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., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0126] The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include a Fab fragment, a F(ab′).sub.2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR). 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.

[0127] Antibodies or fragments described herein can be produced by any method known in the art for the synthesis of antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.

[0128] 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).

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

[0129] Insulin-like growth factor 1 receptor is a transmembrane protein found on the surface of human cells activated by insulin-like growth factor 1 (IGF-1) and 2 (IGF-2). In some embodiments, radioimmunoconjugates comprise antibodies against insulin-like growth factor-1 receptor (IGF-1R). Although not a typical oncogene, IGF-1R promotes initiation and progression of cancer, playing a critical role in mitogenic transformation and maintenance of the transformed phenotype. IGF-1R has been associated with development of multiple common cancers including breast, lung (e.g., non-small lung), liver, prostate, pancreas, ovarian, colon, melanoma, adrenocortical carcinoma, and various types of sarcomas. IGF-1R signaling stimulates tumour cell proliferation and metabolism, supports angiogenesis, and confers protection from apoptosis. It affects metastatic factors (e.g., HIF-1 dependent hypoxia signaling), anchorage independent growth, as well as growth and survival of tumour metastases after extravasation. IGF-1R has also been implicated in the development, maintenance and enrichment of therapeutic resistant cancer stem cell populations.

[0130] Despite the abundance of data implicating IGF-1R’s role in cancer, therapeutics targeting IGF-1R have yet to demonstrate a significant impact on disease. There has been much speculation for this lack of efficacy including the inability to identify appropriate biomarkers for patient identification, complexity and interdependency of the IGF-1/IR signaling pathway and the development of other growth hormone compensatory mechanisms [Beckwith and Yee, Mol Endocrinol, November 2015, 29(11):1549-1557]. Radioimmunotherapy, however, may provide a viable mechanism for treating cancers overexpressing the IGF-1 receptor by utilizing the ability of IGF-1R to undergo antibody triggered internalization and lysosomal degradation to deliver targeted radioisotopes inside cancer cells. Internalization and lysosomal degradation of an IGF-1R targeted radioimmunoconjugate prolongs the residence time of the delivered radioisotope inside cancer cells, thereby maximizing the potential for a cell killing emission to occur. In the case of actinium-225, which yields 4 alpha particles per decay chain, cell death can be accomplished by as little as 1 atom of radionuclide delivered per cell [Sgouros, et al. J Nucl Med. 2010, 51:311-2]. Cell killing due to direct DNA impact and breakage by an alpha particle may occur in the targeted cell or in a radius of 2 or 3 non-targeted cells for a given alpha particle decay. In addition to having very high potential anti-tumour potency, IGF-1R targeted radioimmunoconjugates may not generate mechanistic resistance as they do not rely on blocking ligand binding to the receptor to inhibit the oncologic process, as needed with a therapeutic antibody.

[0131] Several IGF-1R antibodies have been developed and investigated for the treatment of various types of cancers, including figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. After binding to IGF-1R, these antibodies are internalized into the cell and degraded by lysosomal enzymes. The combination of overexpression on tumor cells and internalization offers the possibility of delivering detection agents directly to the tumor site while limiting the exposure of normal tissues to toxic agents.

[0132] The CDRs of the light chain variable region of AVE1642 have the sequences:

TABLE-US-00001 SEQ ID NO: 1 (CDR-L1) RSSQSIVHSNVNTYLE

TABLE-US-00002 SEQ ID NO: 2 (CDR-L2) KVSNRFS

TABLE-US-00003 SEQ ID NO: 3 (CDR-L3) FQGSHVPPT

[0133] The light chain variable region of AVE1642 has the sequence:

TABLE-US-00004 SEQ ID NO: 4 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTY LEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAED LGIYYCFQGSHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAK

[0134] The CDRs of the heavy chain variable region of AVE1642 have the sequences:

TABLE-US-00005 SEQ ID NO: 5 (CDR-H1) SYWMH

TABLE-US-00006 SEQ ID NO: 6 (CDR-H2) GEINPSNGRTNY NQKFQG

TABLE-US-00007 SEQ ID NO: 7 (CDR-H3) GRPDYYGSSKWY FDV

[0135] The heavy chain variable region of AVE1642 has the sequence:

TABLE-US-00008 SEQ ID NO: 8 QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWV KQRPGQGLEWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLT SEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALG

Endosialin (TEM-1) Antibodies

[0136] Endosialin, also known as TEM-1 or CD-248, is an antigen expressed by tumor-associated endothelial cells, stromal cells, and pericytes.

[0137] Examples of endosialin antibodies include hMP-E-8.3 (disclosed in WO 2017/134234, the entire contents of which are incorporated by reference herein) and ontuxizumab (MORAb-004).

