CROSS-REFERENCE TO RELATED APPLICATION

[0001] T his application claims the benefit of priority to US Provisional Application Serial no. 62/729,959, filed oil September 11, 2018, the complete contents of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

1 · Field of the Invention

[0002] T he present invention relates to the field of cancer diagnosis and therapy and more particularly to treating, diagnosing and staging cancers, and, in particular, to cancers

overexpressing the Human Epidermal growth factor Receptor 2 protein (HER2+), and the use of compounds to treat, diagnose and stage cancers.

2. Brief Description of the Related Art

[0003] Breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2017, representing about 25 percent of all cancers in women. HER2+breast cancer is a type of cancer that tests positive for a protein called human epidermal growth factor receptor 2 (HER2+), which promotes the growth of cancer cells. In about 1 of every 5 breast cancers, the cancer cells have a gene mutation that makes an excess of the HER2+ protein.

HER2+ breast cancers tend to be more aggressive than other types of breast cancer. They are less likely to be sensitive to hormone therapy, though many people with HER2+breast cancer can still benefit from hormone therapy. Treatments that specifically target HER2+ are very effective. In marked contrast, these treatments are so effective that the prognosis for HER2+breast cancer is actually quite good. However, HER2+ cancer is not limited to the breast.

[0004] The widely used chemotherapy drug trastuzumab (Herceptm), a standard -of-care, can be life-saving for women with HER2-positive breast cancer, a particularly aggressive form of the disease. But new research now' adds to mounting evidence that the treatment can take a toll on the heart, increasing the risk for heart failure in many cases, the benefits of the chemotherapy still outweigh the risks. Cardiotoxicity in younger women and the risk of heart failure increased with age. Taking other chemotherapy drugs, known as an thracy dines, could also increase the likelihood of heart problems. Comorbidities such as diabetes, which tend to be associated with obesity, w'ere associated with a higher risk of heart failure. Breast cancer patients treated with Herceptin require regular heart monitoring. Heart disease has been considered and reported to be the second leading cause of death among breast cancer survivors mainly due to the toxic effects of some cancer treatments. This underscores the need for novel adjuvant or neoadjuvant therapy to treat both early and late stage HER2+ cancers, alone or together with standard of care, i.e., the focus of this patent. [0005] Receptor tyrosine-protein kinase erbB-2, also known as CD340, proto-oncogene Neu, Erbb2, or ERBB2, is a protein that in humans is encoded by the ERBB2 gene. It is frequently called HER2+ or HER2+/neu. HER2+ neoplasia occurs in a variety of organ sites and tissue types including breast, gastric, gastroesophageal junction adenocarcinoma, and pancreas. HER2+ tumors have a range of morphologic, functional, and behavioral characteristics. Moreover, in addition to the breast, this cancer has the potential to spread to other organs such as the brain, and when they do, they can be especially life threatening and difficult to treat with current modalities.

Heterogeneous tumors originating from HER2+proteins typically begin in the breast but may be distributed throughout the body.

[0006] Some patients have breast tumors, gastric or gastroesophageal junction tumors that contain cancers with higher levels of a protein known as HER2/neu, which is a growth-promoting protein on the outside of breast cancer cells with higher than normal levels of HER2 are called HER2- positive (HER2+). These cancers tend to grow and spread faster than other breast cancers. A biopsy or surgery sample of the cancer is usually tested with either immunohistochemical stains (IHC) or Fluorescent in situ hybridization (FISH). Many breast cancer specialists think that the FISH test is more accurate than IHC. However, it is more expensive and takes longer to get the results. Often the IHC test is done first. If the result is 0 or 1+, the cancer is considered HER2- negative. They do not respond to treatment with drugs that target HER2. If the result is 3+, the cancer is HER2-positive. They are usually treated with drugs that target HER2. If the result is 2+, the HER2 status of the tumor is not clear and is called "equivocal." This means that the HER2 status needs to be tested with FISH to clarify the result. Triple-negative breast tumors do not have much HER2 and also don’t have estrogen or progesterone receptors. They are HER2-, ER-, and PR-negative. These cancers are more common in younger women and in African-American or Hispanic/Latina women. Triple-negative breast cancers grow and spread more quickly than most other types of breast cancer. Because the cancer cells don’t have hormone receptors, hormone therapy is not helpful in treating these cancers. Because they do not have too much HER2, drugs that target HER2 are not helpful, either. Chemotherapy can still be useful, though. Triple-positive breast tumors are HER2-, ER-, and PR-positive. These cancers are treated with hormone drugs as well as drugs that target HER2.

[0007] Normally, HER2+ receptors help control how a healthy breast cell grows, divides, and repairs itself. But in about 25% of breast cancers, the HER2+ gene doesn't work correctly and makes too many copies of itself (known as HER2+ gene amplification). All these

extra HER2 + genes tell breast cells to make too many HER2+ receptors (HER2+ protein overexpression). This makes breast cells grow and divide in an uncontrolled way. [0008] Breast cancers with HER2+ gene amplification or HER2+ protein overexpression are called HER2+ in the pathology report. HER2+breast cancers tend to grow faster and are more likely to spread and come back compared to HER2+-negative breast cancers. But there are medicines specifically for HER2+breast cancers.

[0009] The HER2+ protein can cause normal cells to grow uncontrollably like aggressive cancer cells. If HER2+ is blocked, the growth of HER2+breast and other cancers might be slowed.

The HER2+ protein can cause normal cells to grow uncontrollably like aggressive cancer cells. If HER2+ is blocked, the growth of HER2+breast and other cancers might be slowed. The gene for HER2+ is located on chromosome 17 and has been found to be amplified with an increased copy number in several cancers (Jorgensen 2010). Amplification of HER2+ has been found to promote tumorigenesis and to be involved in the pathogenesis of several human cancers (Moasser 2007).

[0010] It also has been reported that breast cancer in patients treated with an immunotherapeutic, trastuzumab and chemotherapy grew at a slower rate than in patients treated with chemotherapy alone; and that subsequent clinical trials also showed positive outcomes among women with early- stage HER2+ breast cancer. Despite successes with trastuzumab, pertuzumab, other

immunotherapies and chemotherapies such as docetaxel, many women with breast cancer do not benefit from current HER2+ -targeted treatments, or they become resistant to the effects of these drugs after initial treatment. Therefore, researchers continue to test new or modified drug combinations. The FDA approved pertuzumab as a treatment for women with HER2+metastatic breast cancer to be used in combination with trastuzumab and docetaxel, a chemotherapy drug. Other drugs that have been approved for the treatment of HER2+ breast cancer include ado- trastuzumab emtansine, lapatinib ditosylate, and neratinib maleate. In 2017, pertuzumab received approval for use in combination with the same drugs as an adjuvant treatment for patients with HER2+ early breast cancer at high risk of recurrence. Nevertheless, the use of chemotherapy and immunotherapy has side effects including nausea, vomiting, hair loss, bone pain, peripheral neuropathy, skin rashes and because of toxicity, a very high incidence of cardiovascular disease. Nevertheless, there still is a high rate of cancer relapse and metastases.

[0011] Although tumors produce certain proteins, hormones and other chemicals that often cause patients to exhibit symptoms, the non-specific nature of these symptoms can lead to delayed diagnosis or even a misdiagnosis. By the time patients are correctly diagnosed, the cancer has usually metastasized, with regional or distant metastasis observed in approximately 30% of cases. Once they have metastasized, HER2+ tumors may not be effectively treated by surgery alone and generally are not curable. Despite the major advance in the therapy of HER2+ breast cancer represented by treatment protocols containing trastuzumab, a first line treatment, nearly all patients given trastuzumab for advanced breast cancer will ultimately experience progression, while a significant proportion of patients receiving this treatment in the setting of early breast cancer will experience disease recurrence. Some may receive trastuzumab in the metastatic setting, but again, will likely experience progression despite this treatment (Murphy 2012).

[0012] While breast cancer is the most common cancer among women in the United States, not all breast cancers are the same. There are ten different types of breast cancer, which are determined by the specific cells in the breast that are affected, according to the American Cancer Society. HER2+ breast cancer accounts for around 20 percent of breast cancer. It is also associated with an increased recurrence risk and poor five-year survival rates relative to other breast cancers. A woman faced with breast cancer, of course, hopes for a definitive test that can tell her what form of cancer she has, and the best steps to treat it. Presently, genetic testing for breast cancer looks mostly at the genetic changes that come from a person’s parents and grandparents, and the increased risk of being genetically predisposed to some cancers. Only 5 to 10 percent of breast cancers are inherited genetically, according to the American Cancer Society. HER2+ breast cancer is a difficult breast cancer to treat because screening tests do not often“catch it” before it becomes serious and the usual drugs that target estrogen, progesterone, and HER-2 may be ineffective. Trastuzumab (Herceptin) has become the standard of care for treatment of eligible women with stage II and III HER2+ breast cancer, and it is frequently considered for stage I breast cancer as well.

[0013] Studies have been performed using diagnostic testing on women with HER2+ breast cancer. Twenty one cancer genes make breast cancer more likely. The proteins or genes, when altered, that increased the risk of triple-negative breast cancer in the women included those that are involved, there’s a greater than 20 percent lifetime risk for all types of breast cancer. One study establishes which genes are associated with high risks of HER2+ breast cancer, and the findings show this and identify new specific and strong associations between the susceptibility genes and HER2+ breast cancer risk.

Immunotherapy Collateral Effects

[0014] While immunotherapeutics such as Trastuzumab have become a part of the standard-of- care armamentarium for HER2+ cancers, the following are common (occurring in greater than 30%) side effects in patients taking Trastuzumab and/or Avastin for treatment of HER2+ cancer: nausea, vomiting, cardiovascular disease, generalized weakness, muscle aches, pain, unusually weak or tired, poor appetite, constipation, breathing difficulties, chest pain or palpitations, cough, dizziness or fainting, fever or chills, sore throat, skin rash, itching or hives, upper respiratory infection, cough, low white blood cell count and increased risk for infection, proteinuria (kidney problems), nose bleed (bleeding problems), diarrhea, hair loss, mouth sores, headache, swelling of the legs or ankles, impaired fertility by several effects. Some patients do not recover completely or recover very slowly following discontinuation of the drug. Targeted radionuclide therapy (TRNT) does not result in these collateral effects. Salivary gland toxicity can occur in some patients receiving TENT and is treated with Botox. Potential kidney toxicity is ameliorated or treated with an amino acid blocker.