[0138] In some embodiments, the endosialin antibody or antigen-binding fragment thereof recognizes an epitope having an amino acid sequence of SRDHQIPVIAAN (SEQ ID NO: 9).

[0139] In some embodiments, the heavy chain variable region of the endosialin antibody or antibody-binding fragment thereof comprises the complementarity determining regions (CDRs) having the following sequences:

TABLE-US-00009 CDR-H1: GYGVN (SEQ ID NO: 10) or GFSLTGYGVN (SEQ I D NO: 11)

TABLE-US-00010     CDR-H2: MIWVDGSTDYNSALKS (SEQ ID NO: 12)

TABLE-US-00011     CDR-H3: GGYGAMDY (SEQ ID NO: 13)

[0140] In some embodiments, the light chain variable region of the endosialin antibody or antibody-binding fragment thereof comprises the complementarity determining regions (CDRs) having the following sequences:

TABLE-US-00012     CDR-L1: HASQNINVWLT (SEQ ID NO: 14)

TABLE-US-00013     CDR-L2: KASNLHT (SEQ ID NO: 15)

TABLE-US-00014     CDR-L3: QQGQSYPWT (SEQ ID NO: 16)

[0141] In some embodiments, the endosialin antibody or antigen-binding fragment thereof is a humanized antibody.

[0142] In some embodiments, the heavy chain variable region of the endosialin antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, or 20: [0143] Humanized VH1:

TABLE-US-00015 QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGM IWVDGSTDYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSS(SEQ ID NO: 17)

[0144] Humanized VH2:

TABLE-US-00016 QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPEKGLEWIGM IWVDGSTDYNSALKSRVNISVDTSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSS(SEQ ID NO: 18)

[0145] Humanized VH3:

TABLE-US-00017 QLQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGM IWVDGSTDYNSALKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSS(SEQ ID NO: 19)

[0146] Humanized VH4:

TABLE-US-00018 QLQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPEKGLEWIGM IWVDGSTDYNSALKSRVNISVDKSKNQFSLKLSSVTAADTAVYYCARGGY GAMDYWGQGTLVTVSS(SEQ ID NO: 20)

[0147] In some embodiments, the light chain variable region of the endosialin antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, or 24: [0148] Humanized VL1:

TABLE-US-00019 DIQMTQSPSSVSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYK ASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPWTFGG GTKLEIK(SEQ ID NO: 21)

[0149] Humanized VL2:

TABLE-US-00020 DIQMTQSPSTLSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYK ASNLHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGQSYPWTFGG GTKLEIK(SEQ ID NO: 22)

[0150] Humanized VL3:

TABLE-US-00021 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLTWYQQKPGKAPKLLIYK ASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQSYPWTFGG GTKLEIK(SEQ ID NO: 23)

[0151] Humanized VL4:

TABLE-US-00022 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLTWYQQKPEKAPKSLIYK ASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPWTFGG GTKLEIK(SEQ ID NO: 24)

Nanobodies

[0152] Nanobodies are antibody fragments consisting of a single monomeric variable antibody domain. Nanobodies may also be referred to as single-domain antibodies. Like antibodies, nanobodies bind selectively to a specific antigen. Nanobodies may be heavy-chain variable domains or light chain domains. Nanobodies may occur naturally or be the product of biological engineering. Nanobodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

Affibodies

[0153] Affibodies are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may be considered to mimic certain functions of antibodies. Affibodies may be engineered variants of the B-domain in the immunoglobulin-binding region of staphylococcal protein A. Affibodies may be engineered variants of the Z-domain, a B-domain that has lower affinity for the Fab region. Affibodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

[0154] Affibody molecules showing specific binding to a variety of different proteins (e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2 and EGFR) have been generated, demonstrating affinities (K.sub.d) in the .Math.M to pM range.

Fibronectin Type III Domains

[0155] The Fibronectin type III domain is an evolutionarily conserved protein domain found in a wide-variety of extracellular proteins. The Fibronectin type III domain has been used as a molecular scaffold to produce molecules capable of selectively binding a specific antigen. Variants of the Fibronectin type III domains (FN3) that have been engineered for selective-binding may also be referred to as monobodies. FN3 domains may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., CIS-display, phage display, yeast display, bacterial display, mRNA display, ribosome display).

Modified Polypeptides

[0156] Polypeptides used in accordance with the disclosure may have a modified amino acid sequence. Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the reference polypeptide). In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g., binding to IGF-1R or to endosialin). 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.

[0157] 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.

[0158] A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy-terminus of a polypeptide 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).

[0159] Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH.sub.2(CH.sub.2).sub.nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

[0160] Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated “exemplary substitutions” in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened.

TABLE-US-00023 Amino acid substitutions Original residue Exemplary substitution Conservative substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, GIn, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu

[0161] Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

Chelating Moieties and Metal Complexes Thereof

Chelating Moieties

[0162] Examples of suitable chelating moieties include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α‴-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2ʹ,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N‴,N⁗-pentaacetic acid), H.sub.4octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H.sub.2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H.sub.6phospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N‴,N‴-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones), or porphyrins.

[0163] In some embodiments, radioimmunoconjugates 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 4,000 keV), such as any of the radioisotopes and radionuclides discussed herein..sup.2, .sup.2 2,

[0164] In some embodiments, chelating moieties are useful as detection agents, and radioimmunoconjugates comprising such detectable chelating moieties can therefore be used as diagnostic or theranostic agents.

Radioisotopes and Radionuclides

[0165] In some embodiments, the metal complex comprises a radionuclide. Examples of suitable radioisotopes and radionuclides include, but are not limited to, .sup.3H, .sup.14C, .sup.15N, .sup.18F, .sup.35S, .sup.47Sc, .sup.55Co, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.66Ga, .sup.67Ga, .sup.67Cu, .sup.68Ga, .sup.75Br, .sup.76Br, .sup.77Br, .sup.82Rb, .sup.89Zr, .sup.86Y, .sup.87Y, .sup.90Y, .sup.97Ru, .sup.99Tc, .sup.99mTc, .sup.105Rh, .sup.109Pd, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.149Pm, .sup.149Tb, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.117mSn, .sup.186Re, .sup.188Re, .sup.198Au, .sup.199Au, .sup.201Tl, .sup.203Pb, .sup.211At, .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.223Ra, .sup.225Ac, .sup.227Th, and .sup.229Th.

[0166] In some embodiments, the radionuclide is an alpha emitter, e.g., Astatine-211 (.sup.211At), Bismuth-212 (.sup.212Bi), Bismuth-213 (.sup.213Bi), Actinium-225 (.sup.225Ac), Radium-223 (.sup.223Ra), Lead-212 (.sup.212Pb), Thorium-227 (.sup.227Th), or Terbium-149 (.sup.149Tb).

Linkers

[0167] In some embodiments, the linker is as shown within the structure of Formula I-b, as that part of Formula I-b absent A and B:

##STR00005##

(A and B are as defined in Formula I-a.)

[0168] Thus, in some embodiments, the linker is —L.sup.1—(L.sup.2).sub.n—, [0169] wherein L.sup.1 is optionally substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl; [0170] n is 1-5; and [0171] each L.sup.2, independently, has the structure: [0172] wherein is X.sup.1 is C═O(NR.sup.1), C═S(NR.sup.1), OC═O(NR.sup.1), NR.sup.1C═O(O), NR.sup.1C═O(NR.sup.1), CH.sub.2PhC═O(NR.sup.1), —CH.sub.2Ph(NH)C═S(NR.sup.1), O, or NR.sup.1; and each R.sup.1 independently is H or optionally substituted C.sub.1-C.sub.6 alkyl or optionally substituted C.sub.1-C.sub.6 heteroalkyl, substituted aryl or heteroaryl, in which C.sub.1-C.sub.6 alkyl can be substituted by oxo (═O), heteroaryl, or a combination thereof; [0173] L.sup.3 is optionally substituted C.sub.1-C.sub.50 alkyl or optionally substituted C.sub.1-C.sub.50 heteroalkyl or C.sub.5-C.sub.20 polyethylene glycol; Z.sup.1 is CH.sub.2, C═O, C═S, OC═O, NR.sup.1C═O, NR.sup.1 and R.sup.1 is a hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, pyrrolidine-2,5-dione.

Cross-Linking Groups

[0174] In some embodiments, radioimmunoconjugates comprise a cross-linking group instead of or in addition to the targeting moiety or therapeutic moiety (e.g., B in Formula I comprises a cross-linking group).

[0175] A cross-linking group is a reactive group that is able to join two or more molecules by a covalent bond. Cross-linking groups may be used to attach the linker and chelating moiety to a therapeutic or targeting moiety. Cross-linking groups may also be used to attach the linker and chelating moiety to a target in vivo. In some embodiments, the cross-linking group is an amino-reactive, methionine reactive or thiol-reactive cross-linking group, or a sortase-mediated coupling. In some embodiments, the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine. In some embodiments, the sortase recognition sequence may comprise of a terminal glycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence, where X is any amino acid. A person having ordinary skill in the art will understand that the use of cross-linking groups is not limited to the specific constructs disclosed herein, but rather may include other known cross-linking groups.