[0015] Moreover, increased angiogenesis is a marker of aggressiveness in many

cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic.

[0016] Therefore, there is a significant need for therapies for HER2+ tumors, however, there are only a limited number of options currently available for the treatment.

[0017] The cornerstone for treatment of HER2+ tumors is the use of Trastuzumab and

chemotherapy in the neoadjuvant setting. Chemotherapy and targeted therapies have been registered for many cancers while for HER2+ brain metastases only have a limited approval of use. As a result of that, many patients exhaust all the available treatment options, particularly in the advanced disease setting, which represents a high unmet medical need for systemic treatments of metastatic HER2+ tumors.

[0018] At a symptomatic level, integrins are used to control certain clinical syndromes with HER2+ positive tumors.

[0019] Most HER2+ tumors overexpress HER2 positive protein receptors. These protein receptors are expressed in human cells that sense molecules outside of the cell and activate sub-cellular events in response to integrin treatments.

[0020] However, the symptomatic targeting of theranostics is distinct from treating the metastatic HER2+ cancerous cells and tumors themselves. Trastuzumab is a HER2+/neu receptor antagonist indicated for the treatment of HER2+ overexpressing breast cancer and the treatment of HER2+- overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. Based on a partial antiproliferative effect. Trastuzumab, a humanized monoclonal antibody inhibiting vascular endothelial growth factor was approved by the FDA for the treatment of patients with well- differentiated or moderately-differentiated, locally advanced or metastatic HER2+ tumors has been approved in the US for treatment of HER2 cancers.

[0021] These are "antitumoral" therapy so far approved for HER2+ tumors. However, when the tumor becomes resistant or unresponsive to its effects, usually several months after the initiation of treatment, there are no other approved therapies for intervention. These tumors therefore are unbeatable by the currently available therapies.

[0022] While chemotherapeutic agents and surgery and some targeted therapies have become the standard of care for HER2+ tumors, the response rates vary according to tumor aggressiveness but are estimated to be between approximately 30% and 50% for targeted therapies (RT)have been approved for the treatment of HER2+ tumors with limited survival benefit. [0023] Thus, traditional chemotherapy has little place in treatment of HER2+ tumors, since most of these tumors are difficult to treat. A rigorous assessment of the efficacy of chemotherapy in literature is hampered by the prevalence of retrospective studies on heterogeneous series of patients, where toxicity is relevant, and the responses are short-lived and sporadic. Many schemes, including single or multiple agents, have been attempted.

[0024] Integrins are transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cellular signals such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and movement of new receptors to the cell membrane. The presence of integrins allows rapid and flexible responses to events at the cell surface (e.g. signal platelets to initiate an interaction with coagulation factors).

[0025] Integrins are obligate heterodimers, meaning that they have two subunits: a (alpha) and b (beta). Integrins in mammals have eighteen a and eight b subunits, in Drosophila five a and two b subunits, and in Caenorhabditis nematodes two a subunits and one b subunit. The a and b subunits each penetrate the plasma membrane and possess several cytoplasmic domains.

[0026] Integrins can be categorized in multiple ways. For example, some a chains have an additional structural element (or "domain") inserted toward the N-terminal, the alpha-A domain (so called because it has a similar structure to the A-domains found in the protein von Willebrand factor; it is also termed the a-I domains.

[0027] Integrins carrying this domain either bind to collagen (e.g. integrins aΐ bΐ, and a.2 bΐ), or act as cell-cell adhesion molecules (integrins of the b2 family). This a-I domain is the binding site for ligands of such integrins. Those integrins that don't carry this inserted domain also have an A- domain in their ligand binding site, but this A-domain is found on the b subunit.

[0028] Integrins play an important role in cell signaling by modulating the cell signaling pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTK). While the interaction between integrin and receptor tyrosine kinases originally was thought of as unidirectional and supportive, recent studies indicate that integrins have additional, multi-faceted roles in cell signaling. Integrins can regulate the receptor tyrosine kinase signaling by recruiting specific adaptors to the plasma membrane. For example, bΐo integrin recruits Gabl/Shp2 and presents Shp2 to IGF1R, resulting in dephosphorylation of the receptor. In the reverse direction, when a receptor tyrosine kinase is activated, integrins co-localise at focal adhesion with the receptor tyrosine kinases and their associated signaling molecules.

[0029] The anb3 integrin is a receptor for vitronectin [Hermann, P. et al. "The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interaction with soluble CD23" [The Journal of cell biology 144 (1999): 767-75]. It consists of two components, integrin av and b3 (CD61), and is expressed by platelets as well as other cell types. It has been shown that inhibitors of anb3 like etaracizumab may be used as antiangiogenics.

[0030] Integrins are members of a family of heterodimeric transmembrane cell surface receptors that play a crucial role in cell-cell and cell-matrix adhesion processes (Hynes, R. O. Cell 1992, 69, 11-25). These receptors consist of an anb3 and anb5 which non-covalently associate in defined combinations (Eble, J. A. Integrin-Ligand Interaction; Springer: Heidelberg, 1997; pp 1-40). Most of them recognize the Arg-Gly-Asp (RGD) triad found in many extracellular matrix proteins (i.e., vitronectin), Serini, G.; et al. A sticky business. Exp. Cell. Res. 2005, and snake venom

disintegrins (Ruoslahti, E.; Pierschbacher, M. Cell 1986, 44, 517-518; D'Souza, S. E.; et al. Trends Biochem. Sci. 1991, 16, 246-250; Gould, R. J.; et al. Proc. Soc. Exp. Biol. Med. 1990, 195, 168- 171). Even if different integrins recognize different proteins containing the RGD sequence, several studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Despite numerous studies reported in the literature, ligand selectivity toward different integrin subtypes is still a challenging problem mainly because most of the 3D-structures of integrin subtypes remain unknown

(Marinelli, L.; et al. J. Med. Chem. 2004, 47, 4166-4177).

[0031] The anb3. integrin is strongly expressed on activated endothelial and melanoma cells and in contrast, it is weakly expressed in quiescent blood vessels and pre-neoplastic melanomas (Hood, J. D.; Cheresh, D. A. Nat. Rev. Cancer 2002, 2, 91-100). Along with anb3 beta..sub.5 integrin, .AVB3 is reported to be involved in physiological processes including angiogenesis and tissue repair as well as pathological conditions such as tumor induced angiogenesis (Eliceiri, B. P.;

Cheresh, D. A. J. Clin. Invest. 1999, 103, 1227-1230; Kumar, C. C. Curr. Drug Targets 2003, 4, 123-131), tumor cell migration and invasion (Clezardin, P. Cell. Mol. Life. Sci. 1998, 54, 541- 548). Despite the fact that both integrins promote cell attachment to vitronectin and participate in the same processes, they are reported to be structurally designed to respond to different signaling events. Previous studies provided evidence that bFGF-induced angiogenesis is mediated by integrin anb3. whereas VEGF-induced angiogenesis is mediated by anb3 beta sub.5 (Friedlander, M.; et al. Science 1995, 270, 1500-1502). Melanoma cells expressing integrin anb3 .migrate in vitro and metastasize in vivo without the need for exogenous cytokine stimulation (Filardo, E. J.; et al. J Cell Biol. 1995, 130, 441-450). Conversely, tumor cells expressing anb3 beta.. sub.5 integrin require a tyrosine kinase receptor-mediated signaling event for motility on vitronectin and in vivo dissemination (Brooks, P. C.; et al J Clin Invest. 1997, 99, 1390-1398). While anb3 beta.. sub.5 is widely expressed by many malignant tumor cells, .AVB3 has a relatively limited cellular distribution compared with that of anb3 beta.. sub.5 (Pasqualini, R.; et al. J. Cell Science 1993, 105, 101-111 ; Walton, H. L.; et al. J. Cell. Biochem. 2000, 78, 674-680.). Therefore, in order to target anb3 beta sub.3 -mediated processes for diagnostic or therapeutic purposes, the development of new compounds that can discriminate between .AVB3 and anb3 beta.. sub.5 is required.

[0032] anb3 integrin interacts with HER2+ in cancer cells and can regulate HER2+ localization. The combined impacts of an-integrin and HER2+ influence the invasive phenotype of breast cancer cells. Targeting an-integrin in HER2+ breast cancer may slow growth and decrease infiltration in the normal brain (Lai 2015).

[0033] A need remains for further improved targeting of integrins; as well as substances such as small molecules, peptides, other nucleic acids, fluorescent moieties, polymers, alpha, beta or alpha/beta tandem radionuclides to particularly target cancer cells, destroy vascular endothelial growth factor— new blood vessels that feed tumors, target the cytoplasm of such cells and impair the DNA of cancer cells.

SUMM ARY OF THE INVENTION

[0034] The present invention relates to methods of treating a patient affected by a tumor that over expresses receptors comprising administering to said patient a combination of a TRNT and imaging of HER2+cancers.

[0035] This invention concerns peptidomimetic compounds as highly potent and selective antagonists of the anb3 and anb5 integrin receptor which may contain the Arg-Gly-Asp sequence for diagnosis and tumor targeted radionuclide therapy (TRNT) and molecular imaging. The compounds of the invention may be used as a novel class of anticancer drugs and/or diagnostic tracers as suitable tools for TRNT and imaging of HER2+ cancers. Embodiments of the present invention provide a combination therapy approach that for many, could effectively treat HER2+ tumors. Therapeutic compositions of the invention comprise peptidomimetic compounds that are radiolabeled that are employed for use in conjunction with a pharmaceutically acceptable excipient for delivery to a patient to treat a cancer, and more particularly a cancer that has a human epidermal growth factor receptor 2-Positive ( hereinafter referred to as HER2+), or other cancer that overexpress integrin receptors. The compositions are used by delivering dosages of the composition to a patient, preferably intravenously, and over the course of time (the treatment term). The patent is monitored using imaging to determine the effectiveness of the treatment. Use of imaging compositions of the invention also is disclosed, wherein these diagnostic compositions are paired with the therapeutic composition.