Checkpoint Inhibitors

[0176] In some embodiments, a checkpoint inhibitor is co-administered with a radioimmunoconjugate. Generally, suitable checkpoint inhibitors inhibit an immune suppressive checkpoint protein. In some embodiments, the checkpoint inhibitor inhibits a protein selected from the group consisting of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG-3, T cell immunoglobulin mucin 3 (TIM-3), and killer immunoglobulin-like receptors (KIRs).

[0177] For example, in some embodiments, the checkpoint inhibitor is capable of binding to CTLA-4, PD-1, or PD-L1. In some embodiments, the checkpoint inhibitor interferes with the interaction (e.g., interferes with binding) between PD-1 and PD-L1.

[0178] In some embodiments, the checkpoint inhibitor is a small molecule.

[0179] In some embodiments, the checkpoint inhibitor is an antibody or antigen-binding fragment thereof, e.g., a monclonal antibody. In some embodiments, the checkpoint inhibitor is a human or humanized antibody or antigen-binding fragment thereof. In some embodiments, the checkpoint inhibitor is a mouse antibody or antigen-binding fragment thereof.

[0180] In some embodiments, the checkpoint inhibitor is a CTLA-4 antibody. Non-limiting examples of CTLA-4 antibodies include BMS-986218, BMS-986249, ipilimumab, tremelimumab (formerly ticilimumab, CP-675,206), MK-1308, and REGN-4659. An additional example of a CTLA-4 antibody is 4F10-11, a mouse monoclonal antibody.

[0181] In some embodiments, the checkpoint inhibitor is a PD-1 antibody. Non-limiting examples of PD-1 antibodies include camrelizumab, cemiplumab, nivolumab, pembrolizumab, sintilimab, tislelizumab and toripalimab. An additional example of a PD-1 antibody is RMP1-14, a mouse monoclonal antibody.

[0182] In some embodiments, the checkpoint inhibitor is a PD-L1 antibody. Non-limiting examples of PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

[0183] In some embodiments, a combination of more than one checkpoint inhibitor is used. For example, in some embodiments, both a CTLA-4 inhibitor and a PD-1 or PD-L1 inhibitor is used.

Subjects

[0184] In some disclosed methods, a therapy (e.g., comprising a therapeutic agent) is administered to a subject. In some embodiments, the subject is a mammal, e.g., a human.

[0185] In some embodiments, the subject has received or is receiving another therapy. For example, in some embodiments, the subject has received or is receiving a radioimmunoconjugate. In some embodiments, the subject has received or is receiving a checkpoint inhibitor.

[0186] In some embodiments, the subject has cancer or is at risk of developing cancer. For example, the subject may have been diagnosed with cancer. The cancer may be a primary cancer or a metastatic cancer. Subjects may have any stage of cancer, e.g., stage I, stage II, stage III, or stage IV with or without lymph node involvement and with or without metastases. Provided compositions may prevent or reduce further growth of the cancer and/or otherwise ameliorate the cancer (e.g., prevent or reduce metastases).In some embodiments, the subject does not have cancer but has been determined to be at risk of developing cancer, e.g., because of the presence of one or more risk factors such as environmental exposure, presence of one or more genetic mutations or variants, family history, etc. In some embodiments, the subject has not been diagnosed with cancer.

[0187] In some embodiments, the cancer is a solid tumor.

[0188] In some embodiments, the solid tumor cancer is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing’s Sarcoma, multiple myeloma, or acute myeloid leukemia.

[0189] In some embodiments, the cancer is a non-solid (e.g., liquid (e.g., hematologic)) cancer.

Administration and Dosage

Effective and Lower Effective Doses

[0190] The present disclosure provides combination therapies in which the amounts of each therapeutic may or may not be, on their own, therapeutically effective. For example, provided are methods comprising administering a first therapy and a second therapy in amounts that together are effective to treat or ameliorate a disorder, e.g., cancer. In some embodiments, at least one of the first and second therapy is administered to the subject in a lower effective dose. In some embodiments, both the first and the second therapies are administered in lower effective doses.

[0191] In some embodiments, the first therapy comprises a radioimmunoconjugate and the second therapy comprises a checkpoint inhibitor.

[0192] In some embodiments, the first therapy comprises a checkpoint inhibitor and the second therapy comprises a radioimmunoconjugate.

[0193] In some embodiments, therapeutic combinations as disclosed herein are administered to a subject in a manner (e.g., dosing amount and timing) sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. In the context of a single therapy (a “monotherapy”), 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. The “therapeutically effective amount” typically varies depending on the therapeutic. For known therapeutic agents, the relevant therapeutically effective amounts may be known to or readily determined by those of skill in the art.

[0194] For example, in the treatment of cancer, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual. For example, a treatment may be therapeutically effective if it causes a cancer to regress or to slow the cancer’s growth.