[0036] According to preferred embodiments, the method is carried out to deliver a targeted radionuclide therapy (TRNT). According to preferred implementations, the method involves the TRNT by administration to a patient of an effective amount of a radioligand, and more preferably a radioligand comprised of an anti-integrin peptidomimetic, a chelator, and a radionuclide. The administration of the radioligand to a patient may include administration of a radioligand where the radionuclide is [177]Lu or [225] Actinium. In specific embodiments, the TRNT is [l77]Lutetium the integrin peptidomimetic, and more particularly, Lu-l77 DOTAGA IAC: A ligand containing the active moiety, the anb3 integrin antagonist peptidomimetic, 4-[2-(3 ,4,5,6- tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3-aminoneopenta-l-carbamyl)]- aminoethylsulfonyl-amino-P-alanine (IAC) and l-(l-carboxy-3-carbotertbutoxymethyl)- 1,4 ,7,10- tetraazacyclododecane (DOTAGA) (tBu).sub.4 and radiolabeled with Lutetium-l77 for tumor targeted radionuclide therapy.

[0037] In the preferred embodiments, the 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3-aminoneopenta-l-carbamyl)]-aminoeth ylsulfonyl-amino-P-alanine (IAC) is conjugated to the l-(l-carboxy-3-carbotertbutoxymethyl)-l,4,7,l0-tetraazacyclo dodecane (DOTAGA) (tBu).sub.4, and radiolabeled.

[0038] According to embodiments, the radionuclide may comprise one or more of the following: Lutetium-l77, Actinium-225, Bismuth-2l3, Thorium-227, Lead-2l2, Astatine-2l 1, Yttrium-90, Iodine- 131 or other therapeutic radiolabel.

[0039] The present invention relates to methods of treating, diagnosing and staging cancers, in particular overexpressing the Human Epidermal growth factor Receptor 2 protein (HER2+) given rise to in breast, gastric, gastroesophageal, ovarian and pancreatic cancer, and which may be metastatic to the brain or other site. More specifically, the invention provides for Targeted Radionuclide Therapy (TRNT) with the radiolabeled composition of the invention and a companion diagnostic, (such as, for example, Ga-68 labeled ligand, Ga-68-NODAGA IAC), anti- integrin precision medicines for cancers expressing anb3 and anb5 integrins, HER2+, vascular endothelial growth factor, vitronectin, fibronectin, tenascin, reelin, kindlin and talin. TRNT may be administered alone or in combination with standard -of-care; an immunooncologic and/or chemotherapeutic, adjuvantly or neoadjuvantly.

[0040] A preferred diagnostic composition, which may serve as the companion diagnostic, may comprise Ga-68-NODAGA IAC: A ligand containing the active moiety, the anb3 integrin antagonist peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoy l-2-[N- (3-aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino^-alan ine (IAC) and l-(l-carboxy-3- carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-l,4,7-tr iazacyclononane, and radiolabeled with [68]Ga. The diagnostic may be administered to the patient, and the appropriate imaging study or studies carried out to determine the location and density of the marker.

[0041] The 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoy l-2-[N-(3- aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino^-alanine (IAC) preferably is conjugated to 1 -( 1 -carboxy-3-carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethy l)- 1 ,4,7-triazacyclononane, and labeled with a radionuclide.

[0042] Preferred embodiments of the method involve the use of an anb3/anb5 anti-integrin antagonist peptidomimetic for the diagnosis and treatment of cancers comprising administering to a patient a pharmaceutical composition. According to preferred embodiments, the composition includes a therapeutically effective amount of the anb3 and anb5 antagonist peptidomimetic compound, covalently linked by a spacer sequence wherein the compound may be a linear artificial sequence, a chelator such as NODAGA, DOTAGA, DOTATATE, or CHX-A, and a

pharmaceutically acceptable excipient for tumor targeted radionuclide therapy. The radionuclide is attached to the composition, preferably, via the chelator. For example, a peptidomimetic may be synthesized by any suitable production method, including for example, one such exemplary method which comprises a solid-phase method using Fmoc chemistry (e.g., amino acids are coupled according to the HBTU/HOBt/DIPEA procedure (Fields, C. G.; et al Pept. Res. 1991, 4, 95-101); and where final deprotection and cleavage form the resin are achieved with TFA and scavengers; and where purity and the identity of the peptides may be confirmed by analytical RP- HPLC and MALDI-TOF mass spectrometry. Peptidomimetics also may be synthesized on an automated peptide synthesizer using Fmoc solid-phase strategy (0.25 mmol).

[0043] The method involves administering compounds that contain anb3 and anb5 anti-integrin antagonist peptidomimetic, which, unlike cyclical peptides, display highly selective affinity for the HER2+ protein, vascular endothelia growth factor vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combinations thereof for the diagnosis or prognosis of disorders using PET, SPECT and MRI by administering an effective amount of anb3/anb5 anti-integrin antagonist peptidomimetic thereof to a subject, particularly for the diagnosis of diseases related to metastasis and angiogenesis such as breast cancer, lung cancer, ovarian cancer, gastric cancer, esophageal cancer, blood borne cancers, musculoskeletal tumors, melanoma, head and neck cancer, human glioma, vascular restenosis, osteoporosis, rheumatoid arthritis.

[0044] The method may also include providing a diagnostic kit or synthesizer cartridges and administering the pharmaceutical treatment and/or diagnostic composition once the composition has been synthesized or generated from the kit. For example, the compositions may be provided in the form of diagnostic kits or synthesizer cartridges comprising the anb3 and anb5

peptidomimetic antagonist attached to a chelator such as NODAGA, DOTAGA, DOTATATE, or CHX-A or a nanocarrier such as a gold nanoparticle for the early detection, which may be in tissue, bone or in plasma for treatment of pathologies, such as breast cancer, esophageal cancer, lung cancers, ovarian cancer, gastric cancer, musculoskeletal tumors, melanoma, head and neck cancer, human glioma, cervical cancer, vascular restenosis, osteoporosis, rheumatoid arthritis.

[0045] In specific embodiments, the Immuno-Oncology (l-O) therapy comprises administering an inhibitor that inhibits the PD-l /PD-L 1 and CTLA-4 pathway. In other embodiments, the inhibitors of the PD-l /PD-L 1 and CTLA-4 pathway are antibodies that target the PD-l /PD-L 1 axis or the CTLA-4/B7 receptor complex. In still further HER2+embodiments, the inhibitor of the PD- 1 /PD-L 1 pathway is a combination of inhibitors that target both PD- 1 and PD-L 1 . [0046] In particular embodiments, the PD-l /PD-L1 or CTLA-4/B7 pathway inhibitors are selected from the group consisting of Nivolumab, MK-3475, MPDL3280A, MED 14736, ipilimumab, and tremelimumab.

[0047] The invention is particularly defined by embodiments in which the cancer is

a HER2+ tumor. More specifically, said treating comprises one or more of inhibiting the growth of a HER2+ tumor, inhibiting proliferation of HER2+ tumor cells,

inhibiting HER2+ tumor metastases, reducing tumorigenicity of HER2+tumor cells and methods of reducing the frequency of cancer stem cells or tumor initiating cells in a HER2+ tumor.

[0048] Any HER2+ tumor that expresses or overexpressed somatostatin receptors may be treated by the methods of the present invention. Exemplary such tumors include but are not limited to the group consisting of a gastroenteropancreatic HER2+ tumor, carcinoid tumor, pheochromocytoma, paraganglioma, medullary thyroid cancer, pulmonary HER2+ tumor, thymic HER2+ tumor, carcinoid tumor or pancreatic HER2+tumor, pituitary adenoma, adrenal gland tumors, Merkel cell carcinoma, breast cancer, Non-Hodgkins lymphoma, Hodgkin lymphoma, Head & Neck tumor, urothelial carcinoma (bladder), Renal Cell Carcinoma, Hepatocellular Carcinoma, GIST, neuroblastoma, bile duct tumor, cervix tumor, Ewing sarcoma, osteosarcoma, small cell and non small cell lung cancer, prostate cancer, melanoma, meningioma, glioma, medulloblastoma hemangioblastoma, supratentorial primitive, neuroectodermal tumor, and esthesioneuroblastoma.

[0049] The HER2+ tumor is selected from the group consisting of functional carcinoid tumor, insulinoma, gastrinoma, vaso active intestinal peptide (VIP) oma, glucagonoma, serotoninoma, histaminoma, ACTHoma, pheochromocytoma, and somatostatinoma.

[0050] The HER2+ tumors treated by the present invention may be defined by grade, and may be low grade, medium grade, or high grade HER2+ tumors. In specific embodiments, the tumor is a functional HER2+ tumors. Alternatively, the HER2+ tumor is a non-functional HER2+ tumor.

[0051] In particularly preferred embodiments, the cancer to be treated, diagnosed and/or staged is a small cell lung cancer. In other preferred embodiments, the cancer is a progressive

midgut HER2+ tumor. In some embodiments, the invention provides therapy for

such HER2+ tumors that are not responsive to Trastuzumab (Roche), Sandostatin (Novartis) or Somatuline® (Ipsen). In other embodiments, the HER2+ tumor is non-responsive or has a low response to an inhibitor of the PD-l /PD-L 1 pathway.

[0052] The present invention therapeutics, namely, the integrin antagonist peptidomimetic,

[l77Lu] DOTAGA IAC, permits targeting of cancers (HER2+) at locations that are beyond the blood brain barrier. In this manner, metastases that may not be reached by prior treatments, may be addressed by the present method, including the therapeutic treatment and diagnostic agents, and the use of the compounds to provide the therapeutic treatments and diagnosis. [0053] The methods of the present invention also may utilize the therapeutic compounds of the invention in conjunction with a diagnostic, including compounds containing anb3 and anb5 anti- integrin antagonist peptidomimetic, displaying a selective affinity for the HER2+ protein, vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combination thereof for the diagnosis or prognosis of disorders using PET, SPECT and MRI or combinations by administering an effective amount of activity thereof to a subject, particularly for the diagnosis of diseases related to metastasis and angiogenesis such as breast cancer, lung cancer, ovarian cancer, gastric cancer, esophageal cancer, blood borne cancers, musculoskeletal tumors, melanoma, head and neck cancer, human glioma, cervical cancer, vascular restenosis, osteoporosis, rheumatoid arthritis. According to some embodiments, a diagnostic detection method may involve the use of a compound containing the anb3 and anb5 integrin antagonist peptidomimetic, displaying a selective affinity for the HER2+ protein, vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combination thereof, and may comprise a pharmaceutical composition comprising a

therapeutically effective amount of the anb3 and anb5 antagonist peptidomimetic compound, covalently linked by a spacer sequence, wherein the compound may be a linear artificial sequence, a chelator such as NODAGA, DOTAGA, DOTATATE, CHX-A and a pharmaceutically acceptable excipient for targeted radionuclide therapy (TRNT). The compound may also be a radiotracer for nuclear medicine, such as the Gallium-68 or Fluorine- 18, Zirconium-68, Copper-64, or Technetium-99m, covalently bound to a spacer, chelator, directly to the peptidomimetic, to a nanoparticle or to more than one amino acid unit of the peptidomimetic. The diagnostic compound is then administered to the patient, and a scan is carried out using a suitable scanning technique, such as, for example, PET, SPECT and MRI or combinations thereof.

[0054] Methods of treatment, diagnosis and staging of a disorder comprising the administration to an individual suffering from such disorder of a therapeutically effective amount of the anb3 and anb5 integrin antagonist peptidomimetic for targeted radionuclide therapy (TRNT), combination therapy, neoadjuvant therapy or adjuvant therapy. Therapeutic compounds with a strong affinity for the HER2+ protein are overexpressed in breast, esophageal, gastric and ovarian cancers and HER2+ metastases to the brain or other organs. Such compounds are able to decrease or eliminate the proliferation of tumor cells and/or to modulate pathologic angiogenesis via TRNT. Compounds containing anb3 and anb5 anti-integrin antagonist peptidomimetic, displaying a selective affinity for the HER2+ protein, vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combination thereof for the diagnosis or prognosis of disorders using PET, SPECT and MRI or combinations by administering an effective amount of activity thereof to a subject, particularly for the diagnosis of diseases related to metastasis and angiogenesis such as breast cancer, lung cancer, ovarian cancer, gastric cancer, esophageal cancer, blood borne cancers, musculoskeletal tumors, melanoma, head and neck cancer, human glioma, cervical cancer, vascular restenosis, osteoporosis, rheumatoid arthritis. The aforementioned compound demonstrates safety and efficacy in animal and human mammalian models.

[0055] Peptides according to the invention can be used in oncology and nuclear medicine application for the diagnosis and treatment of cancers.

[0056] According to preferred embodiments, the invention accordingly provides a method of treatment of a disorder comprising the administration to an individual suffering from such a disorder of a therapeutically effective amount of the anb3 and anb5 integrin antagonist peptidomimetic for TRNT.

[0057] Examples of specific disorders include pathologies that are related to angiogenesis and metastasis, such as breast cancer, ovarian cancer, lung cancer, gastric cancer, esophageal cancer, musculoskeletal tumors, melanoma, head and neck cancer, blood cancers, human glioma, cervical cancer, vascular restenosis, osteoporosis, rheumatoid arthritis.

[0058] Particularly, the compounds of the invention are able to decrease the proliferation of tumor cells and/or to modulate pathologic angiogenesis via TRNT.

[0059] Even more particularly, the compounds of the invention demonstrate a strong affinity of the HER2+ protein overexpressed in around 20% of all breast, esophageal, gastric and ovarian cancers.

[0060] The inventive method also may implement companion diagnostics, in association with the therapeutic compounds. Companion diagnostic compounds may also be used in the diagnosis or prognosis of disorders using PET, SPECT and MRI or combinations by administering an effective amount thereof to a subject, particularly for the diagnosis of diseases related to metastasis and angiogenesis such as breast cancer, lung cancer, ovarian cancer, gastric cancer, esophageal cancer, blood borne cancers, musculoskeletal tumors, melanoma, head and neck cancer, human glioma, cervical cancer, vascular restenosis, osteoporosis, rheumatoid arthritis.

[0061] The present invention further relates to the field of cancer diagnosis and therapy, theranostics, and the creation of novel molecules; more particularly an integrin peptidomimetic, an isotope and a chelating agent for treating, diagnosing and staging cancers, and, in particular, cancers overexpressing the human epidermal growth factor positive receptor 2 protein (HER2+) and triple negative breast cancer and relates to the synthesis and reaction of potent and selective small molecule integrin antagonists containing appropriate linkers and functional groups for chemical reaction with other molecules which contain reactive nucleophiles such as thiols such that a covalent linkage is formed between a moiety to be conjugated and the targeting entity. The small molecule targeting antagonist anti anb3 binds to cognate receptor systems with high specificity. The covalently linked moiety includes peptidometics preferably but may also be small molecule therapeutics, nanoparticles, polymers, peptides, oligonucleotides, and antineoplastics. [0062] Preferred embodiments of the compositions include a peptide and more particularly a peptidomimetic, linked to a spacer and chelated to a radionuclide, wherein the peptide comprises an anb3 integrin antagonist.

[0063] More particularly, the invention relates to compounds of a formula comprising an anb3 integrin antagonist, and more particularly a peptidomimetic, which is radiolabeled with a radionuclide. According to preferred embodiments the radionuclide is [l77]Lu or [225]Actinium; and according to other embodiments the radionuclide may be Bismuth-213, Thorium-227, Lead- 212, Astatine-2l 1, Yttrium-90, Iodine-l3 l, Gallium-68, Zirconium-68 or other therapeutic radiolabel.

[0064] Embodiments of the invention involve the following therapeutic compositions: [l77Lu] DOTAGA IAC or a ligand containing the active moiety, the anb3 integrin antagonist

peptidomimetic, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3- aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino^-alanine (IAC) and l-(l-carboxy-3- carbotertbutoxymethyl)-l,4,7,l0-tetraazacyclododecane (DOTAGA) (tBu)4; or 4-[4,7-Bis- (carboxymethyl)-[l,4,7]triazonan-l-yl]-4-carboxy-butyryl (NODAGA); DOTATATE, or

C26H34N4O10S (CHX-A), radiolabeled with Lutetium-l77, Actinium-225, Bismuth-2l3, Thorium-227, Lead-2l2, Astatine-2l 1, Yttrium-90, Iodine-l31, Gallium-68 and Zirconium-68 or a combination of radionuclides for tumor targeted radionuclide therapy (TRNT); and of the formula Ga-68-NODAGA IAC or a ligand containing the active moiety, the anb3 integrin antagonist peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoy l-2-[N-(3- aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino^-alanine (IAC) and to l-(l-carboxy-3- carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-l,4,7-tr iazacyclononane. The former composition is preferably used for therapeutic treatment, while the latter composition (i.e., Ga-68 composition) is used for diagnostic imaging. The former and the latter compositions are companion compositions so that the therapeutic administration of the former compound (to a patient) may be carried out and imaging of the applied therapeutic results (to detect cancer) may be carried out using the latter Ga-68 composition (which also is administered to a patient prior to or in connection with imaging).

[0065] The anb3/anb5 anti-integrin antagonist peptidomimetic can be used for the diagnosis and treatment of cancers, and comprises a pharmaceutical composition consisting of a therapeutically effective amount of the anb3 and/or anb5 antagonist peptidomimetic compound, covalently linked by a spacer sequence wherein the compound may be a linear artificial sequence, a chelator such as NODAGA, DOTAGA, DOTATATE, or CHX-A, and a pharmaceutically acceptable excipient for tumor targeted radionuclide therapy (TRNT) using a radionuclide such as Lutetium-l77,

Actinium-225, Bismuth-213, Thorium-227, Lead-212, Astatine-211, Yttrium-90, Iodine- 131, Gallium-68, Zirconium-68, or other therapeutic radiolabel. [0066] The compounds of this invention contain an anb3 and anb5 anti-integrin antagonist peptidomimetic and unlike cyclical peptides display highly selective affinity for the HER2+ protein, vascular endothelial growth factor, vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combinations thereof, and demonstrate safety and efficacy in animal and mammalian models.

[0067] In specific embodiments, a tumor targeted radionuclidic agent contains a [68Ga], [18F], [203 Pb], [99mTc] or [89Zr] as a diagnostic. For tumor targeted therapy the radionuclide may be [l77]Lu, [225 Ac], [90Y], [227Th], more particularly, a [l77Lu] DOTAGA IAC or a ligand containing the active moiety, the anb3 integrin antagonist peptidomimetic, 4-[2-(3 ,4,5,6- tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3-aminoneopenta-l-carbamyl)]- aminoethylsulfonyl-amino^-alanine (IAC) and l-(l-carboxy-3-carbotertbutoxymethyl)- 1,4 ,7,10- tetraazacyclododecane (DOTAGA) (tBu)4, or 4-[4,7-Bis-(carboxymethyl)-[l,4,7]triazonan-l-yl]- 4-carboxy-butyryl (NODAGA), or C26H34N4O10S (CHX-A), to radiolabel with Lutetium-l77, Actinium-225, Bismuth-213, Thorium-227, Lead-212, Astatine-211, Yttrium-90, Iodine- 131, Gallium-68, Zirconium-68, or a combination of radionuclides for tumor targeted radionuclide therapy (TRNT).

[0068] The peptidomimetic compositions may comprise first and second binding regions, including a first region that is an antigen binding region and a second region that is an integrin binding region. The second integrin binding region binds an epitope to a paratope, and the first antigen-binding region binds an epitope on human epidermal growth factor receptor 2 (HER2) and blocks the binding to HER2, optionally soluble HER2, wherein the peptidomimetic comprises an anti-integrin that shows high affinity for integrin anb3/anb3, vascular endothelial growth factor, vitronectin, fibronectin and tenascin, actin, reelin, talin or kindlin.

[0069] Compositions of the invention comprise integrin receptor peptidomimetic antagonists whose molecular structure includes a tetrahydropyridimidinyl aminoethyloxybenzoyl group on a sulfonylamino^-alanine nucleus exhibit increased binding affinity for the anb3/ anb3 receptor when further substituted on the sulfonyl moiety with an N-amino alkycarbamyl group or a butyloxycarbonylamino alkylcarbamoyl group or similar groups.

[0070] In particular, the present invention relates to the compounds of formulas herein for the improved delivery of conjugated moieties such as small molecules, peptidomimetics, peptides, nucleic acids, fluorescent moieties, antineoplastics, nanoparticles and polymers to target cells expressing the . anb3 dimer for various therapeutic and other applications. The present invention also relates to methods of manufacturing and using such compounds.