[0195] The dosage regimen (e.g., amounts of each therapeutic, relative timing of therapies, etc.) that is effective for these uses may depend on the severity of the disease or condition and the weight and general state of the subject. For example, the therapeutically effective amount of a particular composition comprising a therapeutic agent applied to mammals (e.g., humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain conjugates of the present disclosure exhibit an enhanced ability to target cancer cells and residualize, the dosage of these compounds can be lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose of required for a therapeutic effect of the unconjugated agent. Therapeutically effective and/or optimal amounts can also be determined empirically by those of skill in the art. Thus, lower effective doses can also be determined by those of skill in the art.

[0196] Single or multiple administrations of a composition (e.g., a pharmaceutical composition comprising a therapeutic agent) can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

[0197] In the disclosed combination therapy methods, the first and second therapies may be administered sequentially or concurrently to a subject. For example, a first composition comprising a first therapeutic agent and a second composition comprising a second therapeutic agent may be administered sequentially or concurrently to a subject. Alternatively or additionally, a composition comprising a combination of a first therapeutic agent and a second therapeutic agent may be administered to the subject.

[0198] In some embodiments, the radioimmunoconjugate is administered in a single dose. In some embodiments, the radioimmunoconjugate is administered more than once. When the radioimmunoconjugate is administered more than once, the dose of each administration may be the same or different.

[0199] In some embodiments, the checkpoint inhibitor is administered in a single dose. In some embodiments, the checkpoint inhibitor is administered more than once, e.g., at least twice, at least three times, etc. In some embodiments, the checkpoint inhibitor is administered multiple times according to a regular or semi-regular schedule, e.g., once every approximately two weeks, once a week, twice a week, three times a week, or more than three times a week. When the checkpoint inhibitor is administered more than once, the dose of each administration may be the same or different. For example, the checkpoint inhibitor may be administered in an initial dose amount, and then subsequent dosages of the checkpoint inhibitor may be higher or lower than the initial dose amount.

[0200] In some embodiments, the first dose of the checkpoint inhibitor is administered at the same time as the first dose of the radioimmunoconjugate. In some embodiments, the first dose of the checkpoint inhibitor is administered before the first dose of radioimmunoconjugate. In some embodiments, the first dose of the checkpoint inhibitor is administered after the first dose of radioimmunoconjugate. In some embodiments, subsequent doses of the checkpoint inhibitor are administered.

[0201] In some embodiments, radioimmunoconjugates (or a composition thereof) and checkpoint inhibitors (or a composition thereof) are administered within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day(s)) of each other.

[0202] In some embodiments, radioimmunoconjugates (or a composition thereof) and checkpoint inhibitors (or a composition thereof) are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)) of each other. In various embodiments the checkpoint inhibitor is administered at the same time as radioimmunoconjugate. In various embodiments, the checkpoint inhibitor is administered multiple times after the first administration of radioimmunoconjugate.

[0203] In some embodiments, compositions (such as compositions comprising radioimmunoconjugates) are administered for radiation treatment planning or diagnostic purposes. When administered for radiation treatment planning or diagnostic purposes, compositions may be administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose. In some embodiments, a first dose of disclosed conjugate or a composition (e.g., pharmaceutical composition) thereof is administered in an amount effective for radiation treatment planning, followed administration of a combination therapy including a conjugate as disclosed herein and another therapeutic.

[0204] Pharmaceutical compositions comprising one or more agents (e.g., radioimmunoconjugates and/or checkpoint inhibitors) can be formulated for use in accordance with disclosed methods and systems in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Examples of suitable formulations are found in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

Formulations

[0205] Pharmaceutical compositions may be formulated for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. Pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Examples of additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Also specifically contemplated are sustained release administration, by such means as depot injections or erodible implants or components. Suitable compositions include compositions comprising include agents (e.g., compounds as disclosed herein) dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others, e.g., for parenteral administration. Compositions may contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others. In some embodiments, compositions are formulated for oral delivery; for example, compositions may contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule. In some embodiments, compositions are formulated for local administration; for example, compositions may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.

[0206] Compositions may be sterilized, e.g., by conventional sterilization techniques, or sterile filtered. Aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5. In some embodiments, compositions in solid form are packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. In some embodiments, compositions in solid form are packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

Effects

[0207] In some embodiments, methods of the present disclosure result in a therapeutic effect. In some embodiments, the therapeutic effect comprises an immune response, for example, an immune response comprises an increase in T cells, e.g. CD8+ (e.g., IFNy-producing CD8+ cells) and/or CD4+ cells. In some embodiments, the T cells comprise T cells specific for a tumor-associated antigen or tumor-specific antigen expressed on the cancer being treated or ameliorated. In some embodiments, the increase in T cells is observed in the tumor relative to the spleen.