[0071] Integrin receptor antagonists whose molecular structure includes a tetrahydropyridimidinyl aminoethyloxybenzoyl group on a sulfonylamino^-alanine nucleus exhibit increased binding affinity for the anb3 receptor when further substituted on the sulfonyl moiety with an N-amino alkycarbamyl group or a butyloxycarbonylamino alkylcarbamoyl group or similar groups. Embodiments of the present invention also utilize the therapeutic compounds of the invention in conjunction with a diagnostic. The aforementioned compounds demonstrates safety and efficacy theranostically in animal and human mammalian models.

[0072] The compounds referred to may be radiotracers for nuclear medicine, such as the Gallium- 68 or Fluorine- 18, Zirconium-68, Copper-64, or Technetium-99m, covalently bound to a spacer, chelator, directly to the peptidomimetic, to a nanoparticle or to more than one amino acid unit of the peptidomimetic. Other radiotracers are those reported in the publication Radionuclide Peptide Cancer Therapy, edited by M. Chinol and G. Paganelli, Taylor and Francis CRC Press, 2006. Studies have been performed comparing this cyclical anb3 and anb5 integrin antagonist peptidomimetic to a linear anb3 integrin peptide in nude mice using the same chelator and radionuclide. The results for this anb3 and anb5 linear peptidomimetic were three to seven times better affinity for the targeted organs. (See LinnebacHER2+M 1 , Maletzki C.“Tumor-infiltrating B cells: The ignored players in tumor immunology.” Oncoimmunology. 2012 Oct 1 ;l (7): l 186-1 188. 15. Nelson BH. CD20B cells: the other tumor-infiltrating lymphocytes. J Immunol. 2010 Nov 1 ;l85(9):4977-82.) In human studies comparing this cyclical anb3 and anb5 integrin antagonist peptidomimetic to a‘gold standard’ [18F] Fludeoxy glucose PET scan in an HER2+ breast cancer patient, the integrin antagonist peptidomimetic enabled detection of 25 tumors distant from the primary versus only 12 with [18F] Fludeoxy glucose. Detection may be effected using a compound containing the anb3 and anb5 integrin antagonist peptidomimetic, displaying a selective affinity for the HER2+ protein, vitronectin, fibronectin, tenascin, reelin, kindlin, talin or combination thereof, and may comprise a pharmaceutical composition comprising a therapeutically effective amount of the anb3 and anb5 antagonist peptidomimetic compound, covalently linked by a spacer sequence, wherein the compound may be a linear artificial sequence, a chelator such as NODAGA, DOTAGA, CHX-A and a pharmaceutically acceptable excipient for targeted radionuclide therapy (TRNT). The compound may also be a radiotracer for nuclear medicine, such as the Gallium-68 or Fluorine-l8, Zirconium-68, Copper-64, or Technetium-99m, covalently bound to a spacer, chelator, directly to the peptidomimetic, to a nanoparticle or to more than one amino acid unit of the peptidomimetic. Other radiotracers are those reported in the publication Radionuclide Peptide Cancer Therapy, edited by M. Chinol and G. Paganelli, Taylor and Francis CRC Press, 2006.

[0073] The peptide hereinafter referred to as integrin which may be a bifunctional molecule containing a linear RGD motif and a sequence corresponding to a C-terminal connected by a linker.

[0074] Synthesis and Production of Compounds

[0075] This peptidomimetic may be synthesized by a suitable production method, one such exemplary method being the solid-phase method using Fmoc chemistry. Amino acids are coupled according to the HBTU/HOBt/DIPEA procedure (Fields, C. G.; et al Pept. Res. 1991, 4, 95-101). Final deprotection and cleavage from the resin were achieved with TFA and scavengers. The purity and the identity of the peptides are confirmed by analytical RP-HPLC and MALDI-TOF mass spectrometry. Peptidomimetics are synthesized on an automated peptide synthesizer using Fmoc solid-phase strategy (0.25 mmol).

[0076] According to a preferred implementation, the a5b3 integrin peptide mimics that bind a5b3 integrin, may be produced by culturing a host cell so that the anti- a5b3 integrin antibody or antibody is produced. The composition may be attached to a suitable spacer sequence and/or a chelator, the radionuclide being attached to or via the chelator.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention provides methods of inhibiting the growth of a HER2+ tumor, methods of inhibiting proliferation of HER2+ tumor cells, methods of treating or stabilizing a HER2+ cancer, methods of inhibiting HER2+ tumor metastases, methods of reducing tumorigenicity of HER2+ tumor cells and methods of reducing the frequency of cancer stem cells or tumor initiating cells in a HER2+ tumor. More specifically, the methods provided herein comprise administering a combination of Peptide Receptor Radionuclide with an immune- oncology therapy. In some embodiments, the TRNT may be a therapy using a radiolabeled SSA peptide (Somatostatin analogue peptide) with high affinity for somatostatin receptors (SSTR) that carries a radioactive isotope such as Lu-l77 within its overall structure. In the present invention, the TRNT may be combined with standard of care; Trastuzumab and chemotherapy in the neoadjuvant setting but may be used with a PD-l and/or PD-L 1 /CDLA-4 or similar inhibitor. Tumor Targeted Radionuclide Therapy (TRNT)

[0078] The method involves administering a pharmaceutical composition that cytotoxic radiation to cancer cells, and in particular to the HER2+ cells, while preferably minimizing the toxicity to surrounding healthy tissue. The method involves delivering a radioligand that is designed specially to treat HER2+ cancers. The method may involve targeting the HER2+ cancel with the radiopharmaceutical, in particular with the radioligand that comprises the anti-integrin

peptidomimetic, the chelator and radionuclide (such as [l77]Lutetium or [225]Actinium). The anti- integrin peptidomimetic targets the HER2+ proteins or characteristic cell feature and specifically determines the localization of the radiopharmaceutical and the radionuclide carried by the composition is delivered to effect treatment. The methods herein also involve conducting diagnostics of cancers, and in particular HER2+ cancers, through non-invasive imaging by detection of g-rays using positron emission tomography (PET) or single-photon emission computerized tomography (SPECT), and/or as therapeutics for TRNT to deliver cancer cell killing radiation to the targeted tumor cells. A composition is administered to the patient, and the imaging carried out to determine the existence or prognosis of the cancer, or for use in staging. [0079] This < .v[’3inLcgrin antagonist peptidomimetic has high affinity for integrins on the surface of or in cancerous tumors and exquisitely targets the HER2+ protein which is overexpressed in breast cancer. Administering the radiolabeled peptidomimetic targets the HER2+ cancer in a patient or subject.

[0080] TRNT and/or a companion diagnostic for pre-targeting has not been utilized or approved by a regulatory agency for treatment of any of the ten types of breast cancer including HER2+.

[0081] TRNT has advantages over conventional cancer drugs, in particular the absence of collateral effects of chemotherapy or immunotherapy. Unlike external beam therapy, it is organ specific and not systemic and is not dose limiting. There is a reduction in renal excretion and/or hepatic degradation, leading to prolonged circulation times and less accumulation in healthy non target tissues and site-avoidance drug delivery, accumulation at pathological sites and site-specific drug delivery without the side effects of chemotherapy. As well, TRNT results in a high therapeutic index and accumulation at the targeted sites. The compound enables the monitoring drug delivery, drug release and drug efficacy, validation and optimization of combination therapies and to the ability to pre-screen patients, enabling personalized medicine. The efficient targeting capacity and fast clearance offers a high potential for targeted radionuclide therapy (TRNT). The pharmacokinetic and pharmacodynamic properties match the decay properties of the short-lived alpha and beta particle emitting radionuclides, offering a treatment option for solid tumors, metastases, micrometastatic cancer and residual disease.

[0082] A preferred implementation of the method involves treating a patient that has a human epidermal growth factor receptor 2-Positive, hereinafter referred to as HER2+, or other cancer that overexpresses integrin receptors with a pharmaceutical composition that is administered to the patient. The method involves administering to the patient a combination of a pharmacologically effective amount of an antagonist peptide mimetic compound of an integrin whose molecular structure includes a tetrahydropyridimidinyl-aminoethyloxybenzoyl group on a sulfonylamino-b- a!anine nucleus, exhibiting selective, high binding affinity for a5b3, hereinafter referred to as [33 integrin receptor when further substituted on the sulfonyl moiety with an N-amino alkyearbamyl group or a butyloxycarbonylamino aikylcarbamoyi group or similar groups with a chelating agent, and a diagnostic or therapeutic radionuclide for tumor-targeted [33 peptide mimetic receptor radionuclide therapy (hereinafter referred to as TRNT).

[0083] According to a preferred embodiment, the therapeutic compound comprises Lu-177 DOTAGA IAC: A ligand containing the active moiety, the anb3 integrin antagonist

peptidomimetic, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3- aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino^-alanine (IAC) and l-(l-carboxy-3- carbon tert butoxymethyl)-l,4,7,l0-tetraazacyclododecane (DOTAGA) (tBu)4 to radiolabel with Lutetium-l77 for peptide receptor Lutetium therapy. The method may provide the pharmaceutical in a kit form that may generate or comprise a ready-to-inject solution of the radioligand.

[0084] The invention involves administering the compound of the invention to treat certain HER2+‘s by selectively binding to SSTR2 receptors, the most commonly expressed receptors on these types of tumors. The compound of the invention then destroys HER2+ cells in a targeted fashion by delivering a local emission of high-energy electrons. Because The compound of the invention also emits gamma radiation, it can also be useful as a disease management tool, as this kind of emission can be captured with a SPECT camera and thus be used for determining the drug's distribution and pharmacokinetics and also for dosimetric estimations.

[0085] In light of the current limited options and effectiveness for treatment of HER2+ s overall and the lack of treatments for progressive midgut HER2+ s specifically, the invention can meet a significant medical need by potentially improving patient outcomes in the treatment of progressive midgut HER2+ s, as well as other somatostatin-receptor tumors.

[0086] The invention provides what the inventor considers is the first ever TRNT

radiopharmaceutical product being tested in a Phase ½ trial for the treatment of inoperable, progressive, HER2+ cancers.

[0087] An objective of TRNT is to selectively deliver cytotoxic radiation to cancer cells causing minimal toxicity to surrounding healthy tissues. This is achieved by delivering a radioactive payload into the tumor cells. Examples of this approach are Gallium-68 (68Ga), Yttrium-90 (90Y), Lutetium- 177 (l77Lu), Iodine- 131, (l77Lu), Zirconium-68, Actinium-225 (225Ac), Bismuth-213 (2l3Bi) and Lead-2l2 (2l2Pb) as radiolabeled peptides to treat tumors overexpressing specific proteins. In addition, monoclonal antibodies (abs) are also used as vehicles to target tumor- associated antigens providing internal radiotherapy. Regulatory-agency approved radiolabeled agents are 90Y -ibritumomab and 901, and l77Lu Lutathera for treatment of gastroenteropancreatic tumors. Thus, a therapeutic radiopharmaceutical can consist of two parts: a targeting biomolecule that specifically determines the localization of the radiopharmaceutical and a radionuclide that delivers the mechanism of action through its decay. Radiopharmaceuticals are used as diagnostics for non-invasive imaging by detection of g-rays using positron emission tomography (PET) or single-photon emission computerized tomography (SPECT), and/or as therapeutics for TRNT to deliver cancer cell killing radiation to the targeted tumor cells.

[0088] The crucial roles of integrin anb3 in tumor angiogenesis have led to a promising strategy to block signaling by antagonists, as this would theoretically inhibit tumor angiogenesis or enhance the efficacy of other tumor therapeutics. In addition, the high expression of integrin anb3 on tumor new-blood vessels and some tumor cells makes the anti-integrin anb3 a suitable marker for cancer-targeted drug delivery. According to a preferred embodiment, the therapeutic compound comprises Ga-68 DOTAGA and Lu-l77 DOTAGA: A ligand containing the active moiety, the anb3 integrin antagonist peptidomimetic, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3-aminoneopenta-l-carbamyl)]-aminoeth ylsulfonyl-amino-P-alanine (IAC) and l-(l-carboxy-3 -carbon tert butoxymethyl)-l,4,7,l0-tetraazacyclododecane (DOTAGA) (tBu)4 to radiolabel Lutetium-l77 for peptide receptor Lutetium therapy.

[0089] The preferred molecule used for tumor targeted radiotherapy in the invention consists of an integrin antagonist peptidomimetic isotope preferably [l77]Lutetium.

[0090] According to a preferred embodiment, a compound of the invention is comprised of three components. The first component is the integrin antagonist peptidomimetic. This component is the peptidomimetic that targets the HER2+ cells. The second component is a chelator, a compound able to combine radiometals (such as Lu-l77) into complexes through a ring-like structure; and the third optional component is the radionuclide to enhance uptake to the tumor and extend biological half life.

[0091] A potent < .v(’3inLcgrin antagonist peptide mimic targeting agent specific for the HER2+ protein was developed, modified for use in humans and radiolabeled with Gallium-68, Fluorine- 18 for diagnosis using positron emission tomography, or Lutetium- 177 or Actinium-225 for use in humans enabling cancerous tumor detection using positron emission tomography and for tumor targeted radionuclide therapy (TRNT) alone or in combination with standard-of-care;

immunotherapy and chemotherapy in the neoadjuvant setting. The radioligands enable non- invasive diagnosis and treatment of a wide range of cancers, while also reducing harmful side effects such as cardiovascular disease, peripheral neuropathy, and improving survival and quality of life.

Methods of Treatment

[0092] The present invention provides methods of treating HER2+ tumors and include a group with a wide range of morphologic, functional, and behavioral characteristics. These tumors are generally slow growing but have the potential to spread, primarily to the liver or brain, and when they do, they can be life threatening and difficult to treat with current modalities.

[0093] HER2+ tumors have traditionally been classified by the site of their origin. In certain embodiments, the HER2+ is selected from the group consisting of pancreatic HER2+ tumors (pHER2+ s) and tumors of the lung, stomach, duodenum, jejunum, ileum, colon and rectum. In further embodiments, the HER2+ is selected from the group consisting HER2+ tumors of the ovary, thymus, thyroid medulla, adrenal glands (e.g., pheochromocytoma) and paraganglia (paraganglioma). In certain embodiments, the HER2+ treated by the methods described herein is small cell lung cancer (SCLC). In certain alternative embodiments, the HER2+ is a non-small cell lung cancer. In certain embodiments, HER2+ s are pancreatic HER2+ tumors (PETs) or carcinoid tumors. In certain embodiments, the HER2+ is non-small cell lung cancer, pancreatic cancer, or thyroid cancer. [0094] HER2+ tumors are also classified by grade and differentiation. See, e.g., Phan et al., Pancreas, 39(6):784-798 (2012) . In certain embodiments, the HER2+ tumor is a well

differentiated, low grade tumor. In certain embodiments, the HER2+ tumor is a moderately differentiated, intermediate grade tumor. In certain embodiments, the HER2+ tumor is poorly differentiated, high grade tumor. In one embodiment, low grade tumors are characterized by <2 mitoses per 10 HPF (high power fields) and no necrosis. In one embodiment, intermediate grade tumors are characterized by 2-10 mitoses per 10 HPF (high power fields) or foci of necrosis. In one embodiment, high grade tumors are characterized by >10 mitoses per 10 HPF (high power fields).

[0095] In other embodiments HER2+ tumors can be divided based on the WHO classification 2000 and 2010 into HER2+ tumors Grade 1 -Grade 2 (or Well-differentiated endocrine tumor or carcinoma (WDET / WDEC), HER2+ carcinoma Grade 3 or Poorly differentiated endocrine carcinoma/small-cell carcinoma (PDEC), Mixed adenoHER2+ carcinoma (MANEC) and

Hyperplastic and preneoplastic lesions. According to the HER2+ SIWHO/AJCC Classification systems Tumors G1 are those with Ki67 index < 2% or Ml (mitotic count) <2, Tumors G2 are those with Ki67 index within 3 -20% or Ml = 2-20 and tumors G3 are those with Ki67 index >

20% or Ml >20.

[0096] HER2+ tumors are also classified as functional and non-functional HER2+ s. HER2+ s are considered functional when a specific clinical syndrome is induced due to excessive production of hormones by the tumor cells. Examples of functional HER2+ s include, but are not limited to, carcinoid tumors, which can result in carcinoid syndrome, and functional pHER2+ s, for example, insulinomas, gastrinomas, vasoactive intestinal peptide (VIPomas), glucagonomas, and

somatostatinomas.

[0097] Non-functional HER2+ s are not associated with a clinical syndrome due to excessive production of hormones by the tumor cells, but can still produce symptoms related to the presence of the tumor or its metastasis (e.g., abdominal pain or bloating). In certain embodiments, the HER2+ tumor is a functional HER2+ . In certain embodiments, the HER2+ tumor is a non functional HER2+ . In certain embodiments, the HER2+ tumor is selected from the group consisting of functional carcinoid tumor, insulinoma, gastrinoma, vaso active intestinal peptide VIPoma, glucagonoma, serotoninoma, histaminoma, ACTHoma, pheochromocytoma, and somatostatinoma. In certain embodiments, the HER2+ tumor is NSCLC.

[0098] In certain embodiments, the HER2+ tumor is a primary tumor. In alternative embodiments, the HER2+ tumor is metastatic tumor. In certain embodiments, the HER2+ tumor has not spread outside of the wall of the primary organ. In certain embodiments, the HER2+ tumor has spread through the wall of the primary organ and to nearby tissues, such as fat, muscle, or lymph nodes. In certain embodiments, the HER2+ tumor has spread to tissues or organs away from the primary organ, for example, to the liver, bones, or lungs.

[0099] In specific embodiments, it is contemplated that the methods of the present invention will be particularly useful in the treatment of HER2+ cancer or tumor that is refractory to treatment. As a non-limiting example, the cancer or tumor may be chemorefractory (i.e., resistant to one or more forms of chemotherapy). In certain embodiments, the cancer or tumor is resistant to treatment with a somatostatin analog. In certain embodiments, the cancer or tumor is resistant to treatment with a kinase inhibitor. In still other embodiments the cancer or tumor is resistant to treatment with an inhibitor of the PD-l /PD-L 1 /CTLA-4 pathway.

[00100] In certain embodiments, the HER2+ cancer or tumor has metastasized to the liver.

By way of non-limiting example, the HER2+ cancer or tumor is a carcinoid or

pancreatic HER2+ tumor that has metastasized to the liver.

[00101] Compounds useful in connection with the invention may be produced by methods well known to those of skill in the art. Exemplary such methods include those described in US 5,804,157 or US 5,830,431, the complete disclosures of which are herein incorporated by reference.

[00102] The present invention provides for methods of treating HER2+ tumor comprising administering a therapeutically effective amount of a Tumor Targeted Radionuclide Therapy (TRNT) in combination with an inhibitor of the PD-l /PD-L 1 /CTLA-4 pathway to a subject (e.g., a subject in need of treatment). In certain embodiments, the HER2+ tumor is a

pancreatic HER2+ tumor. In certain embodiments, the HER2+ tumor is a carcinoid. In certain embodiments, the HER2+ tumor is HER2+ tumor of the lung. By way of non-limiting example, the HER2+ tumor in the lung may be SCLC. The invention is particularly useful for the treatment of HER2+ tumors that overexpress SSTR in their cellular surface such as (but not limited to) pituitary adenomas, gastrointestinal and pancreatic endocrine carcinomas (GEPNET tumors), pulmonary HER2+ , paragangliomas, pheochromocytomas, small cell lung cancers, medullary thyroid carcinomas, breast cancers, prostate cancer and malignant lymphomas. In certain embodiments, the subject is a human. In certain embodiments, the TRNT is the invention, for example, comprising the compositions and their application to deliver a therapeutically effective treatment to the patient.

[00103] The present invention further provides methods for

inhibiting HER2+ tumor growth using a therapeutically effective amount of a TRNT in combination with an inhibitor of the PD-l /PD-L 1 /CTLA-4 pathway to a subject. In certain embodiments, the method of inhibiting the HER2+ tumor growth comprises contacting

the tumor cell with a therapeutically effective amount of one or both of a TRNT and with an inhibitor of the PD-l /PD-L 1 /CTLA-4 pathway to a subject in vitro. For example, an immortalized HER2+ tumor cell line is cultured in medium to which is added the TRNT to inhibit tumor growth. In some embodiments, HER2+ tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and cultured in medium to which is added the TRNT and/or the inhibitor of the PD-l/PD-L 1 /CTLA-4 pathway to inhibit tumor growth.