[0208] In some embodiments, the step of administering results in at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the total T cell population in a sample in the mammal being specific for the tumor-associated antigen or tumor-specific antigen. In some embodiments, the sample is a tumor sample.

[0209] In some embodiments, the therapeutic effect comprises a decrease in tumor volume, a stable tumor volume, or a reduced rate of increase in tumor volume. In some embodiments, the therapeutic effect comprises a decreased incidence of recurrence or metastasis.

Other Agents

[0210] In some embodiments, disclosed methods further include administering an antiproliferative agent, radiation sensitizer, or an immunoregulatory or immunomodulatory agent.

[0211] By “antiproliferative” or “antiproliferative agent,” as used interchangeably herein, is meant any anticancer agent, including those antiproliferative agents listed in Table 2, any of which can be used in combination with a radioimmunoconjugate to treat a condition or disorder. Antiproliferative agents also include organo-platinum derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.

[0212] By “immunoregulatory agent” or “immunomodulatory agent,” as used interchangeably herein, is meant any immuno-modulator, including those listed in Table 2, any of which can be used in combination with a radioimmunoconjugate.

[0213] As used herein, “radiation sensitizer” includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiation sensitizers may include, but are not limited to, 5-fluorouracil, analogs of platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.

TABLE-US-00024 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazine ifosfamide altretamine hexamethylmelamine estramustine phosphate thiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomide cyclophosphamide Semustine Platinum agents spiroplatin lobaplatin (Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464 (Hoffmann-La Roche) iproplatin Miriplatin picoplatin AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabine Topoisomerase inhibitors amsacrine exatecan mesylate (Daiichi) epirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) Edotecarin rebeccamycin analogue (Exelixis) Cositecan BBR-3576 (Novuspharma) Belotecan rubitecan (SuperGen) hydroxycamptothecin (SN-38) irinotecan (CPT-11) topotecan Antitumor antibiotics valrubicin azonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycin Sabarubicin porfiromycin mitoxantrone (novantrone) Epirubicin amonafide mitoxantrone doxorubicin Antimitotic agents colchicine E7010 (Abbott) vinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma) auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075 (GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin A paclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi) atamestane (BioMedicines) formestane letrozole exemestane anastrazole Thymidylate synthase inhibitors pemetrexed (Eli Lilly) nolatrexed (Eximias) ZD-9331 (BTG) CoFactor™ (BioKeys) DNA antagonists trabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxter International) mafosfamide (Baxter International) albumin + 32P (Isotope Solutions) apaziquone (Spectrum thymectacin (NewBiotics) Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferase inhibitors arglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) lonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (Schering AG) Histone acetyltransferase inhibitors tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan) SAHA (Aton Pharma) depsipeptide (Fujisawa) MS-275 (Schering AG) Metalloproteinase inhibitors Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex) marimastat (British Biotech) BMS-275291 (Celltech) Ribonucleoside reductase inhibitors gallium maltolate (Titan) tezacitabine (Aventis) triapine (Vion) didox (Molecules for Health) TNF alpha agonists/antagonists virulizin (Lorus Therapeutics) revimid (Celgene) CDC-394 (Celgene) Endothelin A receptor antagonist atrasentan (Abbott) YM-598 (Yamanouchi) ZD-4054 (AstraZeneca) Retinoic acid receptor agonists fenretinide (Johnson & Johnson) alitretinoin (Ligand) LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy (Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK (Progenics) Technology) adenocarcinoma vaccine (Biomira) ISF-154 (Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2 (Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira) synchrovax vaccines (CTL Immuno) MGV (Progenics) melanoma vaccine (CTL Immuno) ß-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy (Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCam Biotherapeutics) abatacept (BMS) atezolizumab (Genentech) pembrolizumab (Merck) Hormonal and antihormonal agents estrogens dexamethasone conjugated estrogens prednisone ethinyl estradiol methylprednisolone chlortrianisen prednisolone idenestrol aminoglutethimide hydroxyprogesterone caproate leuprolide medroxyprogesterone octreotide testosterone mitotane testosterone propionate; P-04 (Novogen) fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosterone arzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofine bicalutamide goserelin flutamide Leuporelin nilutamide bicalutamide Photodynamic agents talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda) Theralux (Theratechnologies) Motexafin lutetium motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774 (Tarceva™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B—Raf enzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BCX-1777 (PNP inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2 inhibitor, Phytopharm) CapCell™ (CYP450 stimulant, Bavarian Nordic) tirapazamine (reducing agent, SRI International) GCS-100 (gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent, Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen (NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, Allos Therapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88 (heparanase inhibitor, Progen) tesmilifene (histamine antagonist, YM BioSciences) seocalcitol (vitamin D receptor agonist, Leo) 131-I-TM-601 (DNA antagonist, TransMolecular) histamine (histamine H2 receptor agonist, Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDH inhibitor, Ribapharm) cilengitide (integrin antagonist, Merck KGaA) minodronic acid (osteoclast inhibitor, Yamanouchi) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinase inhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE V inhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst) CP-461 (PDE V inhibitor, Cell Pathways) AG-2037 (GARFT inhibitor, Pfizer) PG2 (hematopoiesis enhancer, Pharmagenesis) WX-UK1 (plasminogen activator inhibitor, Wilex) Immunol™ (triclosan oral rinse, Endo) PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine (uridine prodrug, Wellstat) bortezomib (proteasome inhibitor, Millennium) SRL-172 (T cell stimulant, SR Pharma) SN-4071 (sarcoma agent, Signature BioScience) TLK-286 (glutathione S transferase inhibitor, Telik) TransMID-107™ (immunotoxin, KS Biomedix) PCK-3145 (apoptosis promotor, Procyon) PT-100 (growth factor agonist, Point Therapeutics) doranidazole (apoptosis promotor, Pola) CHS-828 (cytotoxic agent, Leo) midostaurin (PKC inhibitor, Novartis) trans-retinoic acid (differentiator, NIH) bryostatin-1 (PKC stimulant, GPC Biotech) MX6 (apoptosis promotor, MAXIA) CDA-II (apoptosis promotor, Everlife) apomine (apoptosis promotor, ILEX Oncology) SDX-101 (apoptosis promotor, Salmedix) urocidin (apoptosis promotor, Bioniche) rituximab (CD20 antibody, Genentech Ro-31-7453 (apoptosis promotor, La Roche) carmustine brostallicin (apoptosis promotor, Pharmacia) Mitoxantrone β-lapachone Bleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesium oxides caffeic acid BRAF inhibitors, Tyrphostin AG PD-L1 inhibitors PD-1 inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenib angiogenesis inhibitors dabrafenib