[00104] In some embodiments, the method of inhibiting HER2+ tumor growth comprises contacting the HER2+ tumor or tumor cells with combination therapy of the present invention in vivo. In certain embodiments, contacting a HER2+ tumor or tumor cell with a TRNT and an inhibitor of the PD-1 /PD-L l/CTLA-4 pathway is performed in an animal model. For example, a TRNT and an inhibitor of the PD-1 /PD-L 1 /CTLA-4 pathway may be administered to HER2+ tumor xenografts that have been grown in immunocompromised mice (e.g. NOD/SCID mice) to inhibit HER2+ tumor growth. In some embodiments, HER2+ tumor cancer stem cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and injected into immunocompromised mice that are then administered the combination therapy of the present invention (i.e., a TRNT and an inhibitor of the PD-1 /PD-L 1 /CTLA-4 pathway) to inhibit HER2+ tumor cell growth. In some embodiments, the TRNT and/or the PD-1 /PD-L 1 /CTLA-4 pathway inhibitor is administered at the same time or shortly after the introduction of tumorigenic cells into the animal to prevent HER2+ tumor growth. In some embodiments, the TRNT and/or the inhibitor of the PD-1 /PD-L 1 /CTLA-4 pathway is administered as a therapeutic after the tumorigenic cells have grown to a specified size.

[00105] In certain embodiments, the method of inhibiting HER2+ tumor growth comprises administering to a subject a therapeutically effective amount of the TRNT and the inhibitor of the PD-1 /PD-L l/CTLA-4. In certain embodiments, the subject is a human. In certain embodiments, the subject has a HER2+ tumor or has had a tumor removed.

[00106] In certain embodiments, the HER2+ tumor is a pancreatic HER2+ tumor. In certain embodiments, the HER2+ tumor is a carcinoid. In certain embodiments, the HER2+ tumor is HER2+ tumor of the lung. In certain embodiments, the HER2+ tumor is NSCLC.

[00107] In addition, the invention provides a method of reducing the tumorigenicity of a HER2+ tumor in a subject, comprising administering a therapeutically effective amount of a TRNT and an inhibitor of the PD-1 /PD-L 1 to the subject. In certain embodiments, the HER2+ tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the HER2+ tumor is reduced by administration of the agent. In certain embodiments, the TRNT is The invention.

[00108] Thus, the invention also provides a method of reducing the frequency of cancer stem cells in a HER2+ tumor, comprising contacting the tumor with an effective amount of a TRNT and an inhibitor of the PD-1 /PD-L 1 /CTLA-4 pathway. [00109] As noted herein, the PORT is administered in combination with an inhibitor of the

PD-l /PD-L 1 pathway. In such methods, the TRNT may be administered prior to, concurrently with, and/or subsequently to administration of the PD-l /PD-L 1 /CTLA-4 inhibitor.

Pharmaceutical compositions comprising the TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitor are also provided. It is contemplated that the combined treatment with the TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitor has a synergistic effect on the treatment of the HER2+ s.

[00110] It will be appreciated that the combination of a TRNT and the PD- 1 /PD-L 1

/CTLA-4 inhibitor agent may be administered in any order or concurrently. In selected

embodiments, the TRNT and PD-l /PD-L 1 /CTLA-4 will be administered to patients that have previously undergone treatment with other anti-cancer agents. In certain other embodiments, the TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitor agent will be administered substantially simultaneously or concurrently. For example, a subject may be given TRNT while undergoing a course of treatment with the PD-l /PD-L 1 /CTLA-4 inhibitor agent. In addition it is contemplated that the subject has already or may be concurrently receiving other forms of cancer therapy, e.g., chemotherapy. In certain embodiments, the TRNT will be administered within 1 year of the treatment with the PD-l /PD-L 1 /CTLA-4 inhibitor agent. In certain alternative embodiments, the TRNT will be administered within 10, 8, 6, 4, or 2 months of any treatment with the PD-l /PD-L 1 /CTLA-4 inhibitor agent and/or additional anti-cancer agent. In certain other embodiments, the TRNT will be administered within 4, 3, 2, or 1 week of any treatment with the PD-l /PD-L l/CTLA-4 inhibitor agent and/or additional anti-cancer agent. In some embodiments, the TRNT will be administered within 5, 4, 3, 2, or 1 days of any treatment with the PD-l /PD-L 1 /CTLA-4 inhibitor agent and/or additional anti-cancer agent. It will further be appreciated that the TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitor agent and/or additional anti-cancer agent or treatment may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).

[00111] In addition to the administration of the combination of TRNT and at least one PD-l

/PD-L 1 inhibitor, it may be useful to also administer additional anticancer agents. Useful classes of anti-cancer agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin),

anthracyclines, antibiotics, antifolates, anti -metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, performing compounds, purine anti metabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like or may be attached to a nanoparticle which crossed the blood-brain barrier for delivery of this cytotoxic agent to brain tumors. In certain embodiments, the second anti-cancer agent is an antimetabolite, an antimitotic, a topoisomerase inhibitor, or angiogenesis inhibitor. [00112] Anticancer agents that may be administered in combination with the TRNT and

PD-l /PD-L 1 /CTLA-4 inhibitor include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the combined administration of a TRNT and PD- 1 /PD-L 1 /CTLA-4 inhibitor and a chemotherapeutic agent or cocktail of multiple different

chemotherapeutic agents. Treatment with a TRNT can occur prior to, concurrently with, or subsequent to administration of these other therapies. Chemotherapies contemplated by the invention include chemical substances or drugs which are known in the art and are commercially available, such as gemcitabine, irinotecan, doxorubicin, 5-fluorouracil, cytosine arabinoside ("Ara- C"), cyclophosphamide, thiotepa, busulfan, cytoxan, Taxol or taxene, methotrexate, cisplatin, melphalan, vinblastine and carboplatin. Combined administration can include co-administration, eitHER2+in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner.

[00113] In certain embodiments, the treatment involves the combined administration of a

PORT and PD-l /PD-L 1 /CTLA-4 inhibitor described herein and radiation therapy. Treatment with the TRNT can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Any dosing schedule for such radiation therapy can be used as determined by the skilled practitioner.

[00114] In some embodiments, the second anti-cancer agent comprises an antibody. Thus, treatment can involve the combined administration of a TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitor agent with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind to EGFR, ErbB2, HER2+, DLL4, Notch and/or VEGF. In certain

embodiments, the second anti-cancer agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF antibody). In certain embodiments, the second anti-cancer agent is an inhibitor of Notch signaling. In certain embodiments, the second anti-cancer agent is Avastin (Bevacizumab), Trastuzumab (Trastuzumab), Vectibix (Panitumumab), or Erbitux (Cetuximab). Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.

[00115] Furthermore, treatment can include administration of one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of cancer cells or cytoreductive treatments such as (chemo-radio)embolization, radiofrequency ablation and high-intensity focused ultrasound (HIFU) ablation or any other therapy deemed necessary by the treating physician. [00116] Preferably, TRNT with the therapeutic treatment using the compounds of the invention is given in a cycle wherein each cycle comprises 3.7-7.4GBq administered

intravenously. Typically, a therapeutic cumulative activity (dose) of approximately 30 GBq may readily be divided into 4 to 6 cycles, each administered every 5 to 12 weeks. It should be understood that longer intercycle intervals as long as 6 Months or even years can be used.

Moreover, the patient may be treated with multiple rounds of the 4 to 6 cycles after months or years from the first set of treatments. The second and subsequent set of treatments may use the same activities (doses) or may use different activities (doses) depending on the experience of the center and of the characteristics and therapeutic needs of each patient.

[00117] Anti-PD-l nivolumab is typically administered intravenously at doses of about 3 mg/kg every 2-3 weeks, for an initial period of two years. Thereafter, maintenance therapy every 12 weeks after the initial treatment are frequently used. It should be understood that these dosing regimens will vary according to the patient's response to the treatments and at the discretion of the treating clinician. The recommended dose of ipilimumab for the treatment of unresectable or metastatic melanoma is 3 mg/kg administered intravenously over 90 minutes every 3 weeks for a total of four doses.

[00118] The TRNT and the PD-l /PD-L 1 /CTLA-4 inhibitors (e.g., antibodies and soluble receptors) can be formulated into a pharmaceutical composition by any suitable method known in the art. In certain embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable vehicle. The pharmaceutical compositions find use in inhibiting HER2+ tumor growth and treating HER2+ tumor in human patients.

[00119] The methods and therapeutic compositions of the present invention may be used in connection with a diagnostic. The diagnostic may be administered to the patient to identify areas of tumor or cancer cells or growth, and may be used to determine one or more subsequent treatment parameters for a treatment protocol, such as the treatment dosage and extent (e.g., where the treatment involves administering an a treatment compound according to the invention, such as the preferred composition, the integrin peptidomimetic Lu-l77 DOTAGA IAC construct.

According to some embodiments, a preferred diagnostic composition may comprise Ga-68- NODAGA IAC: A ligand containing the active moiety, the anb3 integrin antagonist

peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoy l-2-[N-(3- aminoneopenta-l-carbamyl)]-aminoethylsulfonyl-amino-P-alanin e (IAC) and to l-(l-carboxy-3- carbo-t-butoxypropyl)-4,7-(carbo-tert-butoxymethyl)-l,4,7-tr iazacyclononane.

[00120] The diagnostic and the therapeutic compositions preferably are prepared from components of a kit, where the composition is prepared and radiolabeled with the respective radionuclide prior to administering the composition to a patient/subject, which preferably is done intravenously. [00121] PROPOSED EXAMPLES

[00122] PROPOSED EXAMPLE 1:

[00123] Example 1 : a preferred compound of the invention, comprising Lu-l77 DOTAGA

IAC, is administered to a human patient at a fixed dose of 7.4 GBq (every 12 weeks) up to a cumulative dose which is tolerated by the patient (maximum 29.6 GBq). The preferred compound preferably is administered intravenously. Nivolumab is administered twice for each treatment of the preferred Lu-l77 DOTAGA IAC compound, at a dose of 3 mg/Kg: one administration seven days before (d-7) and the other administration seven days after (d-positive7) administration of the compound of the invention, with the aim of achieving an effective PD-l /PD-L1 blockade, but also in the need not to overlap the anticipated lymphocyte nadir related to the lymphocytopenia-induced effect of the compound of the invention.

[00124] Studies have shown that intravenous administration of amino acids has a renal protective effect. An infusion of amino acids (containing lysine and arginine) could be done 30 to 45 minutes before the administration of l77Lu-DOTATATE and last for 3 to 4 hours.

[00125] PROPOSED EXAMPLE 2:

[00126] Example 2: In a proposed example, the compound of the present invention was administered to a patient suffering from HER2+ breast cancer. The patient was given the compound of the invention (e.g., in an amount of from about 3.7-7.4GBq) administered intravenously, and representing a dosage. One or more, and preferably a plurality of subsequent treatments of a similar amount are dosed to the patient intravenously, a couple to a few weeks from the first dosage. In this example, an additional therapeutic may be administered on either side of a window based on when the patient receives the inventive compound doses, e.g., such as seven days prior to a dose and seven days after a dose.

[00127] PROPOSED EXAMPLE 3:

[00128] Example 3: The patient was treated as in proposed example 1, above, however, prior to treatment with the Lu-l77 DOTAGA IAC, the patient was diagnosed using the preferred diagnostic composition, comprising Ga-68-NODAGA IAC.

[00129] PROPOSED EXAMPLE 4:

[00130] Example 4: The patient was treated as in proposed example 2, above, however, prior to treatment with the Lu-l77 DOTAGA IAC, the patient was diagnosed using the preferred diagnostic composition, comprising Ga-68-NODAGA IAC.

[00131] PROPOSED EXAMPLE 5:

[00132] Example 5: The patient was treated as in Example 1, but with the radionuclide consisting of one or more of: Actinium-225, Bismuth-2l3, Thorium-227, Lead-2l2, Astatine-2l 1, Yttrium-90, and Iodine- 131.

[00133] PROPOSED EXAMPLE 6: Example 6: The patient was treated as in Example 5, but with the chelator comprising one or more of: 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino) ethyloxy]benzoyl-2-[N-(3-aminoneopenta- l-carbamyl)]-aminoethylsulfonyl-amino-P-alanine (IAC), and (ii) at least one of the following:

(a) 1 -( 1 -carboxy-3 -carbotertbutoxy methyl)- 1 ,4,7, 10-tetraazacyclododecane (DOTAGA) (tBu).sub.4; or

(b) 4-[4,7-Bis-(carboxymethyl)-[l,4,7]triazonan-l-yl]-4-carboxy- butyryl

(NODAGA); or

(c) DOT AT ATE; or

(d) C26H34N4O10S (CHX-A).

[00134] Breast cancer metastasis to the brain develops after a clinical latency of years to even decades, suggesting that colonization of the brain is the most challenging step of the metastatic cascade. However, the underlying mechanisms used by breast cancer cells to successfully colonize the brain's microenvironment remain elusive. Reelin is an archetypal extracellular glycoprotein that regulates migration, proliferation, and lamination of neurons. It is epigenetically silenced in various cancers, and its expression in multiple myelomas is linked to poor patient survival. Reelin expression was low in primary breast cancer tissue. However, its expression is significantly higher in HER2+ breast cancers metastasizing to the brain. In particular, Reelin was highly expressed in the tumor periphery adjacent to surrounding astrocytes. This augmented Reelin expression was seen in HER2+ metastases, but not in triple negative (TN) primary tumors or in TN breast to brain metastasis cells co-cultured with astrocytes. The relative growth and rate of spheroids formation derived from HER2+ primary and BBM cells co-cultured with astrocytes were higher than those of TN primary and BBM cells, and knockdown of both Reelin and HER2+ suppressed the astrocyte-induced growth and spheroid forming ability of HER2+ cells. Within the neural niche, astrocytes epigenetically regulate Reelin expression and its interaction with HER2+ leading to increased proliferation and survival fitness.

Kindlin

[00135] Kindlins are a small family of 4. l-ezrin-radixin-moesin (FERM)-containing cytoplasmic proteins. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Kindlin-3 promotes integrin activation, clustering and outside-in signaling. Aberrant expression of kindlin-3 was reported in HER2+ breast cancer. Intriguingly, kindlin-3 positively regulates cancer cell metastasis. The expression of kindlin-3 in HER2+ cancer cells and its role in metastasis has been reported. This data demonstrates that despite its well-established role as a positive regulator of integrin-mediated cell adhesion, aberrant expression of kindlin-3 could lead to imbalanced Rho GTPases signaling that promotes cell migration. Kindlin-2 has been reported to be considered a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Racl and supplying Racl with the Arp2/3 complex. It has been reported that Kindlin 2 markedly downregulates the expression of miR-200 family by inducing CpG island hypermethylation. Mechanistically, Kindlin 2 forms a complex with DNMT3A in the cell nucleus and the two proteins co-occupy the promoter of miRNA- 200b. Functionally, repression of miR- 200b is required for Kindlin 2-induced breast cancer cell invasion and tumor formation. Kindlin 2 plays a novel role in epigenetic repression of miR-200 family, a mechanism that promotes breast cancer invasion. miR-200b is required for Kindlin 2-induced breast cancer cell invasion and tumor formation data suggest a novel mechanism that Kindlin-2 regulates breast cancer progression by inducing genome instability. Kindlin 2 plays a novel role in epigenetic repression of miR-200 family, a mechanism that promotes breast cancer invasion.

[00136] The alpha (5) beta (3) integrin antagonist targets several proteins or any combination thereof including integrins, vitronectin, fibronectin and tenascin. The one or more proteins and mRNAs that also may be targeted can include, but are not limited to Actin, Reelin, Talin, and Kindlin or any combination thereof and can be used as specific biomarkers and targeted in HER2+ positive cancers with a radiolabeled ligand.

[00137] The anti-integrin antagonist that is the subject of this invention adheres to the

HER2+ protein and for some patients may be used in combination with standard of care or alone. This can be a‘break-through’ in treatment and for some, enable targeting of brain metastases, micrometastases or recurrence.

[00138] The action of the HER2+ protein may be blocked using the alpha5 beta 3 integrin antagonist peptidomimetic that attaches to specific proteins and disrupts its function.

[00139] TRNT might improve survival rates for women with stage 2 to 4 HER2+ breast cancer significantly.

[00140] The HER2+ protein is expressed at high levels in several other cancers besides breast cancer. The radioligand enables treatment of some patients with HER2+ gastric, gastroesophageal junction, ovarian or HER2+ cancer that has metastasized to other organs including brain. This radioligand works by blocking HER2+ from sending signals to other proteins that cause cells to grow and replicate and destroying the cancer cells with radiation or impairing the DNA so the cells cannot replicate.

[00141] This anb3 integrin antagonist peptidomimetic has high affinity for integrins on the surface of or in cancerous tumors and exquisitely targets the HER2+ protein which is

overexpressed in breast cancer. Serendipitously, it was discovered that in addition to adhering to integrins, tenascin, vitronectin and fibronectin, that reelin, actin, talin and kindlin are also targeted. These are important targets in tumor and micrometastatic targeting. Potentially, ovarian, gastric gastroesophageal junction, brain metastases and other HER2+ cancers can be treated.

[00142] Reelin, an archetypal extracellular glycoprotein highly expressed in the tumor in

HER2+ breast cancer tumors, enabling precise delivery of the radioligand consisting of an anti- integrin peptidomimetic, a chelator and a radionuclide which may be Lutetium-l77 or Actinium- 225 that can destroy cancerous cells, including micrometastases. Astrocyte induced reelin expression drives proliferation of HER2+ breast cancer metastases. By measuring reelin expression in breast cancer-derived cell lines before and after TRNT treatment, it has been established that reelin plays an important role in invasiveness and metastatic potential of breast cancer cells and has an important role in invasiveness and metastases. Breast cancer metastasis to the brain develops after a clinical latency of years to even decades, suggesting that colonization of the brain is the most challenging step of the metastatic cascade. However, the underlying mechanisms used by breast cancer cells to successfully colonize the brain's microenvironment remain elusive. Reelin is an archetypal extracellular glycoprotein that regulates migration, proliferation, and lamination of neurons. It is epigenetically silenced in various cancers, and its expression in multiple myelomas is linked to poor patient survival. Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. It has been reported that reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Reelin expression was analyzed by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2+ status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P=0.0l) and positive lymph node status (P<0.00l). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, Reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. A syngeneic mouse mammary tumor transplantation model was used to examine the impact of host-derived reelin on breast cancer progression. It was found that transplanted syngeneic tumors grew more slowly in reelin-deficient (rl ^{0rl ;} ) mice and had delayed metastatic colonization of the lungs.

Immunohistochemistry of primary tumors revealed that tumors grown in rl ^{0rl ;} animals had fewer blood vessels and increased macrophage infiltration. Gene expression studies from tumor tissues indicate that loss of host-derived reelin alters the balance of Ml - and M2-associated macrophage markers, suggesting that reelin may influence the polarization of these cells. Consistent with this, rl ^{0rl ;} M 1 -polarized bone marrow-derived macrophages have heightened levels of the M 1 - associated cytokines iNOS and IL-6. Based on these observations, there has been proposed a novel function for the reelin protein in breast cancer progression. It has been concluded that that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation. Reelin expression has been found to be low in primary breast cancer tissue. However, its expression was significantly higher in HER2+ breast cancers metastasizing to the brain. In particular, Reelin was highly expressed in the tumor periphery adjacent to surrounding astrocytes. This augmented Reelin expression was seen in HER2+ metastases, but not in triple negative (TN) primary tumors or in TN breast to brain metastasis cells co-cultured with astrocytes. Furthermore, the elevated expression was sustained in HER2+ cells grown in the presence of the DNA methyltransferase inhibitor 5-azacytidine, indicating epigenetic regulation of Reelin expression. The relative growth and rate of spheroids formation derived from HER2+ primary and BBM cells co-cultured with astrocytes were higher than those of TN primary and BBM cells, and knockdown of both Reelin and HER2+ suppressed the astrocyte-induced growth and spheroid forming ability of HER2+ cells. Within the neural niche, astrocytes epigenetically regulate Reelin expression and its interaction with HER2+ leading to increased proliferation and survival fitness. Reelin/integrin cell adhesion underscores the therapeutic potential of targeting reelin/integrin expression in HER2+ tumors.