EXAMPLES

Example 1. Single Agent Efficacy of Checkpoint Inhibitors in the CT-26 Syngeneic Model was Observed

[0214] A single agent efficacy study of two checkpoint inhibitors (PD-1 and CTLA-4) was conducted in the CT-26 model, a murine colon carcinoma model. It is known that these carcinomas are partially sensitive to α-PD-1 mAbs and sensitive to α-CTLA-4 mAbs. Mice were injected i.p. with either 5 or 15 mg/kg i.p. of either the α-PD-1 mAb or the α-CTLA-4 mAb. The α-PD-1 mAb group was dosed twice a week for four weeks. The α-CTLA-4 mAb group was dosed only 3 times a day, 3 days apart. CTLA-4 treatment was more efficacious than PD-1 treatment, as expected for this model. In both treatment groups, 5 mg/kg appeared to be the most efficacious dose for impairing tumor growth. See FIG. 1. CD8+/CD4+ T cell recruitment following the different treatments is also measured using immunohistochemistry and flow cytometry techniques.

Example 2. [.SUP.177.Lu]-FPI-1755 Biodistribution in the CT-26 Syngeneic Model.

[0215] MAB391, a murine monoclonal antibody against IGF-1R, was conjugated with FPI-1397 (a bifunctional chelate) and radiolabeled with Lu-177 using methods well known in the art to form [.sup.177Lu]-FPI-1755. The ability of [.sup.177Lu]-FPI-1755 to target antigen expressing mouse IGF-1R overexpressing tumors in vivo was demonstrated using the CT-26 syngeneic model. Tumor uptake was steady at 15-17% injected dose/g (ID/g) from 24-96 hours post injection. See FIG. 2.

Example 3. Enhanced Efficacy of [.SUP.225.Ac]-FPI-1792 in Immunocompetent vs. in Immunodeficient Mice

[0216] MAB391, a murine monoclonal antibody against IGF-1R, was conjugated with FPI-1397 (a bifunctional chelate) and radiolabeled with [.sup.225Ac] using standard techniques to form [.sup.225Ac]-FPI-1792. An efficacy study of [.sup.225Ac]-FPI-1792 in immunocompetent and in immunodeficient mice was conducted using a 400 nCi dose of [.sup.225Ac]-FPI-1792. It was found that [.sup.225Ac]-FPI-1792 had enhanced efficacy in reducing tumor volume in mice with an intact immune system relative to mice with no immune system. See FIG. 3.

Example 4. Synergy Between [.SUP.225.Ac]-FPI-1792 and α-CTLA-4/PD-1 Treatment in the CT26 Syngeneic Mouse Model.

[0217] An in vivo synergy study was conducted to test the effect of [.sup.225Ac]-FPI-1792 (as described in Example 3) and checkpoint inhibitors, α-CTLA-4 and α-PD-1 antibodies, on relative tumor volume in the CT26 mouse model. Mice treated either with the CTLA-4 inhibitor alone or the PD-1 inhibitor alone showed modest reductions in relative tumor volume when compared to the vehicle control groups. Mice treated with [.sup.225Ac]-FPI-1792 demonstrated greater reductions in tumor volume relative to the vehicle control group or the groups administered CTLA-4 inhibitor or PD-1 inhibitor alone. However, when [.sup.225Ac]-FPI-1792 was co-administered with ether CTLA-4 or PD-1 or both, a synergistic effect was seen-co-administration resulted in significantly smaller tumor volume when compared to treatment with [.sup.225Ac]-FPI-1792, or when compared to treatment with the CTLA-4 inhibitor or the PD-1 inhibitor alone. See FIG. 4.

Example 5. Development of Protective Immunity in [.SUP.225.Ac]-FPI-1792 Retreated Mice Upon CT26 Re-Challenge

[0218] A re-challenge experiment was conducted to test the development of protective immunity in [.sup.225Ac]-FPI-1792 treated mice upon CT26 re-challenge. Mice had been previously treated with either [.sup.225Ac]-FPI-1792 alone or in combination with an α-CTLA-4 or α-PD-1 antibody. Naïve mice were used as controls. All mice previously treated with [.sup.225Ac]-FPI-1792 +/- an anti-CTLA-4 or anti-PD-1 antibody were protected from tumor challenge, suggesting development of protective T cell immunity. See FIG. 5.

Example 6. Cytokine Response and T-Cell Recruitment After [.SUP.225.Ac]-FPI-1792 Treatment

[0219] Cytokine response and T-cell recruitment after [.sup.225Ac]-FPI-1792 treatment is measured. Mice were inoculated with 1 x 10.sup.6 CT26 cells. Mice were then treated with either [.sup.225Ac]-FPI-1792, the unconjugated MAB391 antibody or vehicle. Samples from the tumor, spleen and blood plasma are analyzed for the presence of cytokines at 24, 48, or 72 hours. Additional samples are taken from the tumor and spleen at 72 hours, 5 days and 8 days for immunohistochemistry to assess the presence of different T-cell types. Finally, at 8 days, tumor-infiltrating lymphocytes are extracted, isolated and quantified using flow cytometry. See FIG. 6.

[0220] CT26 cells were stably transfected with human IGF-1R plasmid. Western blot analysis was conducted for the presence of hIGF-1R for the selection of hIGF-1R expressing clones. See FIG. 7. The best clones are chosen based on both in vitro and in vivo characteristics. The resulting cell lines will be used as an immunocompetent mouse model for testing [.sup.225Ac]-FPI-1434 and other radioimmunoconjugates and additional synergies with immune checkpoint inhibitors.

Example 7. Combination Therapies Result in Increased Tumor-Associated Antigen-Specific CD8+ T Cells in Both the Spleen and Tumor Itself.

[0221] Ac-TAB-199 is a radioimmunoconjugate comprising human monoclonal IGF-1R antibody labeled with .sup.225-Actinium. Combinations with Ac-TAB-199 and checkpoint inhibitors (α-PD-1, α-CTLA-4, or both α-PD-1 and α-CTLA-4) were tested in the CT26 syngeneic mouse model. Mice were re-challenged with CT26 cells at day 28 after initial tumor inoculation.

[0222] CD8+ and CD4+ T cell populations were assessed in both the spleen and the tumor after re-challenge. In mice treated with Ac-TAB-199 and checkpoint inhibitors, both the spleen and the tumor exhibited the presence of CD8+ T-cells. Importantly, an increase in the CD8+ T-cell frequency in the tumor, relative to controls, was observed. These results suggest that these combination treatments lead to improved levels of therapeutically effective CD8+ T cells.

[0223] Antigen-specific T-cells were detected and enumerated using an MHC class I tetramer assay. In this assay, MHC I molecules presenting an epitope specific to CT26 cells are labelled with biotin. In the presence of streptavidin, these MHC I molecules tetramerize. CD8+ T cells specific for the CD26 epitope are thereby labelled when their T-cell receptors bind to MHC I/CT26 epitope complexes within tetramers. Based on tetramer analysis, approximately 35%, 62%, and 75% of the CD8+ T cells were antigen-specific in mice treated with Ac-TAB-199/α-CTLA-4, Ac-TAB-199/α-PD-1, and Ac-TAB-199/α-CTLA-4/α-PD-1, respectively.

EQUIVALENTS/ OTHER EMBODIMENTS

[0224] 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.